Yee‐Joo Tan

Article24 June 2020Open Access Transparent process Mechanism of baricitinib supports artificial intelligence-predicted testing in COVID-19 patients Justin Stebbing Justin Stebbing orcid.org/0000-0002-1117-6947 Department of Surgery and Cancer, Imperial College, London, UK Search for more papers by this author Venkatesh Krishnan Corresponding Author Venkatesh Krishnan [email protected] orcid.org/0000-0002-3295-6598 Eli Lilly and Company, Indianapolis, IN, USA Search for more papers by this author Stephanie de Bono Stephanie de Bono Eli Lilly and Company, Indianapolis, IN, USA Search for more papers by this author Silvia Ottaviani Silvia Ottaviani Department of Surgery and Cancer, Imperial College, London, UK Search for more papers by this author Giacomo Casalini Giacomo Casalini Luigi Sacco, Department of Clinical and Biomedical Sciences, University of Milan, Milan, Italy Search for more papers by this author Peter J Richardson Peter J Richardson BenevolentAI, London, UK Search for more papers by this author Vanessa Monteil Vanessa Monteil Unit of Clinical Microbiology, Department of Laboratory Medicine, Karolinska Institutet and Karolinska University Hospital, Stockholm, Sweden Department of Physiology and Pharmacology, Karolinska Institutet and Karolinska University Hospital, Stockholm, Sweden Search for more papers by this author Volker M Lauschke Volker M Lauschke Unit of Clinical Microbiology, Department of Laboratory Medicine, Karolinska Institutet and Karolinska University Hospital, Stockholm, Sweden Department of Physiology and Pharmacology, Karolinska Institutet and Karolinska University Hospital, Stockholm, Sweden Search for more papers by this author Ali Mirazimi Ali Mirazimi Unit of Clinical Microbiology, Department of Laboratory Medicine, Karolinska Institutet and Karolinska University Hospital, Stockholm, Sweden Department of Physiology and Pharmacology, Karolinska Institutet and Karolinska University Hospital, Stockholm, Sweden Search for more papers by this author Sonia Youhanna Sonia Youhanna Unit of Clinical Microbiology, Department of Laboratory Medicine, Karolinska Institutet and Karolinska University Hospital, Stockholm, Sweden Department of Physiology and Pharmacology, Karolinska Institutet and Karolinska University Hospital, Stockholm, Sweden Search for more papers by this author Yee-Joo Tan Yee-Joo Tan Infectious Diseases Programme, Immunology Programme, Department of Microbiology and Immunology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore City, Singapore Institute of Molecular and Cell Biology (IMCB), A*STAR (Agency for Science, Technology and Research), Singapore, Singapore Search for more papers by this author Fausto Baldanti Fausto Baldanti Department of Clinical, Surgical, Diagnostics and Pediatric Sciences, University of Pavia, Pavia, Italy Molecular Virology Unit, Fondazione IRCCS Policlinico San Matteo, Pavia, Italy Search for more papers by this author Antonella Sarasini Antonella Sarasini Department of Clinical, Surgical, Diagnostics and Pediatric Sciences, University of Pavia, Pavia, Italy Molecular Virology Unit, Fondazione IRCCS Policlinico San Matteo, Pavia, Italy Search for more papers by this author Jorge A Ross Terres Jorge A Ross Terres Eli Lilly and Company, Indianapolis, IN, USA Search for more papers by this author Brian J Nickoloff Brian J Nickoloff Eli Lilly and Company, Indianapolis, IN, USA Search for more papers by this author Richard E Higgs Richard E Higgs Eli Lilly and Company, Indianapolis, IN, USA Search for more papers by this author Guilherme Rocha Guilherme Rocha Eli Lilly and Company, Indianapolis, IN, USA Search for more papers by this author Nicole L Byers Nicole L Byers Eli Lilly and Company, Indianapolis, IN, USA Search for more papers by this author Douglas E Schlichting Douglas E Schlichting Eli Lilly and Company, Indianapolis, IN, USA Search for more papers by this author Ajay Nirula Ajay Nirula Eli Lilly and Company, Indianapolis, IN, USA Search for more papers by this author Anabela Cardoso Anabela Cardoso Eli Lilly and Company, Indianapolis, IN, USA Search for more papers by this author Mario Corbellino Mario Corbellino Division of Infectious Diseases, ASST Fatebenefratelli Sacco, Milan, Italy Search for more papers by this author on behalf of the Sacco Baricitinib Study Group the Sacco Baricitinib Study GroupSacco Baricitinib Study Group members are listed in the AppendixSearch for more papers by this author Justin Stebbing Justin Stebbing orcid.org/0000-0002-1117-6947 Department of Surgery and Cancer, Imperial College, London, UK Search for more papers by this author Venkatesh Krishnan Corresponding Author Venkatesh Krishnan [email protected] orcid.org/0000-0002-3295-6598 Eli Lilly and Company, Indianapolis, IN, USA Search for more papers by this author Stephanie de Bono Stephanie de Bono Eli Lilly and Company, Indianapolis, IN, USA Search for more papers by this author Silvia Ottaviani Silvia Ottaviani Department of Surgery and Cancer, Imperial College, London, UK Search for more papers by this author Giacomo Casalini Giacomo Casalini Luigi Sacco, Department of Clinical and Biomedical Sciences, University of Milan, Milan, Italy Search for more papers by this author Peter J Richardson Peter J Richardson BenevolentAI, London, UK Search for more papers by this author Vanessa Monteil Vanessa Monteil Unit of Clinical Microbiology, Department of Laboratory Medicine, Karolinska Institutet and Karolinska University Hospital, Stockholm, Sweden Department of Physiology and Pharmacology, Karolinska Institutet and Karolinska University Hospital, Stockholm, Sweden Search for more papers by this author Volker M Lauschke Volker M Lauschke Unit of Clinical Microbiology, Department of Laboratory Medicine, Karolinska Institutet and Karolinska University Hospital, Stockholm, Sweden Department of Physiology and Pharmacology, Karolinska Institutet and Karolinska University Hospital, Stockholm, Sweden Search for more papers by this author Ali Mirazimi Ali Mirazimi Unit of Clinical Microbiology, Department of Laboratory Medicine, Karolinska Institutet and Karolinska University Hospital, Stockholm, Sweden Department of Physiology and Pharmacology, Karolinska Institutet and Karolinska University Hospital, Stockholm, Sweden Search for more papers by this author Sonia Youhanna Sonia Youhanna Unit of Clinical Microbiology, Department of Laboratory Medicine, Karolinska Institutet and Karolinska University Hospital, Stockholm, Sweden Department of Physiology and Pharmacology, Karolinska Institutet and Karolinska University Hospital, Stockholm, Sweden Search for more papers by this author Yee-Joo Tan Yee-Joo Tan Infectious Diseases Programme, Immunology Programme, Department of Microbiology and Immunology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore City, Singapore Institute of Molecular and Cell Biology (IMCB), A*STAR (Agency for Science, Technology and Research), Singapore, Singapore Search for more papers by this author Fausto Baldanti Fausto Baldanti Department of Clinical, Surgical, Diagnostics and Pediatric Sciences, University of Pavia, Pavia, Italy Molecular Virology Unit, Fondazione IRCCS Policlinico San Matteo, Pavia, Italy Search for more papers by this author Antonella Sarasini Antonella Sarasini Department of Clinical, Surgical, Diagnostics and Pediatric Sciences, University of Pavia, Pavia, Italy Molecular Virology Unit, Fondazione IRCCS Policlinico San Matteo, Pavia, Italy Search for more papers by this author Jorge A Ross Terres Jorge A Ross Terres Eli Lilly and Company, Indianapolis, IN, USA Search for more papers by this author Brian J Nickoloff Brian J Nickoloff Eli Lilly and Company, Indianapolis, IN, USA Search for more papers by this author Richard E Higgs Richard E Higgs Eli Lilly and Company, Indianapolis, IN, USA Search for more papers by this author Guilherme Rocha Guilherme Rocha Eli Lilly and Company, Indianapolis, IN, USA Search for more papers by this author Nicole L Byers Nicole L Byers Eli Lilly and Company, Indianapolis, IN, USA Search for more papers by this author Douglas E Schlichting Douglas E Schlichting Eli Lilly and Company, Indianapolis, IN, USA Search for more papers by this author Ajay Nirula Ajay Nirula Eli Lilly and Company, Indianapolis, IN, USA Search for more papers by this author Anabela Cardoso Anabela Cardoso Eli Lilly and Company, Indianapolis, IN, USA Search for more papers by this author Mario Corbellino Mario Corbellino Division of Infectious Diseases, ASST Fatebenefratelli Sacco, Milan, Italy Search for more papers by this author on behalf of the Sacco Baricitinib Study Group the Sacco Baricitinib Study GroupSacco Baricitinib Study Group members are listed in the AppendixSearch for more papers by this author Author Information Justin Stebbing1,‡, Venkatesh Krishnan *,2,‡, Stephanie Bono2, Silvia Ottaviani1, Giacomo Casalini3, Peter J Richardson4, Vanessa Monteil5,6, Volker M Lauschke5,6, Ali Mirazimi5,6, Sonia Youhanna5,6, Yee-Joo Tan7,8, Fausto Baldanti9,10, Antonella Sarasini9,10, Jorge A Ross Terres2, Brian J Nickoloff2, Richard E Higgs2, Guilherme Rocha2, Nicole L Byers2, Douglas E Schlichting2, Ajay Nirula2, Anabela Cardoso2,‡, Mario Corbellino11,‡ and 1Department of Surgery and Cancer, Imperial College, London, UK 2Eli Lilly and Company, Indianapolis, IN, USA 3Luigi Sacco, Department of Clinical and Biomedical Sciences, University of Milan, Milan, Italy 4BenevolentAI, London, UK 5Unit of Clinical Microbiology, Department of Laboratory Medicine, Karolinska Institutet and Karolinska University Hospital, Stockholm, Sweden 6Department of Physiology and Pharmacology, Karolinska Institutet and Karolinska University Hospital, Stockholm, Sweden 7Infectious Diseases Programme, Immunology Programme, Department of Microbiology and Immunology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore City, Singapore 8Institute of Molecular and Cell Biology (IMCB), A*STAR (Agency for Science, Technology and Research), Singapore, Singapore 9Department of Clinical, Surgical, Diagnostics and Pediatric Sciences, University of Pavia, Pavia, Italy 10Molecular Virology Unit, Fondazione IRCCS Policlinico San Matteo, Pavia, Italy 11Division of Infectious Diseases, ASST Fatebenefratelli Sacco, Milan, Italy ‡These authors contributed equally to this work as first authors ‡These authors contributed equally to this work as senior authors *Corresponding author. Tel: +1 317 985 3662; E-mail: [email protected] EMBO Mol Med (2020)12:e12697https://doi.org/10.15252/emmm.202012697 See also: MB Schultz et al (August 2020) PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Baricitinib is an oral Janus kinase (JAK)1/JAK2 inhibitor approved for the treatment of rheumatoid arthritis (RA) that was independently predicted, using artificial intelligence (AI) algorithms, to be useful for COVID-19 infection via proposed anti-cytokine effects and as an inhibitor of host cell viral propagation. We evaluated the in vitro pharmacology of baricitinib across relevant leukocyte subpopulations coupled to its in vivo pharmacokinetics and showed it inhibited signaling of cytokines implicated in COVID-19 infection. We validated the AI-predicted biochemical inhibitory effects of baricitinib on human numb-associated kinase (hNAK) members measuring nanomolar affinities for AAK1, BIKE, and GAK. Inhibition of NAKs led to reduced viral infectivity with baricitinib using human primary liver spheroids. These effects occurred at exposure levels seen clinically. In a case series of patients with bilateral COVID-19 pneumonia, baricitinib treatment was associated with clinical and radiologic recovery, a rapid decline in SARS-CoV-2 viral load, inflammatory markers, and IL-6 levels. Collectively, these data support further evaluation of the anti-cytokine and anti-viral activity of baricitinib and support its assessment in randomized trials in hospitalized COVID-19 patients. Synopsis This study provides biochemical and cellular evidence confirming artificial intelligence (AI)-predictions focused on anti-cytokine signaling and potential anti-viral effects for baricitinib, along with a case series, supporting its potential utility in hospitalized COVID-19 patients. Baricitinib, an oral Janus kinase (JAK)1/JAK2 inhibitor used to treat rheumatoid arthritis, was hypothesised using AI to be useful in COVID-19. Baricitinib-mediated inhibition of numb associated kinases utilized by SARS-CoV-2 for its propagation, led to reduced viral infectivity in primary liver spheroids. Baricitinib reduces levels of cytokines implicated in COVID-19 and inhibits their signaling. In patients with bilateral COVID-19 pneumonia, baricitinib treatment was associated with clinical and radiologic recovery, a rapid decline in SARS-CoV-2 viral load, inflammatory markers, and IL-6 levels. The paper explained Problem There are few drugs that are useful to treat COVID-19, the largest medical crisis of this century. To try to solve this problem, using AI, we found that an oral, once-daily medicine, baricitinib, normally used to treat adult rheumatoid arthritis (RA), may be useful in both reducing viral propagation in cells, and to mitigate the cytokine signaling seen in the hyper-inflammatory stage of the disease. We wished to understand this further in various models and in a case series of patients. Results We found that baricitinib inhibited the signaling of cytokines we typically see as being present in hospitalized patients with COVID-19. Using samples from a previous randomized trial in RA, we showed statistically significant declines in IL-6 levels with baricitinib treatment. In liver spheroids designed to investigate SARS-CoV-2 infectivity and separately in kinase assays, we showed that baricitinib could reduce cellular infection by blockade of numb-associated kinase members used in viral propagation. In a small case series of patients in northern Italy with bilateral COVID-19 pneumonia, baricitinib therapy was associated with improvement in clinical, radiologic, and viral parameters along with a rapid decline in CRP and plasma IL-6 levels. Impact Finding new drugs in our armamentarium to treat COVID-19 would be enormously valuable. Here, we stitched together the anti-cytokine and anti-viral activity of baricitinib and studied it in a small number of hospitalized patients. This study represents rapid repurposing from AI to the laboratory to a potential bedside therapeutic and supports the testing of baricitinib in randomized controlled trials in COVID-19 patients. Introduction The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is currently the biggest public health challenge to the biomedical community. Despite multiple public health measures, there remains an urgent need for pharmacologic therapies to treat infected patients, minimize mortality, and optimally decrease viral shedding and subsequent transmission. Artificial intelligence (AI) allows for rapid drug development (Schneider et al, 2020) including repurposing existing drugs. Algorithms were used to search for approved drugs capable of inhibiting both the inflammatory damage and infectivity associated with SARS-CoV-2 (Richardson et al, 2020a; Stebbing et al, 2020). Baricitinib, an oral inhibitor of Janus kinase (JAK)1 and JAK2 (Fridman et al, 2010) approved for the treatment of moderately-to-severely active rheumatoid arthritis (RA) in adults, was independently hypothesized to be a therapeutic option for COVID-19. It was considered among all molecules studied to have a unique role by virtue of its potential to both inhibit relevant cytokine signaling and have activity against the numb-associated kinases (NAKs), AAK1 and GAK (Richardson et al, 2020a; Stebbing et al, 2020), which stimulate AP-2-mediated host viral propagation (Inoue et al, 2007; Bekerman et al, 2017; Owczarek et al, 2018). Infection by pathogenic coronaviruses (e.g., SARS and SARS-CoV-2) often results in excessive cytokine and chemokine action with the development of acute respiratory distress syndrome (ARDS; Huang et al, 2005, 2020; Cameron et al, 2007; Ruan et al, 2020; Siddiqi & Mehra, 2020; Zhou et al, 2020). In patients with moderate-to-severe forms of these diseases, anti-viral cytokine signaling is maintained at inappropriate levels (perhaps due to incomplete viral clearance) causing acute lung injury (Nicholls et al, 2003), persistent interferon (IFN) activity, and impaired T-cell and antibody responses (Cui et al, 2003; Huang et al, 2005; Shi et al, 2020). In the COVID-19 setting, ARDS is the leading cause of death and is associated with high levels of interleukin-6 (IL-6), which appears to be a predictor of mortality (Ruan et al, 2020). Therefore, treating hospitalized patients requires both improved viral clearance and restriction of the inflammatory response, with the potential to improve outcomes such as mortality and reduce admissions to intensive care units. The anti-inflammatory benefit of baricitinib has been previously demonstrated through a reduction in a range of JAK-STAT-dependent cytokines (Fridman et al, 2010; Shi et al, 2014; McInnes et al, 2019). Phase 3 clinical trials providing safety and efficacy data have been conducted or are ongoing for baricitinib treatment in patients with autoimmune diseases including RA, atopic dermatitis, systemic lupus erythematosus, alopecia areata, juvenile idiopathic arthritis, and chronic atypical neutrophilic dermatosis with lipodystrophy and elevated temperature (CANDLE). Now, we provide biochemical and cellular evidence confirming predictions focused on anti-cytokine signaling and potential anti-viral effects for baricitinib, along with a case series, supporting its potential utility in hospitalized COVID-19 patients. Results Anti-cytokine activity for baricitinib The anti-cytokine and anti-inflammatory activity of baricitinib was evaluated with a focus on cytokines relevant to COVID-19 infection. We evaluated the in vitro pharmacology of baricitinib across relevant leukocyte subpopulations coupled to its in vivo pharmacokinetics to determine its effect on distinct cytokine pathways. Concentration–response curves combined with exposure data from baricitinib-treated healthy volunteers demonstrated that baricitinib affects cytokine-dependent phosphorylated STAT (pSTAT) inhibition to varying degrees (Fig 1A). Baricitinib inhibited signaling of cytokines implicated in COVID-19 infection, including IL-2, IL-6, IL-10, IFN-γ, and G-CSF, with lower half-maximum inhibitory concentration (IC50) values translating to a greater overall inhibition of cytokine-induced JAK/STAT signaling during the dosing interval (Fig 1A). Furthermore, baricitinib treatment resulted in a significant reduction (P < 0.05) from baseline in plasma IL-6 at week 12 in patients with active RA who had an inadequate response to methotrexate from a phase 2b (Tanaka et al, 2016), randomized, placebo-controlled, dose-ranging study (Fig 1B). Figure 1. Anti-cytokine activity of baricitinib A. IC50 values for baricitinib (orange) in cytokine-stimulated human CD4+ T cells, CD8+ T cells, NK cells, and monocytes are shown for IL-2/pSTAT5, IL-4/pSTAT6, IL-3/pSTAT5, IL-6/pSTAT3, IL-10/pSTAT3, IL-15/pSTAT5, IL-21/pSTAT3, G-CSF/pSTAT3, GM-CSF/pSTAT5, and IFN-γ/pSTAT1. Average daily percent STAT inhibition was calculated using in vitro concentration–response curves and published human exposure data. Cytokine treatments that did not result in sufficient pSTAT stimulation are denoted as “NS” in the radar plots. B. Fold change ± standard error in baseline IL-6 plasma levels at week 12 from RA patients treated with placebo (n = 47), and baricitinib 1 mg (n = 23), 2 mg (n = 24), 4 mg (n = 23), and 8 mg (n = 23) in the phase 2b randomized, placebo-controlled study NCT01469013. P values are for comparisons of baricitinib 1, 2, 4, and 8 mg change from baseline compared with the placebo change from baseline. Treatment effects were estimated using a mixed-effects repeated-measures model (SAS Proc Mixed) using log10-transformed IL-6 levels with an unstructured covariance matrix. Data information: IC50, half-maximum inhibitory concentration; NK, natural killer; pSTAT, phosphorylated signal transducer and activator of transcription. Download figure Download PowerPoint Anti-viral activity for baricitinib Next, we validated the proposed biochemical inhibitory activity of baricitinib on the NAK family members AAK1, BIKE, GAK, and STK16, some of which are hypothesized to facilitate viral propagation of coronavirus in epithelial cells (Bekerman et al, 2017; Owczarek et al, 2018; Richardson et al, 2020a). Baricitinib activity demonstrated affinity against AAK1 (8.2 nM), BIKE (20 nM), and GAK (120 nM; Fig 2A and B); these values are within the exposure range of the approved 2 mg (US, EU) and 4 mg (EU) once-daily doses of baricitinib for the treatment of RA (Shi et al, 2014). The pharmacokinetics of baricitinib show the unbound fraction of free bioavailable drug in RA patient sera as being 326 nM area under the curve (AUC) and 652 nM (AUC) for baricitinib 2 and 4 mg, respectively (data on file). We compared the relative binding affinities of different JAK inhibitors (JAKis) for these NAKs. Notably, among JAKis approved for the treatment of RA, baricitinib uniquely demonstrated high affinity for AAK1, BIKE, and GAK, whereas tofacitinib and upadacitinib did not demonstrate high affinity for these kinases (Fig 2B). The binding affinity of baricitinib for AAK1 and GAK is similar to the binding affinity of baricitinib for JAK1 (5.9 nM) and JAK2 (5.7 nM) (Fridman et al, 2010). Figure 2. NAK-binding affinities of baricitinib and other comparators A, B. Binding affinities in equilibrium affinity constant (Kd) are shown for baricitinib (A and B), tofacitinib, upadacitinib, AZD7762 (positive control), and broad-spectrum tyrosine kinase inhibitors sunitinib and erlotinib (B). Kd values were estimated from an 11-point concentration–response curve with fixed top parameters and a four-parameter logistic model (n = 3). C. Protein and mRNA expression of relevant numb-associated kinases in primary human liver organoids (n = 3) (P < 0.05). The immunofluorescence shows the juxtaposition of 1A9 (detecting spike protein from the SARS-CoV-2) in red and ACE2 in green along with the spheroid nuclei in blue as a DAPI stain (n = 1). Colocalization of spike and ACE2 is depicted with an arrow. D, E. Time course of days (one, three, and six days) post-infection (dpi) using liver spheroids and SARS-CoV-2 at 0.1 and 1.0 multiplicity of infection (MOI) (n = 1). F. Viability of spheroids was quantified by measuring intracellular ATP concentrations. Note that no significant (n.s.; Student's t-test) decrease in cell viability was observed for any concentration tested (n = 10). G. Schematic showing the timeline for the experiment shown in panel H. H. Viral load in infected (MOI 0.1) spheroids as measured by qPCR after treatment with 400 and 800 nM of baricitinib 48 h after infection (*P < 0.05; Student's t-test). Data are mean ± standard error of mean (n = 3). Download figure Download PowerPoint To extend these activities on NAKs, we evaluated the effect of baricitinib in reducing viral infectivity in 3D primary human liver spheroids (Bell et al, 2016) infected with purified SARS-CoV-2 and treated with baricitinib. Specifically, we used a 3D spheroid model of primary human liver cells in which hepatocytes retain their transcriptomic, proteomic, and metabolomic phenotype and functionality for multiple weeks (Bell et al, 2016, 2017; Vorrink et al, 2017), including expression of AAK1 and GAK (Fig 2C). The overlay of the viral antigen spike (as detected with 1A9 antibody) with the ACE-2 protein as detected by immunofluorescence, after 5 days of SARS-CoV-2 infection in these spheroids, provides a visual confirmation of virus infectivity in this system (Fig 2C). SARS-CoV-2 was able to infect human primary liver spheroids in this experimental paradigm, as demonstrated by a day post-infection (dpi) dependent increase in viral RNA (Fig 2D and E). Baricitinib did not result in liver cell injury up to concentrations approximately eightfold higher than the clinically observed AUC values at 4 mg (AUC = 652 nM; Fig 2F). Importantly, pretreatment of spheroids with physiologically relevant concentrations of baricitinib (400 and 800 nM) significantly (P < 0.05) reduced viral load by 30–40%, corroborating the proposed inhibitory effects of baricitinib on AAK1 and GAK-mediated viral propagation (Fig 2G and H). These results suggest that host cells that express these NAKs may serve as a target for baricitinib-mediated reduction in viral propagation. Clinical case series using baricitinib Following the recent publications by Richardson et al (2020a) and Stebbing et al (2020), COVID-19 patients were treated with baricitinib in a pilot study in Milan, Italy. Four patients with bilateral COVID-19 pneumonia, who presented with varying degrees of disease severity (Table 1), were included in this pilot study; three individuals (Patients B, C, and D) were clinically unstable with moderate-to-severe disease. All four patients were admitted to the ward from the emergency department in March 2020. As shown in Table 1, Patient A was a female nurse, aged 29. Patient B, a 76-year-old male, had significant co-morbidities (a former smoker, arterial hypertension (AH), chronic obstructive pulmonary disease [COPD], coronary artery disease (CAD), and had undergone an aortic aneurysm Endurant II graft repair on February 25, 2020). Patient C, a 57-year-old male, had co-morbidities including AH and COPD (non-smoker); he suddenly deteriorated a few hours following hospitalization, and was placed on continuous positive airway pressure at the time baricitinib was initiated. Patient D, a 51-year-old male, had a high body mass index (BMI) of 35. All four patients had detectable plasma IL-6 levels (Fig 3A) with markedly raised inflammatory markers (C-reactive protein [CRP]), as would be expected in patients with COVID-19 pneumonia. Table 1. Clinical characteristics of patients in baricitinib COVID-19 case study Patient A Patient B Patient C Patient D Age (years) 29 76 57 51 Gender Female Male Male Male Coexisting conditions None AH, COPD, CAD AH, COPD Obesity BMI 22 28 29 35 Medications Combined oral contraception Losartan, propranolol, omeprazole, aspirin, LMW heparin Losartan, hydrochlorothiazide, inhaled beclomethasone None Smoking No Former No No Data at presentation Symptoms Fever, dry cough, and myalgias Fever, dry cough Fever, dry cough, dyspnea Fever, dry cough, headache Blood oxygen levels PaO2 (mmHg) 111 81 62 80 Alveolar oxygen gradient (KPa) 0 4.7 12.9 4.9 SpO2 (%) 98 97 94 91 RR/min 26 32 28 28 Baricitinib dose/days on treatment 4 mg for 10 days 2 mg for 10 days 4 mg for 12 days 4 mg for 10 days Antibiotic therapy None Ceftriaxone, tazocin, fosfomycin Ceftriaxone, azithromycin None Study note: According to the eligibility criteria, Patient A tested negative for pregnancy and all four patients tested negative for HIV-1/2, and tuberculosis using QuantiFERON-TB Gold Plus. AH, arterial hypertension; BMI, body mass index; CAD, coronary artery disease; COPD, chronic obstructive pulmonary disease; RR, respiratory rate. Figure 3. Viral detection and immunological features of four COVID-19 patients treated with baricitinib A. Detection of SARS-CoV-2 in nasopharyngeal swabs and peripheral blood using the GeneFinder™ COVID-19 Plus RealAmp Kit assay (ELITechGroup S.p.A., Turin, Italy). Three viral target genes, RdRp, N, and E, together with the housekeeping gene GAPDH, were simultaneously amplified. Here, the most sensitive target gene, N, is shown. Samples with Ct values > 40 were defined as negative. A dashed horizontal line indicates the cutoff. IL-6 values are shown in the same graph. Time of baricitinib treatment is highlighted (Patients A, B, and D for 10 days, Patient C for 12 days) with total time of evaluation on the x-axis. Fever and cough symptoms are indicated. The bars in gray indicate that cough has improved but has not resolved. B. Levels of CD3+, CD4+, CD8+, B cells (CD19+), and NK cells (CD3−, CD16+, CD56+) were determined using the AQUIOS CL Flow Cytometry System (Beckman Coulter). C. Total serum IgG, IgA, and IgM levels are shown for all patients. Data information: Ct, cycle threshold; E, envelope membrane; ED, emergency department; N, nucleocapsid protein; NK, natural killer; RdRp, RNA-dependent RNA polymerase. Download figure Download PowerPoint As shown in Fig 3A, all four patients showed improvement with baricitinib treatment in signs and symptoms such as cough, fever, and reduction in plasma IL-6 levels, along with a reduction in the SARS-CoV-2 RNA viral load, as detected by the real-time reverse transcriptase–polymerase chain reaction (RT–PCR) signal from the nasopharyngeal carriage. Stringent criteria were used for RNA detection of SARS-CoV-2 in nasopharyngeal carriage and peripheral blood. Real-time RT–PCR was performed on three distinct viral gene targets (Corman et al, 2020; Appendix Table S1), using the most sensitive target (N gene) and a cutoff using Ct values > 40 for the analyses illustrated in Fig 3A, in contrast to o

The dual anticytokine and antiviral actions of baricitinib reduce SARS-CoV-2 infectivity in organoids and morbidity in people.

In mammalian cells, IFN responses that occur during RNA and DNA virus infections are activated by distinct signaling pathways. The RIG-I-like-receptors (RLRs) bind viral RNA and engage the adaptor MAVS (mitochondrial antiviral signaling) to promote IFN expression, whereas cGAS (cGMP-AMP synthase) binds viral DNA and activates an analogous pathway via the protein STING (stimulator of IFN genes). In this study, we confirm that STING is not necessary to induce IFN expression during RNA virus infection but also find that STING is required to restrict the replication of diverse RNA viruses. The antiviral activities of STING were not linked to its ability to regulate basal expression of IFN-stimulated genes, activate transcription, or autophagy. Using vesicular stomatitis virus as a model, we identified a requirement of STING to inhibit translation during infection and upon transfection of synthetic RLR ligands. This inhibition occurs at the level of translation initiation and restricts the production of viral and host proteins. The inability to restrict translation rendered STING-deficient cells 100 times more likely to support productive viral infections than wild-type counterparts. Genetic analysis linked RNA sensing by RLRs to STING-dependent translation inhibition, independent of MAVS. Thus, STING has dual functions in host defense, regulating protein synthesis to prevent RNA virus infection and regulating IFN expression to restrict DNA viruses.

Nitric oxide is an important molecule playing a key role in a broad range of biological process such as neurotransmission, vasodilatation and immune responses. While the anti-microbiological properties of nitric oxide-derived reactive nitrogen intermediates (RNI) such as peroxynitrite, are known, the mechanism of these effects are as yet poorly studied. Severe Acute Respiratory Syndrome coronavirus (SARS-CoV) belongs to the family Coronaviridae, was first identified during 2002-2003. Mortality in SARS patients ranges from between 6 to 55%. We have previously shown that nitric oxide inhibits the replication cycle of SARS-CoV in vitro by an unknown mechanism. In this study, we have further investigated the mechanism of the inhibition process of nitric oxide against SARS-CoV. We found that peroxynitrite, an intermediate product of nitric oxide in solution formed by the reaction of NO with superoxide, has no effect on the replication cycle of SARS-CoV, suggesting that the inhibition is either directly effected by NO or a derivative other than peroxynitrite. Most interestingly, we found that NO inhibits the replication of SARS-CoV by two distinct mechanisms. Firstly, NO or its derivatives cause a reduction in the palmitoylation of nascently expressed spike (S) protein which affects the fusion between the S protein and its cognate receptor, angiotensin converting enzyme 2. Secondly, NO or its derivatives cause a reduction in viral RNA production in the early steps of viral replication, and this could possibly be due to an effect on one or both of the cysteine proteases encoded in Orf1a of SARS-CoV.

A new coronavirus (severe acute respiratory syndrome coronavirus [SARS-CoV]) has been identified to be the etiological agent of severe acute respiratory syndrome. Given the highly contagious and acute nature of the disease, there is an urgent need for the development of diagnostic assays that can detect SARS-CoV infection. For determination of which of the viral proteins encoded by the SARS-CoV genome may be exploited as diagnostic antigens for serological assays, the viral proteins were expressed individually in mammalian and/or bacterial cells and tested for reactivity with sera from SARS-CoV-infected patients by Western blot analysis. A total of 81 sera, including 67 from convalescent patients and seven pairs from two time points of infection, were analyzed, and all showed immunoreactivity towards the nucleocapsid protein (N). Sera from some of the patients also showed immunoreactivity to U274 (59 of 81 [73%]), a protein that is unique to SARS-CoV. In addition, all of the convalescent-phase sera showed immunoreactivity to the spike (S) protein when analyzed by an immunofluorescence method utilizing mammalian cells stably expressing S. However, samples from the acute phase (2 to 9 days after the onset of illness) did not react with S, suggesting that antibodies to N may appear earlier than antibodies to S. Alternatively, this could be due to the difference in the sensitivities of the two methods. The immunoreactivities to these recombinant viral proteins are highly specific, as sera from 100 healthy donors did not react with any of them. These results suggest that recombinant N, S, and U274 proteins may be used as antigens for the development of serological assays for SARS-CoV.

The severe acute respiratory syndrome (SARS) epidemic started in late 2002 and swiftly spread across 5 continents with a mortality rate of around 10%. Although the epidemic was eventually controlled through the implementation of strict quarantine measures, there continues a need to investigate the SARS coronavirus (SARS-CoV) and develop interventions should it re-emerge. Numerous studies have shown that neutralizing antibodies against the virus can be found in patients infected with SARS-CoV within days upon the onset of illness and lasting up to several months. In contrast, there is little data on the kinetics of T cell responses during SARS-CoV infection and little is known about their role in the recovery process. However, recent studies in mice suggest the importance of T cells in viral clearance during SARS-CoV infection. Moreover, a growing number of studies have investigated the memory T cell responses in recovered SARS patients. This review covers the available literature on the emerging importance of T cell responses in SARS-CoV infection, particularly on the mapping of cytotoxic T lymphocyte (CTL) epitopes, longevity, polyfunctionality and human leukocyte antigen (HLA) association as well as their potential implications on treatment and vaccine development.

BackgroundA novel coronavirus, SARS-CoV-2, which emerged at the end of 2019 and causes COVID-19, has resulted in worldwide human infections. While genetically distinct, SARS-CoV-1, the aetiological agent responsible for an outbreak of severe acute respiratory syndrome (SARS) in 2002-2003, utilises the same host cell receptor as SARS-CoV-2 for entry: angiotensin-converting enzyme 2 (ACE2). Parts of the SARS-CoV-1 spike glycoprotein (S protein), which interacts with ACE2, appear conserved in SARS-CoV-2.AimThe cross-reactivity with SARS-CoV-2 of monoclonal antibodies (mAbs) previously generated against the S protein of SARS-CoV-1 was assessed.MethodsThe SARS-CoV-2 S protein sequence was aligned to those of SARS-CoV-1, Middle East respiratory syndrome (MERS) and common-cold coronaviruses. Abilities of mAbs generated against SARS-CoV-1 S protein to bind SARS-CoV-2 or its S protein were tested with SARS-CoV-2 infected cells as well as cells expressing either the full length protein or a fragment of its S2 subunit. Quantitative ELISA was also performed to compare binding of mAbs to recombinant S protein.ResultsAn immunogenic domain in the S2 subunit of SARS-CoV-1 S protein is highly conserved in SARS-CoV-2 but not in MERS and human common-cold coronaviruses. Four murine mAbs raised against this immunogenic fragment could recognise SARS-CoV-2 S protein expressed in mammalian cell lines. In particular, mAb 1A9 was demonstrated to detect S protein in SARS-CoV-2-infected cells and is suitable for use in a sandwich ELISA format.ConclusionThe cross-reactive mAbs may serve as useful tools for SARS-CoV-2 research and for the development of diagnostic assays for COVID-19.

Previous experimental and theoretical studies have produced high-resolution descriptions of the native and folding transition states of chymotrypsin inhibitor 2 (CI2). In similar fashion, here we use a combination of NMR experiments and molecular dynamics simulations to examine the conformations populated by CI2 in the denatured state. The denatured state is highly unfolded, but there is some residual native helical structure along with hydrophobic clustering in the center of the chain. The lack of persistent nonnative structure in the denatured state reduces barriers that must be overcome, leading to fast folding through a nucleation–condensation mechanism. With the characterization of the denatured state, we have now completed our description of the folding/unfolding pathway of CI2 at atomic resolution.

According to landscape theory proteins do not fold by localised pathways, but find their native conformation by a progressive organisation of an ensemble of partly folded structures down a folding funnel. Here, we use kinetics and protein engineering to investigate the shape of the free-energy profile for two-state folding, which is the macroscopic view of the funnel process for small and rapidly folding proteins. Our experiments are based mainly on structural changes of the transition state of chymotrypsin inhibitor 2 (CI2) upon destabilisation with temperature and GdnHCl. The transition state ensemble of CI2 is a localised feature in the free-energy profile that is sharply higher than the other parts of the activation barrier. The relatively fixed position of the CI2 transition state on the reaction coordinate makes it easy to characterise but contributes also to overshadow the rest of the free-energy profile, the shape of which is inaccessible for analysis. Results from mutants of CI2 and comparison with other two-state proteins, however, point at the possibility that the barrier for folding is generally broad and that localised transition states result from minor ripples in the free-energy profile. Accordingly, variabilities in the folding kinetics may not indicate different folding mechanisms, but could be accounted for by various degrees of ruggedness on top of very broad activation barriers for folding. The concept is attractive since it summarises a wide range of folding data which have previously seemed unrelated. It is also supported by theory. Consistent with experiment, broad barriers predict that new transition state ensembles are exposed upon extreme destabilisation or radical mutations.

We show in this study that the ionisation equilibria of denatured proteins in pure water are inconsistent with the "fully-unfolded" conformation being an extended coil where the residues are isolated from one another by the intervening solvent. The effects of acid and salt on the stability of the barley chymotrypsin inhibitor 2 (CI2) were investigated and the pKA-values of all carboxylate residues in the native protein were determined by NMR. A comparison of the experimentally determined pH-dependence of the protein stability and that calculated using observed pKA-values in the native state, reveals that the pKA-values in the denatured state are, on average, 0.3 pH units lower than those of model compounds. An increase in ionic strength eliminates these pKAshifts in the dentured state. This shows that there are electrostatic interactions in the denatured state of CI2. Since previous studies on barnase and the Ovomucoid Third Domain also report anomalous titration behaviours of the denatured states, it appears that perturbed pKA-values in the denatured state is a general phenomenon, indicating that the unfolded conformation in pure water is a fairly compact species. In addition, we used a mutational approach to determine the pKA-values of a carboxylate group in both the native and denatured states. The pKA-value in the native state obtained by this method is in precise agreement with that obtained by NMR.

A new disease, termed severe acute respiratory syndrome (SARS), emerged at the end of 2002 and caused profound disturbances in over 30 countries worldwide in 2003. A novel coronavirus was identified as the aetiological agent of SARS and the 30 kb viral genome was deciphered with unprecedented speed in a coordinated manner by the global community. Since then, much progress has been made in the virological and molecular characterization of the proteins encoded by SARS-coronavirus (SARS-CoV) genome, which contains 14 potential open reading frames (ORFs). These investigations can be broadly classified into three groups: (a) studies on the replicase 1a/1b gene products which are important for viral replication, (b) studies on the structural proteins, spike, nucleocapsid, membrane and envelope, which have homologues in all coronaviruses, and are important for viral assembly and (c) expression and functional studies of the "accessory" proteins that are specifically encoded by SARS-CoV. A comparison of the properties of these three groups of SARS-CoV proteins with the knowledge that coronavirologists have generated over more than 30 years of research can help us in the prevention and treatment of SARS in the event of the re-emergence of this new infectious disease.

Severe acute respiratory syndrome (SARS) is a highly contagious and life threatening disease, with a fatality rate of almost 10%. The etiologic agent is a novel coronavirus, severe acute respiratory syndrome coronavirus (SARS-CoV), with animal reservoirs found in bats and other wild animals and thus the possibility of reemergence. In this study, we first investigated at 6 years postinfection whether SARS-specific memory T cells persist in SARS-recovered individuals, demonstrating that these subjects still possess polyfunctional SARS-specific memory CD4+ and CD8+ T cells. A dominant memory CD8+ T cell response against SARS-CoV nucleocaspid protein (NP; amino acids 216 to 225) was then defined in SARS-recovered individuals carrying HLA-B*40:01, a HLA-B molecule present in approximately one-quarter of subjects of Asian ethnicities. To reconstitute such a CD8+ T cell response, we isolated the alpha and beta T cell receptors of the HLA-B*40:01-restricted SARS-specific CD8+ T cells. Using T cell receptor gene transfer, we generated SARS-specific redirected T cells from the lymphocytes of normal individuals. These engineered CD8+ T cells displayed avidity and functionality similar to that of natural SARS-specific memory CD8+ T cells. They were able to degranulate and produce gamma interferon, tumor necrosis factor alpha, and macrophage inflammatory proteins 1α and 1β after antigenic stimulation. Since there is no effective treatment against SARS, these transduced T cells specific for an immunodominant SARS epitope may provide a new avenue for treatment during a SARS outbreak.

Crimean-Congo hemorrhagic fever, a severe hemorrhagic disease found throughout Africa, Europe, and Asia, is caused by the tick-borne Crimean-Congo hemorrhagic fever virus (CCHFV). CCHFV is a negative-sense single-stranded RNA (ssRNA) virus belonging to the Nairovirus genus of the Bunyaviridae family. Its genome of three single-stranded RNA segments is encapsidated by the nucleocapsid protein (CCHFV N) to form the ribonucleoprotein complex. This ribonucleoprotein complex is required during replication and transcription of the viral genomic RNA. Here, we present the crystal structures of the CCHFV N in two distinct forms, an oligomeric form comprised of double antiparallel superhelices and a monomeric form. The head-to-tail interaction of the stalk region of one CCHFV N subunit with the base of the globular body of the adjacent subunit stabilizes the helical organization of the oligomeric form of CCHFV N. It also masks the conserved caspase-3 cleavage site present at the tip of the stalk region from host cell caspase-3 interaction and cleavage. By incubation with primer-length ssRNAs, we also obtained the crystal structure of CCHFV N in its monomeric form, which is similar to a recently published structure. The conformational change of CCHFV N upon deoligomerization results in the exposure of the caspase-3 cleavage site and subjects CCHFV N to caspase-3 cleavage. Mutations of this cleavage site inhibit cleavage by caspase-3 and result in enhanced viral polymerase activity. Thus, cleavage of CCHFV N by host cell caspase-3 appears to be crucial for controlling viral RNA synthesis and represents an important host defense mechanism against CCHFV infection.

Viruses have developed distinct strategies to overcome the host defense system. Regulation of apoptosis in response to viral infection is important for virus survival and dissemination. Like other viruses, Crimean-Congo hemorrhagic fever virus (CCHFV) is known to regulate apoptosis. This study, for the first time, suggests that the non-structural protein NSs of CCHFV, a member of the genus Nairovirus, induces apoptosis. In this report, we demonstrated the expression of CCHFV NSs, which contains 150 amino acid residues, in CCHFV-infected cells. CCHFV NSs undergoes active degradation during infection. We further demonstrated that ectopic expression of CCHFV NSs induces apoptosis, as reflected by caspase-3/7 activity and cleaved poly(ADP-ribose) polymerase, in different cell lines that support CCHFV replication. Using specific inhibitors, we showed that CCHFV NSs induces apoptosis via both intrinsic and extrinsic pathways. The minimal active region of the CCHFV NSs protein was determined to be 93–140 amino acid residues. Using alanine scanning, we demonstrated that Leu-127 and Leu-135 are the key residues for NSs-induced apoptosis. Interestingly, CCHFV NSs co-localizes in mitochondria and also disrupts the mitochondrial membrane potential. We also demonstrated that Leu-127 and Leu-135 are important residues for disruption of the mitochondrial membrane potential by NSs. Therefore, these results indicate that the C terminus of CCHFV NSs triggers mitochondrial membrane permeabilization, leading to activation of caspases, which, ultimately, leads to apoptosis. Given that multiple factors contribute to apoptosis during CCHFV infection, further studies are needed to define the involvement of CCHFV NSs in regulating apoptosis in infected cells. Viruses have developed distinct strategies to overcome the host defense system. Regulation of apoptosis in response to viral infection is important for virus survival and dissemination. Like other viruses, Crimean-Congo hemorrhagic fever virus (CCHFV) is known to regulate apoptosis. This study, for the first time, suggests that the non-structural protein NSs of CCHFV, a member of the genus Nairovirus, induces apoptosis. In this report, we demonstrated the expression of CCHFV NSs, which contains 150 amino acid residues, in CCHFV-infected cells. CCHFV NSs undergoes active degradation during infection. We further demonstrated that ectopic expression of CCHFV NSs induces apoptosis, as reflected by caspase-3/7 activity and cleaved poly(ADP-ribose) polymerase, in different cell lines that support CCHFV replication. Using specific inhibitors, we showed that CCHFV NSs induces apoptosis via both intrinsic and extrinsic pathways. The minimal active region of the CCHFV NSs protein was determined to be 93–140 amino acid residues. Using alanine scanning, we demonstrated that Leu-127 and Leu-135 are the key residues for NSs-induced apoptosis. Interestingly, CCHFV NSs co-localizes in mitochondria and also disrupts the mitochondrial membrane potential. We also demonstrated that Leu-127 and Leu-135 are important residues for disruption of the mitochondrial membrane potential by NSs. Therefore, these results indicate that the C terminus of CCHFV NSs triggers mitochondrial membrane permeabilization, leading to activation of caspases, which, ultimately, leads to apoptosis. Given that multiple factors contribute to apoptosis during CCHFV infection, further studies are needed to define the involvement of CCHFV NSs in regulating apoptosis in infected cells.

Multiple Omicron sub-lineages have emerged, with Omicron XBB and XBB.1.5 subvariants becoming the dominant variants globally at the time of this study. The key feature of new variants is their ability to escape humoral immunity despite the fact that there are limited genetic changes from their preceding variants. This raises the question of whether Omicron should be regarded as a separate serotype from viruses serologically clustered with the ancestral SARS-CoV-2 virus. Here, we present cross-neutralization data based on pseudovirus neutralization test using convalescent sera from naïve individuals who had recovered from primary infection by SARS-CoV-1 and SARS-CoV-2 strains/variants including the ancestral virus and variants Beta, Delta, Omicron BA.1, Omicron BA.2, and Omicron BA.5, respectively. The results revealed no significant cross-neutralization in any of the three-way testing for SARS-CoV-1, ancestral SARS-CoV-2, and SARS-CoV-2 Omicron subvariants. The data argue for the assignment of three distinct serotypes for the currently known human-infecting SARS-related coronaviruses.

The severe acute respiratory syndrome coronavirus (SARS-CoV) 3a protein is one of the opening reading frames in the viral genome with no homologue in other known coronaviruses. Expression of the 3a protein has been demonstrated during both in vitro and in vivo infection. Here we present biochemical data to show that 3a is a novel coronavirus structural protein. 3a was detected in virions purified from SARS-CoV infected Vero E6 cells although two truncated products were present predominantly instead of the full-length protein. In Vero E6 cells transiently transfected with a cDNA construct for expressing 3a, a similar cleavage was observed. Furthermore, co-expression of 3a, membrane and envelope proteins using the baculovirus system showed that both full-length and truncated 3a can be assembled into virus-like particles. This is the first report that demonstrated the incorporation of 3a into virion and showed that the SARS-CoV encodes a novel coronavirus structural protein.

CI2 folds and unfolds as a single cooperative unit by simple two-state kinetics, which enables the properties of the transition state to be measured from both the forward and backward rate constants. We have examined how the free energy of the transition state for the folding of chymotrypsin inhibitor 2 (CI2) changes with pH and temperature. In addition to the standard thermodynamic quantities, we have measured the overall acid-titration properties of the transition state and its heat capacity relative to both the denatured and native states. We were able to determine the latter by a method analogous to a well-established procedure for measuring the change in heat capacity for equilibrium unfolding: the enthalpy of activation of unfolding at different values of acid pH were plotted against the average temperature of each determination. Our results show that the transition state of CI2 has lost most of the electrostatic and van der Waals' interactions that are found in the native state, but it remains compact and this prevents water molecules from entering some parts of the hydrophobic core. The properties of the transition state of CI2 are then compared with the major folding transition state of the larger protein barnase, which folds by a multi-state mechanism, with the accumulation of a partly structured intermediate (Dphysor I). CI2 folds from a largely unstructured denatured state under physiological conditionsviaa transition state which is compact but relatively uniformly unstructured, with tertiary and secondary structure being formed in parallel. We term this an expanded pathway. Conversely, barnase folds from a largely structured denatured state in which elements of structure are well formed through a transition state that has islands of folded elements of structure. We term this a compact pathway. These two pathways may correspond to the two extreme ends of a continuous spectrum of protein folding mechanisms. Although the properties of the two transition states are very different, the activation barrier for folding (Dphys→‡ ) is very similar for both proteins.

Regulation of apoptosis during infection has been observed for several viral pathogens. Programmed cell death and regulation of apoptosis in response to a viral infection are important factors for host or virus survival. It is not known whether Crimean-Congo hemorrhagic fever virus (CCHFV) infection regulates the apoptosis process in vitro. This study for the first time suggests that CCHFV induces apoptosis, which may be dependent on caspase-3 activation. This study also shows that the coding sequence of the S segment of CCHFV contains a proteolytic cleavage site, DEVD, which is conserved in all CCHFV strains. By using different recombinant expression systems and site-directed mutagenesis, we demonstrated that this motif is subject to caspase cleavage. We also demonstrate that CCHFV nucleocapsid protein (NP) is cleaved into a 30-kDa fragment at the same time as caspase activity is induced during infection. Using caspase inhibitors and cells lacking caspase-3, we clearly demonstrate that the cleavage of NP is caspase-3-dependent. We also show that the inhibition of apoptosis induced progeny viral titers of ∼80-90%. Thus, caspase-3-dependent cleavage of NP may represent a host defense mechanism against lytic CCHFV infection. Taken together, these data suggest that the most abundant protein of CCHFV, which has several essential functions such as protection of viral RNA and participation in various processes in the replication cycle, can be subjected to cleavage by host cell caspases.

ABSTRACT The spread of the recently emerged, highly pathogenic H5N1 avian influenza virus has raised concern. Preclinical studies suggest that passive immunotherapy could be a new form of treatment for H5N1 virus infection. Here, a neutralizing monoclonal antibody (MAb) against the hemagglutinin (HA) of the influenza A/chicken/Hatay/2004 H5N1 virus, MAb 9F4, was generated and characterized. MAb 9F4 binds both the denatured and native forms of HA. It was shown to recognize the HA proteins of three heterologous strains of H5N1 viruses belonging to clades 1, 2.1, and 2.2, respectively. By use of lentiviral pseudotyped particles carrying HA on the surface, MAb 9F4 was shown to effectively neutralize the homologous strain, Hatay04, and another clade 1 strain, VN04, at a neutralization titer of 8 ng/ml. Furthermore, MAb 9F4 also neutralized two clade 2 viruses at a neutralizing titer of 40 ng/ml. The broad cross-neutralizing activity of MAb 9F4 was confirmed by its ability to neutralize live H5N1 viruses of clade 2.2.2. Epitope-mapping analysis revealed that MAb 9F4 binds a previously uncharacterized epitope below the globular head of the HA1 subunit. Consistently, this epitope is well conserved among the different clades of H5N1 viruses. MAb 9F4 does not block the interaction between HA and its receptor but prevents the pH-mediated conformational change of HA. MAb 9F4 was also found to be protective, both prophylactically and therapeutically, against a lethal viral challenge of mice. Taken together, our results showed that MAb 9F4 is a neutralizing MAb that binds a novel and well-conserved epitope in the HA1 subunit of H5N1 viruses.

In the midst of the unceasing COVID-19 pandemic, the identification of immunogenic epitopes in the SARS-CoV-2 spike (S) glycoprotein plays a vital role in the advancement and development of intervention strategies. S is expressed on the exterior of the SARS-CoV-2 virion and contains two subunits, namely the N-terminal S1 and C-terminal S2. It is the key element for mediating viral entry as well as a crucial antigenic determinant capable of stimulating protective immune response through elicitation of anti-SARS-CoV-2 antibodies and activation of CD4+ and CD8+ cells in COVID-19 patients. Given that S2 is highly conserved in comparison to the S1, here, we provide a review of the latest findings on the SARS-CoV-2 S2 subunit and further discuss its potential as an attractive and promising target for the development of prophylactic vaccines and therapeutic agents against COVID-19.

Severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV) caused a severe outbreak in several regions of the world in 2003. The SARS-CoV genome is predicted to contain 14 functional open reading frames (ORFs). The first ORF (1a and 1b) encodes a large polyprotein that is cleaved into nonstructural proteins (nsp). The other ORFs encode for four structural proteins (spike, membrane, nucleocapsid and envelope) as well as eight SARS-CoV-specific accessory proteins (3a, 3b, 6, 7a, 7b, 8a, 8b and 9b). In this report we have cloned the predicted nsp8 gene and the ORF6 gene of the SARS-CoV and studied their abilities to interact with each other. We expressed the two proteins as fusion proteins in the yeast two-hybrid system to demonstrate protein–protein interactions and tested the same using a yeast genetic cross. Further the strength of the interaction was measured by challenging growth of the positive interaction clones on increasing gradients of 2-amino trizole. The interaction was then verified by expressing both proteins separately in-vitro in a coupled-transcription translation system and by coimmunoprecipitation in mammalian cells. Finally, colocalization experiments were performed in SARS-CoV infected Vero E6 mammalian cells to confirm the nsp8–ORF6 interaction. To the best of our knowledge, this is the first report of the interaction between a SARS-CoV accessory protein and nsp8 and our findings suggest that ORF6 protein may play a role in virus replication.

A novel coronavirus, termed the severe acute respiratory syndrome coronavirus (SARS-CoV), infected humans in Guangdong, China, in November 2002 and the subsequent efficient human-to-human transmissions of this virus caused profound disturbances in over 30 countries worldwide in 2003. Eventually, this epidemic was controlled by isolation and there has been no human infection reported since January 2004. However, research on different aspects of the SARS-CoV is not waning, as it is not known if this virus will re-emerge, especially since its origins and potential reservoir(s) are unresolved. The SARS-CoV genome is nearly 30 kb in length and contains 14 potential open reading frames (ORFs). Some of these ORFs encode for genes that are homologous to proteins found in all known coronaviruses, namely the replicase genes (ORFs 1a and 1b) and the four structural proteins: nucleocapsid, spike, membrane and envelope, and these proteins are expected to be essential for the replication of the virus. The remaining eight ORFs encodes for accessory proteins, varying in length from 39 to 274 amino acids, which are unique to SARS-CoV. This review will summarize the expeditious research on these accessory viral proteins in three major areas: (i) the detection of antibodies against accessory proteins in the serum of infected patients, (ii) the expression, processing and cellular localization of the accessory proteins, and (iii) the effects of the accessory proteins on cellular functions. These in-depth molecular and biochemical characterizations of the SARS-CoV accessory proteins, which have no homologues in other coronaviruses, may offer clues as to why the SARS-CoV causes such a severe and rapid attack in humans, while other coronaviruses that infect humans seem to be more forgiving.

The severe acute respiratory syndrome coronavirus (SARS CoV) genome has 14 potential open reading frames (ORFs). The first ORF is translated from the full-length genomic mRNA while the remaining ORFs are translated from eight subgeomic RNAs (sgRNAs). In this study, we designed small interference RNAs (siRNAs) targeting sgRNA 2, 3 and 7 and tested their efficiency and specificity in silencing the protein translated from the targeted sgRNA. Our results demonstrated that siRNA 7 could inhibit sgRNA 7, which showed 19/19 nucleotides (nt) matching, and sgRNA 8, which showed 18/19 nt matching; but, it did not inhibit the full-length genomic mRNA which showed 17/19 nt matching. Overall, each of the siRNAs can inhibit the targeted sgRNA without affecting the full-length genomic mRNA or the other sgRNAs that showed mismatch of two or more nt. Thus, siRNA could be designed so as to knockdown the expression of viral protein(s) from a targeted sgRNA during viral infection, thereby allowing the contribution of individual viral proteins to viral infection to be delineated. When Vero E6 cells expressing siRNA 2, 3 or 7 were infected with SARS-CoV, a significant reduction in the yield of progeny virus was observed. Indirect immunofluorescence assays showed that in the infected cells expressing each of the siRNAs, there was aspecific silencing of S, 3a and 7a, respectively, but the expression of nucleocapsid protein was not affected. Thus, our data suggests that the accessory proteins, i.e. 3a and 7a, could play an important role during the replication cycle of the SARS-CoV.

Conditional ligands have enabled the high-throughput production of human leukocyte antigen (HLA) libraries that present defined peptides. Immunomonitoring platforms typically concentrate on restriction elements associated with European ancestry, and such tools are scarce for Asian HLA variants. We report 30 novel irradiation-sensitive ligands, specifically targeting South East Asian populations, which provide 93, 63, and 79% coverage for HLA-A, -B, and -C, respectively. Unique ligands for all 16 HLA types were constructed to provide the desired soluble HLA product in sufficient yield. Peptide exchange was accomplished for all variants as demonstrated by an ELISA-based MHC stability assay. HLA tetramers with redirected specificity could detect antigen-specific CD8(+) T-cell responses against human cytomegalovirus, hepatitis B (HBV), dengue virus (DENV), and Epstein-Barr virus (EBV) infections. The potential of this population-centric HLA library was demonstrated with the characterization of seven novel T-cell epitopes from severe acute respiratory syndrome coronavirus, HBV, and DENV. Posthoc analysis revealed that the majority of responses would be more readily identified by our unbiased discovery approach than through the application of state-of-the-art epitope prediction. This flow cytometry-based technology therefore holds considerable promise for monitoring clinically relevant antigen-specific T-cell responses in populations of distinct ethnicity.

Hepatitis B virus (HBV) infection is a major health problem affecting about 300 million people globally. Although successful administration of a prophylactic vaccine has reduced new infections, a cure for chronic hepatitis B (CHB) is still unavailable. Current anti-HBV therapies slow down disease progression but are not curative as they cannot eliminate or permanently silence HBV covalently closed circular DNA (cccDNA). The cccDNA minichromosome persists in the nuclei of infected hepatocytes where it forms the template for all viral transcription. Interactions between host factors and cccDNA are crucial for its formation, stability, and transcriptional activity. Here, we summarize the reported interactions between HBV cccDNA and various host factors and their implications on HBV replication. While the virus hijacks certain cellular processes to complete its life cycle, there are also host factors that restrict HBV infection. Therefore, we review both positive and negative regulation of HBV cccDNA by host factors and the use of small molecule drugs or sequence-specific nucleases to target these interactions or cccDNA directly. We also discuss several reporter-based surrogate systems that mimic cccDNA biology which can be used for drug library screening of cccDNA-targeting compounds as well as identification of cccDNA-related targets.

Human enterovirus A71 (HEVA71) causes hand, foot, and mouth disease (HFMD) in young children and is considered a major neurotropic pathogen but lacks effective antivirals. To identify potential therapeutic agents against HFMD, we screened a 502-compound flavonoid library for compounds targeting the HEVA71 internal ribosome entry site (IRES) that facilitates translation of the HEVA71 genome and is vital for the production of HEVA71 viral particles. We validated hits using cell viability and viral plaque assays and found that prunin was the most potent inhibitor of HEVA71. Downstream assays affirmed that prunin disrupted viral protein and RNA synthesis and acted as a narrow-spectrum antiviral against enteroviruses A and B, but not enterovirus C, rhinovirus A, herpes simplex 1, or chikungunya virus. Continuous HEVA71 passaging with prunin yielded HEVA71-resistant mutants with five mutations that mapped to the viral IRES. Knockdown studies showed that the mutations allowed HEVA71 to overcome treatment-induced suppression by differentially regulating recruitment of the IRES trans-acting factors Sam68 and hnRNPK without affecting the hnRNPA1-IRES interaction required for IRES translation. Furthermore, prunin effectively reduced HEVA71-associated clinical symptoms and mortality in HEVA71-infected BALB/c mice and suppressed hepatitis C virus at higher concentrations, suggesting a similar mechanism of prunin-mediated IRES inhibition for both viruses. These studies establish prunin as a candidate for further development as a HEVA71 therapeutic agent.

IDentif.AI-x, a clinically actionable artificial intelligence platform, was used to rapidly pinpoint and prioritize optimal combination therapies against COVID-19 by pairing a prospective, experimental validation of multi-drug efficacy on a SARS-CoV-2 live virus and Vero E6 assay with a quadratic optimization workflow. A starting pool of 12 candidate drugs developed in collaboration with a community of infectious disease clinicians was first narrowed down to a six-drug pool and then interrogated in 50 combination regimens at three dosing levels per drug, representing 729 possible combinations. IDentif.AI-x revealed EIDD-1931 to be a strong candidate upon which multiple drug combinations can be derived, and pinpointed a number of clinically actionable drug interactions, which were further reconfirmed in SARS-CoV-2 variants B.1.351 (Beta) and B.1.617.2 (Delta). IDentif.AI-x prioritized promising drug combinations for clinical translation and can be immediately adjusted and re-executed with a new pool of promising therapies in an actionable path towards rapidly optimizing combination therapy following pandemic emergence.

Recent results on the 102 residue protein U1A show that protein aggregation is not always slow and irreversible but may take place transiently in refolding studies on a millisecond time scale. In this study we observe a similar aggregation behavior with the classical two-state protein CI2. Since both U1A and CI2 appear to fold directly from the coil at low protein concentrations, it is likely that the aggregates also form directly from the coil. This is in contrast to the behavior of larger multistate proteins where aggregation occurs in connection to "sticky" intermediates.

An enzyme-linked immunosorbent assay (ELISA) and a rapid immunochromatographic test for detection of immunoglobulin G (IgG) antibodies in severe acute respiratory syndrome (SARS) patients were developed by utilizing the well-characterized recombinant proteins Gst-N and Gst-U274. The ELISA detected IgG antibodies to SARS-CoV in all 74 convalescent-phase samples from SARS patients while weakly cross-reacting to only 1 of the 210 control sera from healthy donors. This finding thus led to a kit sensitivity, specificity, and accuracy of 100, 99.5, and 99.6%, respectively. The test thus provided a positive predictive value (PPV) of 98.7% and a negative predictive value (NPV) of 100%. In addition, the ELISA gave a positive delta of 5.4 and a negative delta of 3.6, indicating an excellent differentiation between positives and negatives. The same recombinant proteins were also applied to a newly developed platform for the development of a 15-min rapid test. The resulting rapid test has an excellent agreement of 99.6%, with a kappa value of 1.00, with the ELISA. Again, this rapid test was able to detect 100% of the samples tested (n = 42) while maintaining a specificity of 99.0% (n = 210). The PPV and NPV for the rapid test thus reached 95.3 and 100%, respectively.

The severe acute respiratory syndrome coronavirus (SARS-CoV), isolated from humans infected during the peak of epidemic, encodes two accessory proteins termed as 8a and 8b. Interestingly, the SARS-CoV isolated from animals contains an extra 29-nucleotide in this region such that these proteins are fused to become a single protein, 8ab. Here, we compared the cellular properties of the 8a, 8b and 8ab proteins by examining their cellular localizations and their abilities to interact with other SARS-CoV proteins. These results may suggest that the conformations of 8a and 8b are different from 8ab although nearly all the amino acids in 8a and 8b are found in 8ab. In addition, the expression of the structural protein, envelope (E), was down-regulated by 8b but not 8a or 8ab. Consequently, E was not detectable in SARS-CoV-infected cells that were expressing high levels of 8b. These findings suggest that 8b may modulate viral replication and/or pathogenesis.

The small glutamine-rich tetratricopeptide repeat protein (SGT) belongs to a family of cochaperones that interacts with both Hsp70 and Hsp90 via the so-called TPR domain. Here, we present the crystal structure of the TPR domain of human SGT (SGT-TPR), which shows that it contains typical features found in the structures of other TPR domains. Previous studies show that full-length SGT can bind to both Vpu and Gag of human immunodeficiency virus type 1 (HIV-1) and the overexpression of SGT in cells reduces the efficiency of HIV-1 particle release. We show that SGT-TPR can bind Vpu and reduce the amount of HIV-1 p24, which is the viral capsid, secreted from cells transfected with the HIV-1 proviral construct, albeit at a lower efficiency than full-length SGT. This indicates that the TPR domain of SGT is sufficient for the inhibition of HIV-1 particle release but the N- and/or C-terminus also have some contributions. The SGT binding site in Vpu was also identified by using peptide array and confirmed by GST pull-down assay.

In June 2009, we conducted a prospective study in Singapore on 51 individuals to determine their serologic responses before and following receipt of the 2009 Southern Hemisphere seasonal influenza vaccine. Paired serum samples were obtained before and 3–4 weeks after vaccination. Virus microneutralization assays were performed to quantify antibodies against A/Brisbane/59/2007 vaccine, pandemic H1N1-2009 and A/Puerto Rico/08/34 H1N1 strains. Post-vaccination, 43%, 12% and 24% of subjects displayed a 4-fold or greater rise in neutralizing antibody titers against the three strains, respectively. There was a positive correlation among individuals who showed increased titers to both pandemic H1N1-2009 and A/Puerto Rico/08/34 (p < 0.001). However, this correlation was not observed for A/Brisbane/59/2007 with either strain. The relative conservation and accessibility of predicted B-cell epitopes may explain the limited cross-reactivity of the antibodies directed against common H1N1 epitopes. These results suggest that seasonal influenza vaccination confers a certain degree of cross-protection to other H1N1 strains. The correlation in cross-reactive antibody titers to A/Puerto Rico/08/34 and pandemic H1N1-2009 implies that previous exposure to pre-1957 H1N1 strains may confer some protection against the 2009 pandemic strain.

Viroporins are small, hydrophobic trans-membrane viral proteins that oligomerize to form hydrophilic pores in the host cell membranes. These proteins are crucial for the pathogenicity and replication of viruses as they aid in various stages of the viral life cycle, from genome uncoating to viral release. In addition, the ion channel activity of viroporin causes disruption in the cellular ion homeostasis, in particular the calcium ion. Fluctuation in the calcium level triggers the activation of the host defensive programmed cell death pathways as well as the inflammasome, which in turn are being subverted for the viruses' replication benefits. This review article summarizes recent developments in the functional investigation of viroporins from various viruses and their contributions to viral replication and virulence.

The Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) is the etiological agent for the infectious disease, SARS, which first emerged 10 years ago. SARS-CoV is a zoonotic virus that has crossed the species barriers to infect humans. Bats, which harbour a diverse pool of SARS-like CoVs (SL-CoVs), are believed to be the natural reservoir. The SARS-CoV surface Spike (S) protein is a major antigenic determinant in eliciting neutralizing antibody production during SARS-CoV infection. In our previous work, we showed that a panel of murine monoclonal antibodies (mAbs) that target the S2 subunit of the S protein are capable of neutralizing SARS-CoV infection in vitro (Lip KM et al, J Virol. 2006 Jan; 80(2): 941-50). In this study, we report our findings on the characterization of one of these mAbs, known as 1A9, which binds to the S protein at a novel epitope within the S2 subunit at amino acids 1111-1130. MAb 1A9 is a broadly neutralizing mAb that prevents viral entry mediated by the S proteins of human and civet SARS-CoVs as well as bat SL-CoVs. By generating mutant SARS-CoV that escapes the neutralization by mAb 1A9, the residue D1128 in S was found to be crucial for its interaction with mAb 1A9. S protein containing the substitution of D1128 with alanine (D1128A) exhibited a significant decrease in binding capability to mAb 1A9 compared to wild-type S protein. By using a pseudotyped viral entry assay, it was shown that the D1128A substitution in the escape virus allows it to overcome the viral entry blockage by mAb 1A9. In addition, the D1128A mutation was found to exert no effects on the S protein cell surface expression and incorporation into virion particles, suggesting that the escape virus retains the same viral entry property as the wild-type virus.

Chronic infection by HCV is closely correlated with liver diseases such as cirrhosis, steatosis, and hepatocellular carcinoma. To understand how long-term interaction between HCV and the host leads to pathogenesis, we identified cellular proteins that interact with NS5A and NS5B using a biochemical approach. Stable cell lines that express flag-NS5A or flag-NS5B under tetracycline induction were generated. The induced flag-tagged proteins were immunoprecipitated (IP'd) and associated proteins separated on 2D gels. Protein spots that specifically co-IP'd with NS5A or NS5B were identified by mass spectrometry. HSP27 was identified as a protein that specifically co-IP'd with NS5A but not with NS5B. The N-terminal regions of NS5A (a.a. 1-181) and HSP27 (a.a. 1-122) were defined to be the domains that interact with each other. HSP27 is generally distributed in the cytoplasm. When heat shocked, HSP27 is concentrated in the ER where NS5A is co-localized.

Abstract The hemagglutinin (HA) of influenza A virus plays an essential role in mediating the entry of the virus into host cells. Here, recombinant full‐length HA5 protein from a H5N1 isolate (A/chicken/hatay/2004(H5N1)) was expressed and purified from the baculovirus‐insect cell system. As expected, full‐length HA5 elicits strong neutralizing antibodies, as evaluated in micro‐neutralization tests using HA5 pseudotyped lentiviral particles. In addition, two fragments of HA5 were expressed in bacteria and the N‐terminal fragment, covering the ectodomain before the HA1/HA2 polybasic cleavage site, was found to elicit neutralizing antibodies. But the C‐terminal fragment, which covers the remaining portion of the ectodomain, did not. Neutralizing titer of the anti‐serum against the N‐terminal fragment is only four times lower than the anti‐serum against the full‐length HA5 protein. Using a novel membrane fusion assay, the abilities of these antibodies to block membrane fusion were found to correlate well with the neutralization activities. J. Med. Virol. 80:1972–1983, 2008. © 2008 Wiley‐Liss, Inc.

Both apoptosis and necrosis have been observed in cells infected by various coronaviruses, suggesting that the regulation of cell death is important for viral replication and/or pathogenesis. Expeditious research on the severe acute respiratory syndrome (SARS) coronavirus, one of the latest discovered coronaviruses that infect humans, has provided valuable insights into the molecular aspects of cell-death regulation during infection. Apoptosis was observed in vitro, while both apoptosis and necrosis were observed in tissues obtained from SARS patients. Viral proteins that can regulate apoptosis have been identified, and many of these also have the abilities to interfere with cellular functions. Occurrence of cell death in host cells during infection by other coronaviruses, such as the mouse hepatitis virus and transmissible porcine gastroenteritis virus, has also being extensively studied. The diverse cellular responses to infection revealed the complex manner by which coronaviruses affect cellular homeostasis and modulate cell death. As a result of the complex interplay between virus and host, infection of different cell types by the same virus does not necessarily activate the same cell-death pathway. Continuing research will lead to a better understanding of the regulation of cell death during viral infection and the identification of novel antiviral targets.

The ORF6 protein is one of the eight accessory proteins of the severe acute respiratory syndrome coronavirus (SARS-CoV). Numerous properties of ORF6 have been documented and this study focuses on two of these, namely, its ability to suppress the expression of co-transfected expression constructs and its subcellular localization to vesicular structures.Using a transient transfection system, ORF6's ability to suppress the expression of co-transfected expression constructs was measured in a quantitative manner. While ORF6 does not have a global effect on protein synthesis, quantitative real-time PCR revealed that it down-regulated the mRNA level of the co-transfected myc-nsp8 gene. Furthermore, alanine substitution of a diacidic cluster motif (aa53-56) in the ORF6 gene caused a reduction in the suppression of expression of co-transfected myc-nsp8 gene. Our previous study revealed that ORF6 localized to vesicular structures in SARS-CoV infected Vero E6 cells. Here, ORF6 was observed to be localized to similar vesicular structures in Vero E6 cells which have been transiently transfected with a mammalian expression plasmid encoding for untagged ORF6. ORF6 showed partial colocalization with cellular proteins CD63 and Lamp1, suggesting that the vesicular structures may be a subpopulation of endosomal/lysosomal vesicles. The alanine substitution of the diacidic cluster motif also altered the subcellular localization of the ORF6 protein, indicating a potential relationship between the subcellular localization of the ORF6 protein and its ability to suppress the expression of co-transfected expression constructs.By combining quantitative real-time PCR and transient transfection system, a simple and safe method is established to measure ORF6's ability to suppress the expression of co-transfected myc-nsp8. In addition, immunofluorescence analysis revealed that the subcellular localization of ORF6 when expressed on its own is similar to that observed in SARS-CoV infected cells. Through the use of these two assays, a putative diacidic motif in the ORF6 protein was found to influence its subcellular localization and ability to suppress the expression of co-transfected expression constructs.

Most viruses encode proteins that modulate cell-death signaling by the host. For hepatitis C virus (HCV) infection, apoptosis and other forms of cell-death have been observed in vitro and in vivo but the detailed understanding of this intricate viral-host interplay is unclear. This study examined the role played by the HCV p7 protein in the induction of cell-death. By measuring caspase-3/7 activation and cleavage of endogenous PARP, two hallmarks of apoptosis, the overexpression of p7 protein was shown to induce apoptosis in Huh7.5 cells. Furthermore, p7-induced apoptosis is caspase-dependent and involves both the intrinsic and extrinsic pathways. Similar to the M2 protein of influenza A virus, p7-induced apoptosis is independent of its ion channel activity. Coimmunoprecipitation experiments further showed that both M2 and p7 interact with the essential autophagy protein Beclin-1. However, only the M2 protein could cause an increase in the level of LC3-II, which is an indicator of autophagic activity. Thus, although the p7 protein is functionally similar to the well-characterized M2 protein, they differ in their activation of autophagic cell-death. Taken together, these results shed more light on the relationship between the HCV p7 ion channel protein and cell-death induction in host cells.

Chronic hepatitis B virus (HBV) infection can lead to liver cirrhosis and hepatocellular carcinoma. HBV reactivation during or after chemotherapy is a potentially fatal complication for cancer patients with chronic HBV infection. Transcription of HBV is a critical intermediate step of the HBV life cycle. However, factors controlling HBV transcription remain largely unknown. Here, we found that different P-TEFb complexes are involved in the transcription of the HBV viral genome. Both BRD4 and the super elongation complex (SEC) bind to the HBV genome. The treatment of bromodomain inhibitor JQ1 stimulates HBV transcription and increases the occupancy of BRD4 on the HBV genome, suggesting the bromodomain-independent recruitment of BRD4 to the HBV genome. JQ1 also leads to the increased binding of SEC to the HBV genome, and SEC is required for JQ1-induced HBV transcription. These findings reveal a novel mechanism by which the HBV genome hijacks the host P-TEFb-containing complexes to promote its own transcription. Our findings also point out an important clinical implication, that is, the potential risk of HBV reactivation during therapy with a BRD4 inhibitor, such as JQ1 or its analogues, which are a potential treatment for acute myeloid leukemia.

We are investigating compounds that could be useful in the treatment of neoplastic lesions of the cervix by acting on the oncoprotein E6 of human papillomavirus-16. The E6 protein contains two potential zinc-binding domains that are required for most of its functions. We have published tests that measure (i) the release of zinc ions after chemical alteration of the cysteine groups of these zinc-binding domains (TSQ assay), (ii) the interaction of E6 with the cellular proteins E6AP and E6BP (BIACORE assay), and (iii) the viability of tumor cell lines that require the continuous expression of HPV oncoproteins (WST1 assay). Based on these tests, we identified 4,4′-dithiodimorpholine as a potential lead compound. In this study we examined whether the dithiobisamine moiety of 4,4′-dithiodimorpholine may be an important molecular prerequisite for further drug development in this system. We have evaluated 59 new substances including organic disulfides and those containing the dithiobisamine moiety, as well as structural analogues. The compounds with significant reactivity in all three assays were observed only for dithiobisamine derivatives with saturated cyclic amines and aryl substituted piperazines. The identity of these substances suggests that the N–S–S–N moiety is necessary but not sufficient for reactivity in our assays, and that dithiobisamine based substances are useful as lead compounds that target the cysteine groups of HPV-16 E6 zinc fingers.

NS3, a non-structural protein of the HCV (hepatitis C virus), contains a protease and a helicase domain and plays essential roles in the processing of the viral polyprotein, viral RNA replication and translation. LMP7 (low-molecular-mass protein 7), a component of the immunoproteasome, was identified as an NS3-binding protein from yeast two-hybrid screens, and this interaction was confirmed by in vitro binding and co-immunoprecipitation analysis. The minimal domain of interaction was defined to be between the pro-sequence region of LMP7 (amino acids 1-40) and the protease domain of NS3. To elucidate the biological importance of this interaction, we studied the effect of this interaction on NS3 protease activity and on LMP7 immunoproteasome activity. Recombinant LMP7 did not have any effect on NS3 protease activity in vitro. The peptidase activities of LMP7 immunoproteasomes, however, were markedly reduced when tested in a stable cell line containing a HCV subgenomic replicon. The down-regulation of proteasome peptidase activities could interfere with the processing of viral antigens for presentation by MHC class I molecules, and may thus protect HCV from host immune surveillance mechanisms to allow persistent infection by the virus.

Abstract Background A recent publication reported that a tyrosine-dependent sorting signal, present in cytoplasmic tail of the spike protein of most coronaviruses, mediates the intracellular retention of the spike protein. This motif is missing from the spike protein of the severe acute respiratory syndrome-coronavirus (SARS-CoV), resulting in high level of surface expression of the spike protein when it is expressed on its own in vitro . Presentation of the hypothesis It has been shown that the severe acute respiratory syndrome-coronavirus genome contains open reading frames that encode for proteins with no homologue in other coronaviruses. One of them is the 3a protein, which is expressed during infection in vitro and in vivo . The 3a protein, which contains a tyrosine-dependent sorting signal in its cytoplasmic domain, is expressed on the cell surface and can undergo internalization. In addition, 3a can bind to the spike protein and through this interaction, it may be able to cause the spike protein to become internalized, resulting in a decrease in its surface expression. Testing the hypothesis The effects of 3a on the internalization of cell surface spike protein can be examined biochemically and the significance of the interplay between these two viral proteins during viral infection can be studied using reverse genetics methodology. Implication of the hypothesis If this hypothesis is proven, it will indicate that the severe acute respiratory syndrome-coronavirus modulates the surface expression of the spike protein via a different mechanism from other coronaviruses. The interaction between 3a and S, which are expressed from separate subgenomic RNA, would be important for controlling the trafficking properties of S. The cell surface expression of S in infected cells significantly impacts viral assembly, viral spread and viral pathogenesis. Modulation by this unique pathway could confer certain advantages during the replication of the severe acute respiratory syndrome-coronavirus.

The most striking difference between the subgenomic mRNA8 of severe acute respiratory syndrome coronavirus isolated from human and some animal species is the deletion of 29 nucleotides, resulting in splitting of a single ORF (ORF8) into two ORFs (ORF8a and ORF8b). ORF8a and ORF8b are predicted to encode two small proteins, 8a and 8b, and ORF8 a single protein, 8ab (a fusion form of 8a and 8b). To understand the functions of these proteins, we cloned cDNA fragments covering these ORFs into expression plasmids, and expressed the constructs in both in vitro and in vivo systems. Expression of a construct containing ORF8a and ORF8b generated only a single protein, 8a; no 8b protein expression was obtained. Expression of a construct containing ORF8 generated the 8ab fusion protein. Site-directed mutagenesis and enzymatic treatment revealed that protein 8ab is modified by N-linked glycosylation on the N81 residue and by ubiquitination. In the absence of the 8a region, protein 8b undergoes rapid degradation by proteasomes, and addition of proteasome inhibitors inhibits the degradation of protein 8b as well as the protein 8b-induced rapid degradation of the severe acute respiratory syndrome coronavirus E protein. Glycosylation could also stabilize protein 8ab. More interestingly, the two proteins could bind to monoubiquitin and polyubiquitin, suggesting the potential involvement of these proteins in the pathogenesis of severe acute respiratory syndrome coronavirus.

The hepatitis C virus (HCV) core protein is known to modulate apoptosis and contribute to viral replication and pathogenesis. In this study, we have identified a Bcl-2 homology 3 (BH3) domain in the core protein that is essential for its proapoptotic property. Coimmunoprecipitation experiments showed that the core protein interacts specifically with the human myeloid cell factor 1 (Mcl-1), a prosurvival member of the Bcl-2 family, but not with other prosurvival members (Bcl-X(L) and Bcl-w). Moreover, the overexpression of Mcl-1 protects against core-induced apoptosis. By using peptide mimetics, core was found to release cytochrome c from isolated mitochondria when complemented with Bad. Thus, core is a bona fide BH3-only protein having properties similar to those of Noxa, a BH3-only member of the Bcl-2 family that binds preferentially to Mcl-1. There are three critical hydrophobic residues in the BH3 domain of the core protein, and they are essential for the proapoptotic property of the core protein. Furthermore, the genotype 1b core protein is more effective than the genotype 2a core protein in inducing apoptosis due to a single-amino-acid difference at one of these hydrophobic residues (residue 119). Replacing this residue in the J6/JFH-1 infectious clone (genotype 2a) with the corresponding amino acid in the genotype 1b core protein produced a mutant virus, J6/JFH-1(V119L), which induced significantly higher levels of apoptosis in the infected cells than the parental J6/JFH-1 virus. Furthermore, the core protein of J6/JFH-1(V119L), but not that of J6/JFH-1, interacted with Mcl-1 in virus-infected cells. Taken together, the core protein is a novel BH3-only viral homologue that contributes to the induction of apoptosis during HCV infection.

Pathogenesis of viral haemorrhagic fevers is associated with alteration of vascular barrier function and haemorrhage. To date, the specific mechanism behind this is unknown. Programmed cell death and regulation of apoptosis in response to viral infection is an important factor for host or virus survival but this has not been well-studied in the case of Crimean-Congo hemorrhagic fever virus (CCHFV). In this study, we demonstrated that CCHFV infection suppresses cleavage of poly(ADP-ribose) polymerase (PARP), triggered by staurosporine early post-infection. We also demonstrated that CCHFV infection suppresses activation of caspase-3 and caspase-9. Most interestingly, we found that CCHFV N can suppress induction of apoptosis by Bax and inhibit the release of cytochrome c from the inner membrane of mitochondria to cytosol. However, CCHFV infection induces activation of Bid late post-infection, suggesting activation of extrinsic apoptotic signalling. Consistently, supernatant from cells stimulated late post-infection was found to induce PARP cleavage, most probably through the TNF-α death receptor pathway. In summary, we found that CCHFV has strategies to interplay with apoptosis pathways and thereby regulate caspase cascades. We suggest that CCHFV suppresses caspase activation at early stages of the CCHFV replication cycle, which perhaps benefits the establishment of infection. Furthermore, we suggest that the host cellular response at late stages post-infection induces host cellular pro-apoptotic molecules through the death receptor pathway.

A synthetic peptide corresponding to amino acids (aa) 15-28 of the severe acute respiratory syndrome coronavirus (SARS-CoV) 3a protein was used to raise polyclonal antibodies in rabbits. This anti-3a N-terminal antibody could detect 3a protein in infected cells, as did an anti-3a C-terminal antibody previously described. The latter targeted the C-terminal cytoplasmic domain of 3a (aa 134-274). The anti-3a N-terminal antibody could detect intracellular 3a as well as 3a expressed on the cell surface. Interestingly, only the anti-3a N-terminal antibody can inhibit SARS-CoV propagation in Vero E6 culture although the binding affinity of the anti-3a N-terminal antibody was lower than the anti-3a C-terminal antibody.

The causative agent of severe acute respiratory syndrome, SARS coronavirus (SARS-CoV) genome encodes several unique group specific accessory proteins with unknown functions. Among them, accessory protein 3b (also known as ORF4) was lately identified as one of the viral interferon antagonist. Recently our lab uncovered a new role for 3b in upregulation of AP-1 transcriptional activity and its downstream genes. Thus, we believe that 3b might play an important role in SARS-CoV pathogenesis and therefore is of considerable interest. The current study aims at identifying novel host cellular interactors of the 3b protein.In this study, using yeast two-hybrid and co-immunoprecipitation techniques, we have identified a host transcription factor RUNX1b (Runt related transcription factor, isoform b) as a novel interacting partner for SARS-CoV 3b protein. Chromatin immunoprecipitaion (ChIP) and reporter gene assays in 3b expressing jurkat cells showed recruitment of 3b on the RUNX1 binding element that led to an increase in RUNX1b transactivation potential on the IL2 promoter. Kinase assay and pharmacological inhibitor treatment implied that 3b also affect RUNX1b transcriptional activity by regulating its ERK dependent phosphorylation levels. Additionally, mRNA levels of MIP-1α, a RUNX1b target gene upregulated in SARS-CoV infected monocyte-derived dendritic cells, were found to be elevated in 3b expressing U937 monocyte cells.These results unveil a novel interaction of SARS-CoV 3b with the host factor, RUNX1b, and speculate its physiological relevance in upregulating cytokines and chemokine levels in state of SARS virus infection.

Chikungunya virus (CHIKV) is a medically important human viral pathogen that causes Chikungunya fever accompanied with debilitating and persistent joint pain. Host-elicited or passively-transferred monoclonal antibodies (mAb) are essential mediators of CHIKV clearance. Therefore, this study aimed to generate and characterize a panel of mAbs for their neutralization efficacy against CHIKV infection in a cell-based and murine model. To evaluate their antigenicity and neutralization profile, indirect enzyme-linked immunosorbent assay (ELISA), an immunofluorescence assay (IFA) and a plaque reduction neutralization test were performed on mAbs of IgM isotype. CHIKV escape mutants against mAb 3E7b neutralization were generated, and reverse genetics techniques were then used to create an infectious CHIKV clone with a single mutation. 3E7b was also administered to neonate mice prior or after CHIKV infection. The survival rate, CHIKV burden in tissues and histopathology of the limb muscles were evaluated. Both IgM 3E7b and 8A2c bind strongly to native CHIKV surface and potently neutralize CHIKV replication. Further analyses of 3E7b binding and neutralization of CHIKV single-mutant clones revealed that N218 of CHIKV E2 protein is a potent neutralizing epitope. In a pre-binding neutralization assay, 3E7b blocks CHIKV attachment to permissive cells, possibly by binding to the surface-accessible E2-N218 residue. Prophylactic administration of 3E7b to neonate mice markedly reduced viremia and protected against CHIKV pathogenesis in various mice tissues. Given therapeutically at 4 h post-infection, 3E7b conferred 100% survival rate and similarly reduced CHIKV load in most mice tissues except the limb muscles. Collectively, these findings highlight the usefulness of 3E7b for future prophylactic or epitope-based vaccine design.

The overexpression of Bax, a member of the Bcl-2 family, promotes cell death and the dimerization (or oligomerization) of Bax has been shown to be important for its function. Using size-exclusion chromatography andin vitrocross-linking experiments, we demonstrated that Bax exists mainly as a large oligomer of approximately 30 monomeric units. Furthermore, several binding assays demonstrated that Bcl-XL, an anti-apoptotic member of the Bcl-2 family, can bind to the oligomeric form of Bax without requiring Bax to dissociate to monomers.

The severe acute respiratory syndrome coronavirus (SARS-CoV) 8b protein, which is not expressed by other known coronaviruses, can down-regulate the envelope (E) protein via a proteasome-dependent pathway. Here, we showed that the down-regulation of E is not dependent on the lysine residues on 8b and the reduction of polyubiquitination of E mutants is not correlated with their down-regulation by 8b, suggesting an ubiquitin-independent proteasome pathway is involved. A time-course study revealed that 8b was expressed at late-stages of SARS-CoV infection. By using Vero E6 cells stably expressing green fluorescence protein-tagged 8b, ectopic expression of 8b was shown to significantly reduce the production of progeny virus and down-regulate E expression. Taken together, these results suggest that 8b negatively modulates virus replication by down-regulating E via an ubiquitin-independent proteasome pathway.

Middle East respiratory syndrome coronavirus (MERS-CoV) causes respiratory diseases in humans and has a high mortality rate. During infection, MERS-CoV regulates several host cellular processes including antiviral response genes. In order to determine if the nucleocapsid protein of MERS-CoV (MERS-N) plays a role in viral-host interactions, a murine monoclonal antibody was generated so as to allow detection of the protein in infected cells as well as in overexpression system. Then, MERS-N was stably overexpressed in A549 cells, and a PCR array containing 84 genes was used to screen for genes transcriptionally regulated by it. Several up-regulated antiviral genes, namely TNF, IL6, IL8, and CXCL10, were selected for independent validation in transiently transfected 293FT cells. Out of these, the overexpression of MERS-N was found to up-regulate CXCL10 at both transcriptional and translational levels. Interestingly, CXCL10 has been reported to be up-regulated in MERS-CoV infected airway epithelial cells and lung fibroblast cells, as well as monocyte-derived macrophages and dendritic cells. High secretions and persistent increase of CXCL10 in MERS-CoV patients have been also associated with severity of disease. To our knowledge, this is the first report showing that the MERS-N protein is one of the contributing factors for CXCL10 up-regulation during infection. In addition, our results showed that a fragment consisting of residues 196-413 in MERS-N is sufficient to up-regulate CXCL10, while the N-terminal domain and serine-arginine (SR)-rich motif of MERS-N do not play a role in this up-regulation.

The non-structural protein NS1 of the influenza A virus is a good target for the development of diagnostic assays. In this study, three NS1 monoclonal antibodies (mAbs) were generated by using recombinant NS1 protein of H5N1 virus and found to bind both the native and denatured forms of NS1. Two of the mAbs, 6A4 and 2H6, bind NS1 of three different strains of influenza A virus, namely H1N1, H3N2, and H5N1. Epitope mapping revealed that residues 42-53 of H5N1 NS1 are essential for the interaction with both mAbs. Between the three strains, there is only one amino acid difference in this domain, which is consistent with the observed cross-reactivities. On the other hand, mAb 1G1 binds to residues 206-215 of H5N1 NS1 and does not bind NS1 of H1N1 or H3N2. Furthermore, all three mAbs detected NS1 proteins expressed in virus infected MDCK cells and indirect immunofluorescence staining with mAbs 6A4 and 2H6 provided an alternative method for viral titer determination. Quantifying the numbers of fluorescent foci units yielded viral titers for three different isolates of H5N1 virus that are highly comparable to that obtained by observing cytopathic effect induced by virus infection. Importantly, this alternative method yields results at 1 day post-infection while the conventional method using cytopathic effect yields results at 3 days post-infection. The results showed that this new panel of NS1 antibodies can detect NS1 protein expressed during viral infection and can be used for fast and easy titration of influenza A virus. J. Med. Virol. 82:467-475, 2010. (c) 2010 Wiley-Liss, Inc.

Initial attempts to develop monoclonal antibodies as therapeutics to resolve influenza infections focused mainly on searching for antibodies with the potential to neutralise the virus in vitro with classical haemagglutination inhibition and microneutralisation assays. This led to the identification of many antibodies that bind to the head domain of haemagglutinin (HA), which generally have potent neutralisation capabilities that block viral entry or viral membrane fusion. However, this class of antibodies has a narrow breadth of protection in that they are usually strain-specific. This led to the emphasis on stalk-targeting antibodies, which are able to bind a broad range of viral targets that span across different influenza subtypes. Recently, a third class of antibodies targeting the vestigial esterase (VE) domain have been characterised. In this review, we describe the key features of neutralising VE-targeting antibodies and compare them with head- and stalk-class antibodies.

RNA helicases of the DEAD (Asp-Glu-Ala-Asp)-box family of proteins are involved in many aspects of RNA metabolism from transcription to RNA decay, but most of them have also been shown to be multifunctional. The DEAD-box helicase DDX5 of host cells has been shown to interact with the RNA-dependent RNA polymerase (NS5B) of HCV (hepatitis C virus). In the present study, we report the presence of two independent NS5B-binding sites in DDX5, one located at the N-terminus and another at the C-terminus. The N-terminal fragment of DDX5, which consists of the first 305 amino acids and shall be referred as DDX5-N, was expressed and crystallized. The crystal structure shows that domain 1 (residues 79–303) of DDX5 contains the typical features found in the structures of other DEAD-box helicases. DDX5-N also contains the highly variable NTR (N-terminal region) of unknown function and the crystal structure reveals structural elements in part of the NTR, namely residues 52–78. This region forms an extensive loop and an α-helix. From co-immunoprecipitation experiments, the NTR of DDX5-N was observed to auto-inhibit its interaction with NS5B. Interestingly, the α-helix in NTR is essential for this auto-inhibition and seems to mediate the interaction between the highly flexible 1–51 residues in NTR and the NS5B-binding site in DDX5-N. Furthermore, NMR investigations reveal that there is a direct interaction between DDX5 and NS5B in vitro.

Nearly 200 million people are infected by hepatitis C virus (HCV) worldwide. For replicating the HCV genome, the membrane-associated machinery needs to be formed by both HCV non-structural proteins (including NS5B) and human host factors such as VAPB. Recently, the 99-residue VAPC, a splicing variant of VAPB, was demonstrated to inhibit HCV replication via binding to NS5B, thus acting as an endogenous inhibitor of HCV infection. So far, the structure of VAPC remains unknown, and its interaction with NS5B has not been biophysically characterized. In this study, we conducted extensive CD and NMR investigations on VAPC which led to several striking findings: 1) although the N-terminal 70 residues are identical in VAPC and VAPB, they constitute the characteristic β-barrel MSP fold in VAPB, while VAPC is entirely unstructured in solution, only with helical-like conformations weakly populated. 2) VAPC is indeed capable of binding to NS5B, with an average dissociation constant (Kd) of ∼20 µM. Intriguingly, VAPC remains dynamic even in the complex, suggesting that the VAPC-NS5B is a "fuzzy complex". 3) NMR mapping revealed that the major binding region for NS5B is located over the C-terminal half of VAPC, which is composed of three discrete clusters, of which only the first contains the region identical in VAPC and VAPB. The second region containing ∼12 residues appears to play a key role in binding since mutation of 4 residues within this region leads to almost complete loss of the binding activity. 4) A 14-residue mimetic, VAPC-14 containing the second region, only has a ∼3-fold reduction of the affinity. Our study not only provides critical insights into how a human factor mediates the formation of the HCV replication machinery, but also leads to design of VAPC-14 which may be further used to explore the function of VAPC and to develop anti-HCV molecules.

Hepatitis C virus (HCV) induces cytopathic effects in the form of hepatocytes apoptosis thought to be resulted from the interaction between viral proteins and host factors. Using pathway specific PCR array, we identified 9 apoptosis-related genes that are dysregulated during HCV infection, of which the BH3-only pro-apoptotic Bcl-2 family protein, BIK, was consistently up-regulated at the mRNA and protein levels. Depletion of BIK protected host cells from HCV-induced caspase-3/7 activation but not the inhibitory effect of HCV on cell viability. Furthermore, viral RNA replication and release were significantly suppressed in BIK-depleted cells and over-expression of the RNA-dependent RNA polymerase, NS5B, was able to induce BIK expression. Immunofluorescence and co-immunoprecipitation assays showed co-localization and interaction of BIK and NS5B, suggesting that BIK may be interacting with the HCV replication complex through NS5B. These results imply that BIK is essential for HCV replication and that NS5B is able to induce BIK expression.

The persistent evolution and circulation of highly pathogenic avian influenza H5N1 viruses pose a serious threat to global heath and hamper pandemic preparedness through conventional vaccine strategies. Combination passive immunotherapy using non-competing neutralizing antibodies has been proposed as a viable alternative to provide broad protection against drift variants. This necessitates the pre-pandemic production and characterization of potently neutralizing monoclonal antibodies (MAbs). One such antibody, MAb 9F4 was shown to provide heterologous protection against multiple H5N1 clade viruses, including one of the recently designated subclades, namely 2.3.4, through binding to a novel epitope, warranting its further development and characterization as a therapeutic candidate. In this study, the conversion of MAb 9F4 from mouse IgG2b to mouse-human chimeric (xi) IgG1 and IgA1 was achieved. These chimeric MAb versions were found to retain high degrees of binding and neutralizing activity against H5N1. The demonstration that xi-IgA1-9F4 retains a fairly high level of neutralizing activity, which is ∼10-fold lower than the corresponding xi-IgG1 isotype, suggests that this MAb could be further developed and engineered for intranasal administration.

The Zika virus (ZIKV) is a mosquito-borne flavivirus that causes neonatal abnormalities and other disorders. Antibodies to the ZIKV envelope (E) protein can block infection. In this study, next-generation sequencing (NGS) of immunoglobulin heavy chain (IgH) mRNA transcripts was combined with single-cell PCR cloning of E-binding monoclonal antibodies for analysing antibody response in a patient from the early stages of infection to more than one year after the clearance of the virus. The patient's IgH repertoire 14 and 64 days after symptom onset showed dramatic dominant clonal expansion but low clonal diversity. IgH repertoire 6 months after disease-free status had few dominant clones but increased diversity. E-binding antibodies appeared abundantly in the repertoire during the early stages of infection but quickly declined after clearance of the virus. Certain VH genes such as VH5-10-1 and VH4-39 appeared to be preferentially enlisted for a rapid antibody response to ZIKV infection. Most of these antibodies require relatively few somatic hypermutations to acquire the ability to bind to the E protein, pointing to a possible mechanism for rapid defence against ZIKV infection. This study provides a unique and holistic view of the dynamic changes and characteristics of the antibody response to ZIKV infection.

There are four peptidyl-proline bonds in the 64-residue protein chymotrypsin inhibitor 2 (CI2), all of which are in the trans conformation in the native structure. The isomerisation of one or more of these peptidyl-proline bonds to the cis conformation in the denatured state gives rise to heterogeneity, leading to both fast and slow-folding species. The refolding of the fast-folding species, which has all trans peptidyl-proline bonds, is much faster than that of the slow-folding species, which have one or more cis peptidyl-proline bonds. In CI2, the slow-folding species can be classified into two groups by their rates of refolding, temperature-dependence, pH-dependence and [GdmCl]-dependence of the rate constants and the effect of peptidyl-prolyl isomerase on the rate constants. The replacement of Pro6 by Ala removes one of the slow refolding phases, suggesting that the cis peptidyl-Pro6 conformation is solely responsible for one of the slow-folding species. Pro6 is located in a region of the protein where non-random interactions have been found in a series of N-terminal fragments of CI2 (residues 1 to 13, 1 to 25, 1 to 28 and 1 to 40). In addition, NMR studies on a mutant fragment, (1-40)T3A, have confirmed that this non-native interaction is associated with the bulky side-chain of Trp5. The atypical rate of cis to trans isomerisation of the peptidyl-Pro bond is indicative of the presence of a similar hydrophobic cluster in the physiological denatured state of intact CI2.

Passive immunotherapy has mainly been used as a therapy against cancer and inflammatory conditions. Recent studies have shown that monoclonal antibody-(mAb-) based passive immunotherapy is a promising approach to combat virus infection. Specific mouse mAbs can be routinely generated in large amounts with the use of hybridoma technology but these cannot be used for therapy in human beings due to their immunogenicity. Therefore, the development of chimeric and humanized mAbs is important for therapeutic purpose. This is facilitated by a variety of molecular techniques like recombinant DNA technology and the better understanding of the structure and function of antibody. The human-mouse chimeric forms allow detailed analysis of the mechanism of inhibition and the potential for therapeutic applications. Here, a step-by-step description of the conversion process will be described. The commercial availability of the reagents required in each step means that this experimentation can be easily set up in research laboratories.

A novel coronavirus was identified as the aetiological agent for the global outbreak of severe acute respiratory syndrome (SARS) at the beginning of the twenty-first century. The SARS coronavirus genome encodes for proteins that are common to all members of the coronavirus, i.e. replicase polyproteins (pp1a and pp1b) and structural proteins (spike, membrane, nucleocapsid and envelope), as well as eight accessory proteins. The accessory proteins have been designated as open reading frames (ORF) 3a, 3b, 6, 7a, 7b, 8a, 8b and 9b, and they do not show significant homology to viral proteins of other known coronaviruses. Epidemiological studies have revealed that the part of the viral genome that encodes for ORF8a and ORF8b showed major variations and the animal isolates contain an additional 29-nucleotide sequence which is absent in most of the human isolates. As a result, ORF8a and ORF8b in the human isolates become one ORF, termed ORF8ab. In this chapter, we will discuss the genetic variation in the ORF8 region, expression of ORF8a, ORF8b and ORF8ab during infection, cellular localization and posttranslational modification of ORF8a, ORF8b and ORF8ab, participation of ORF8a, ORF8b and ORF8ab in viral–viral interactions, their effects on other viral proteins and impact on viral replication and/or pathogenesis.

Saffold virus (SAFV), a newly discovered human cardiovirus of the Picornaviridae family, causes widespread infection among children, as shown by previous seroprevalence studies. To determine the host cell range of SAFV and its cytopathogenicity, eight mammalian cell lines that were available in the laboratory were screened for productive SAFV infection by a laboratory-adapted SAFV of genotype 3. Five of the cell lines (Neuro2A, CHO-K1, NIH/3T3, Vero and HEp-2) were found to be permissible. The time required for SAFV to induce complete lysis as a cytopathic effect (CPE) in these permissibly infected cells and the resultant end point virus titer differed for each cell type. HEp-2 exhibited the shortest time frame to reach full CPE compared to the others. All infected cell lines produced a high virus titer at 72 h post-infection. In addition to causing lytic cell death, SAFV also induced apoptotic cell death in host cells through both extrinsic and intrinsic pathways, although the apoptotic events in HEp-2 cells appeared to have been blocked between the early and late stages. In conclusion, laboratory-adapted SAFV is able to productively infect a number of mammalian cell lines and induce apoptosis in the infected host cells. However, apoptosis in HEp-2 cells is blocked before the end stage.

The emergence of resistant influenza A viruses highlights the continuous requirement of new antiviral drugs that can treat the viral infection. Non-structural 1 (NS1) protein, an indispensable component for efficient virus replication, can be used as a potential target for generating new antiviral agents. Here, we study the interaction of 2H6 monoclonal antibody with NS1 protein and also determine whether influenza virus replication can be inhibited by blocking NS1. The 2H6-antigen binding fragment (Fab) forms a multimeric complex with the NS1 RNA-binding domain (RBD). T49, a residue which forms a direct hydrogen bond with double stranded RNA, in NS1 protein was found to be critical for its interaction with 2H6 antibody. NS1(RBD) has high affinity to 2H6 with KD of 43.5 ± 4.24 nM whereas NS1(RBD)-T49A has more than 250 times lower affinity towards 2H6. Interestingly, the intracellular expression of 2H6-single-chain variable fragment (scFv) in mammalian cells caused a reduction in viral growth and the M1 viral protein level was significantly reduced in 2H6-scFv transfected cells in comparison to vector transfected cells at 12 h post infection. These results indicate that the tight binding of 2H6 to NS1 could lead to reduction in viral replication and release of progeny virus. In future, 2H6 antibody in combination with other neutralizing antibodies can be used to increase the potency of viral inhibition.

The sporadic outbreaks of highly pathogenic H5N1 avian influenza virus have raised public health concerns. Monoclonal antibodies (MAbs) against hemagglutinin (HA) have been increasingly used successfully for therapeutic purposes. Previously, MAb 9F4, generated against clade 1 H5N1 HA, was observed to have cross-clade neutralizing efficacy and inhibited viral entry by preventing the pH-mediated conformational change of HA. Furthermore, mouse-human chimeric MAb 9F4 was found to retain high degrees of neutralizing activity. In this study, through escape mutant generation and in-silico prediction, it was revealed that MAb 9F4 binds to a novel epitope in the vestigial esterase sub-domain of HA comprising at least three non-continuous amino acid residues, arginine (R) at position 62, tryptophan (W) at position 69 and phenylalanine (F) at position 79, which interacted with MAb 9F4 in a conformation-dependent manner. Binding and neutralization studies suggested that R62 is the critical residue for MAb 9F4 binding whereas W69 and F79 seem to cooperate with R62 to stabilize the epitope. Mutation of either R62 or W69 did not affect replicative fitness of the virus in vitro. Interestingly, MAb 9F4 retained neutralizing efficacy against a clade 2.3.2.1a H5N1 virus consisting of an arginine to lysine substitution at position 62 in HA.

Rhesus macaques (Macaca mulatta) are used as a human-relevant animal species for the evaluation of vaccines and as a source for cloning monoclonal antibodies (mAbs) that are highly similar to human-derived antibodies. Although antibody-secreting plasmablasts in humans are well-defined and can be easily isolated for mAb cloning, it remains unclear whether the same phenotypic markers could be applied for isolating antibody-secreting plasmablasts from Chinese rhesus macaques. In this study, we evaluated a series of cell surface and intracellular markers and identified the phenotypic markers of plasmablasts in Chinese rhesus macaques as CD3-CD14-CD56-CD19-CD27-CD20-/lowCD80+HLA-DR+CD95+. After influenza virus vaccination, the plasmablasts in peripheral blood mononuclear cells (PBMCs) increased transiently, peaked at day 4-7 after booster vaccination and returned to nearly undetectable levels by day 14. Antigen-specific enzyme-linked immunosorbent spot (ELISPOT) assays confirmed that the majority of the plasmablasts could produce influenza virus-specific antibodies. These plasmablasts showed transcriptional characteristics similar to those of human plasmablasts. Using single-cell PCR for immunoglobulin heavy and light chains, most mAbs cloned from the CD3-CD14-CD56-CD19-CD27-CD20-/lowCD80+HLA-DR+CD95+ plasmablasts after vaccination exhibited specific binding to influenza virus. This study defined the phenotypic markers for isolating antibody-secreting plasmablasts from Chinese rhesus macaques, which has implications for efficient cloning of mAbs and for the evaluation of plasmablast response after vaccination or infection in Chinese rhesus macaques.

In recent years, there has been an alarming increase in the number of cases of coinfection with the human immunodeficiency virus type 1 (HIV-1) and the hepatitis C virus (HCV). It is now known that coinfection of HIV-1 patients by HCV can complicate the treatment of these patients with highly active antiretroviral therapy and the interactions between anti-HIV-1 and anti-HCV medications can also affect treatment efficacy and efficiency. Equally concerning, the bidirectional interferences between the two viruses are complex and can modify the natural history of both infections. This review aims to summarize the findings of numerous scientific investigations in the area of HIV/HCV coinfection. These investigations can be broadly classified into 3 groups; (a) immune evasion mechanisms (b) viral evolution and quasispecies diversity and (c) functions of viral proteins and their interactions with host factors. Our cumulative knowledge in this area and future research on the interplay between these two viruses will be important to the development of better antiviral therapeutics.

The highly pathogenic avian influenza A (H5N6) virus has caused sporadic human infections with a high case fatality rate. Due to the continuous evolution of this virus subtype and its ability to transmit to humans, there is an urgent need to develop effective antiviral therapeutics. In this study, a murine monoclonal antibody 9F4 was shown to display broad binding affinity against H5Nx viruses. Furthermore, 9F4 can neutralize H5N6 pseudotyped particles and prevent entry into host cells. Additionally, ADCC/ADCP deficient L234A, L235A (LALA) and CDC deficient K322A mutants were generated and displayed comparable binding affinity and neutralizing activity as wild type 9F4 (9F4-WT). Notably, 9F4-WT, 9F4-LALA and 9F4-K322A exhibit in vivo protective efficacies against H5N6 infections in that they were able to reduce viral loads in mice. However, only 9F4-WT and 9F4-K322A but not 9F4-LALA were able to reduce viral pathogenesis in H5N6 challenged mice. Furthermore, depletion of phagocytic cells in mice lungs nullifies 9F4-WT's protection against H5N6 infections, suggesting a crucial role of the host's immune cells in 9F4 antiviral activity. Collectively, these findings reveal the importance of ADCC/ADCP function for 9F4-WT protection against HPAIV H5N6 and demonstrate the potential of 9F4 to confer protection against the reassortant H5-subtype HPAIVs.

The HI assay is a standard method for profiling the antigenic characterization of influenza viruses. Suspected antigenic changes based on HI divergency in H7N9 viruses during the 2016-2017 wave prompted the recommendation of new H7N9 candidate vaccine viruses (CVVs). In this study, we found that the L226Q substitution in HA of A/Guangdong/17SF003/2016 (H7/GD16) increased the viral receptor-binding avidity to red blood cells with no impact on the antigenicity of H7N9 virus. Although immune sera raised by an earlier vaccine strain (H7/AH13) showed poor HI titers against H7/GD16, the H7/AH13 immune sera had potent cross-neutralizing antibody titers against H7/GD16 and could provide complete passive protection against H7N9/GD16 virus challenge in mice. Our study highlights that receptor-binding avidity might lead to biased antigenic evaluation by using the HI assay. Other serological assays, such as the microneutralization (MN) assay, should be considered a complementary indicator for analysis of antigenic variation and selection of influenza CVVs.

The DEAD-box RNA helicase DDX5 is involved in many aspects of RNA processing and has been implicated in a number of cellular processes involving alteration of RNA secondary structure. The N-terminal region of DDX5, which contains the conserved domain 1 of the DEAD-box helicases, has been cloned and expressed in Escherichia coli and purified. Here, the crystallization and preliminary diffraction analysis of this region is reported. X-ray diffraction data were processed to a resolution of 2.7 Å. The crystals belonged to space group I222, with unit-cell parameters a = 66.18, b = 73.80, c = 104.00 Å, α = β = γ = 90°.

Hepatitis B virus (HBV) infection persists as a major global health problem despite the availability of HBV vaccines for disease prevention. However, vaccination rates remains low in some regions of the world, driving the need for novel strategies to minimise infections and prevent disease progression. Thus, understanding of perturbed molecular signaling events during early phases of HBV infection is required. Phosphosignaling is known to be involved in the HBV infection processes, yet systems-level changes in phosphosignaling pathways in the host during infection remain unclear. To this end, we performed phosphoproteome profiling on HBV-infected HepG2-NTCP cells. Our results showed that HBV infection drastically altered the host phosphoproteome and its associated proteins, including kinases. Computational analysis of this phosphoproteome revealed dysregulation of the pathways involved in immune responses, cell cycle processes, and RNA processing during HBV infection. Kinase Substrate Enrichment Analysis (KSEA) identified the dysregulated activities of important kinases, including those from CMGC (CDK, MAPK, GSK, and CLK), AGC (protein kinase A, G, and C), and TK (Tyrosine Kinase) families. Of note, the inhibition of CLKs significantly reduced HBV infection in HepG2-NTCP cells. In all, our study unravelled the aberrated phosphosignaling pathways and the associated kinases, presenting potential entry points for developing novel therapeutic strategies for HBV treatment.

We have previously shown that a non-structural protein 1 (NS1)-binding monoclonal antibody, termed as 2H6, can significantly reduce influenza A virus (IAV) replication when expressed intracellularly. In this study, we further showed that 2H6 binds stronger to the NS1 of H5N1 than A/Puerto Rico/8/1934(H1N1) because of an amino acid difference at residue 48. A crystal structure of 2H6 fragment antigen-binding (Fab) has also been solved and docked onto the NS1 structure to reveal the contacts between specific residues at the interface of antibody-antigen complex. In one of the models, the predicted molecular contacts between residues in NS1 and 2H6-Fab correlate well with biochemical results. Taken together, residues N48 and T49 in H5N1 NS1 act cooperatively to maintain a strong interaction with mAb 2H6 by forming hydrogen bonds with residues found in the heavy chain of the antibody. Interestingly, the pandemic H1N1-2009 and the majority of seasonal H3N2 circulating in humans since 1968 has N48 in NS1, suggesting that mAb 2H6 could bind to most of the currently circulating seasonal influenza A virus strains. Consistent with the involvement of residue T49, which is well-conserved, in RNA binding, mAb 2H6 was also found to inhibit the interaction between NS1 and double-stranded RNA.

The membrane fusion process mediated by severe acute respiratory syndrome coronavirus (SARS-CoV) S protein and its cellular receptor angiotensin-converting enzyme 2 (ACE2) had been reconstituted using two Chinese hamster ovary (CHO) cell lines that constitutively express these recombinant proteins separately. This system was applied to develop a quantitative measurement of cell-cell fusion using hepatitis C virus (HCV) NS3/4A protease and a secretion-blocked EGFP-4A/4B-SEAP (EGFP: enhanced green fluorescent protein; 4A/4B: a decapeptide substrate of NS3/4A protease; SEAP: secreted alkaline phosphatase) fusion gene. Both genes were transiently expressed in either of the CHO cell lines, followed by fusion treatment. Significant SEAP activity could be detected in the culture medium only after cell-cell fusion occurred. Cell-cell fusion provides an environment in which the protease encounters its substrate 4A/4B, thereby releasing SEAP from the fusion protein. In this study, we developed a simple, sensitive, and quantitative assay to study the membrane fusion process by measuring the extracellular activity of SEAP.

Human small glutamine-rich tetratricopeptide-repeat protein (hSGT) is a 35 kDa protein implicated in a number of biological processes that include apoptosis, cell division and intracellular cell transport. The tetratricopeptide-repeat (TPR) domain of hSGT has been cloned and expressed in Escherichia coli and purified. Here, the crystallization and preliminary diffraction analysis of the TPR domain of hSGT is reported. X-ray diffraction data were processed to a resolution of 2.4 A. Crystals belong to space group P2(1)2(1)2, with unit-cell parameters a = 67.82, b = 81.93, c = 55.92 A, alpha = beta = gamma = 90 degrees .

Over 250 million people are living with chronic infection caused by the hepatitis B virus (HBV). HBV has three surface proteins, namely small (SHBs), medium (MHBs) and large (LHBs), and they play different roles in the virus life cycle. The approved hepatitis B vaccine only contains the SHBs protein and many studies have focused on characterising the functional domains in SHBs. Although the LHBs protein is less studied, recent studies have shown that it plays important roles in mediating viral entry, replication and assembly. Over the years, there have been major advancements in monoclonal antibody (mAb) discovery tools and multiple mAbs have been developed to specifically target the preS1 domain in LHBs. We summarise the HBV infection systems and antibody discovery strategies that have been utilised by various research groups to assess the potential use of anti-preS1 mAbs as therapeutic antibodies against HBV or in the development of new diagnostic assays.

The Saffold virus (SAFV) genome is translated as a single long polyprotein precursor and co-translationally cleaved to yield 12 separate viral proteins. Little is known about the activities of SAFV proteins although their homologs in other picornaviruses have already been described. To further support research on functions and activities of respective viral proteins, we investigated the spatio-temporal distribution of SAFV proteins in Vero and HEp-2 cells that had been either transfected with plasmids that express individual viral proteins or infected with live SAFV. Our results revealed that, with the exception of the Leader (L) protein, all viral proteins were localized in the cytoplasm at all the time points assayed. The L protein was found in the cytoplasm at an early time point but was subsequently translocated to the nucleus of HEp-2, but not Vero, cells. This was observed in both transfected and infected cells. Further mutational analysis of L protein revealed that Threonine 58 of the Ser/Thr-rich domain of L protein is crucial for protein trafficking between the cytoplasm and nucleus in HEp-2 cells. These findings contribute to a deeper understanding and stimulate investigation of the differetial cellular responses of HEp-2 cells in comparison to other mammalian cell lines during SAFV infection.

C2C12 myoblasts serve as well-established model system to study myogenesis, as they fuse to form multinucleated myotubes.Severe acute respiratory syndrome coronavirus (SARS-CoV) spike (S) protein plays a crucial role in viral entry.Exogenous expression of S protein in C2C12 myoblasts inhibits the formation of myotubes.Global changes in gene expression were studied in C2C12 cells expressing S protein using oligonucleotide microarray analysis.The expression profile showed that, most of the myogenic marker genes were downregulated.Next, we used RT-PCR analysis to reexamine some downregulated and upregulated genes.To further study the antimyogenic effects induced by the S protein, we introduced antisense Plf (proliferin), an upregulated gene, into the antimyogenic cells.Antisense Ace2 (angiotensin-converting enzyme 2), the cellular receptor of S protein, was also introduced into C2C12 myoblasts.Results indicated that antimyogenic effect induced by S protein was partially rescued in cells expressing antisense Plf, while C2C12 cells expressing antisense Ace2 showed upregulation of Plf.

In April 2003, the Collaborative Antiviral Research group started to work on the SARS coronavirus (SARS-CoV). Firstly, we profiled the antibody responses against SARS-CoV proteins during infection. In 74 convalescent sera obtained from confirmed SARS cases in Singapore, we found that all of them contained antibodies against the major structural proteins, nucleocapsid and spike (S), and 70% contained antibodies against one of the accessory proteins termed 3a. The N-terminus of the 3a protein, which is also incorporated into the virion, elicits antibodies that can inhibit the replication of the SARS-CoV in Vero E6, a green African monkey cell-line. A panel of rabbit polyclonal antibodies targeting the S protein was also tested for viral neutralizing activities and it was found that the amino acids 1029–1192 of S contain neutralizing epitopes. Four groups of mouse monoclonal antibodies were also produced using this region of S and they were found to have viral neutralizing activities. By using an in vitro cell–cell fusion assay, it was demonstrated that these anti-S antibodies can block membrane fusion, suggesting that the mechanism of inhibition is likely to be through the blocking of viral–cell or cell-cell fusion during the SARS-CoV infection.

Several human monoclonal Abs for treating Influenza have been evaluated in clinical trials with limited success despite demonstrating superiority in preclinical animal models including mice. To conduct efficacy studies in mice, human monoclonal Abs are genetically engineered to contain mouse heavy chain constant domain to facilitate the engagement of Fc-receptors on mouse immune effector cells. Although studies have consistently reported discrepancies in Ab effectiveness following genetic engineering, the structural and mechanistic basis for these inconsistencies remain uncharacterized. Here, we use homology modeling to predict variable region (VR) analogous monoclonal Abs possessing human IgG1, mouse IgG1, and mouse IgG2a heavy chain constant domains. We then examine predicted 3D structures for variations in the spatial location and orientation of corresponding paratope amino acid residues. By structurally aligning crystal structures of Fabs in complex with hemagglutinin (HA), we show that corresponding paratope amino acid residues for VR-analogous human IgG1, mouse IgG1, and mouse IgG2a monoclonal Abs interact differentially with HA suggesting that their epitopes might not be identical. To demonstrate that variations in the paratope 3D fine architecture have implications for Ab specificity and effectiveness, we genetically engineered VR-analogous human IgG1, human IgG4, mouse IgG1, and mouse IgG2a monoclonal Abs and explored their specificity and effectiveness in protecting MDCK cells from infection by pandemic H1N1 and H3N2 Influenza viruses. We found that VR-analogous monoclonal Abs placed on mouse heavy chain constant domains were more efficacious at protecting MDCK cells from Influenza virus infection relative to those on human heavy chain constant domains. Interestingly, mouse but not human heavy chain constant domains increased target breadth in some monoclonal Abs. These data suggest that heavy chain constant domain sequences play a role in shaping Ab repertoires that go beyond class or sub-class differences in immune effector recruitment. This represents a facet of Ab biology that can potentially be exploited to improve the scope and utilization of current therapeutic or prophylactic candidates for influenza.

The non-structural protein 1 (NS1) of influenza A virus (IAV) is a multifunctional protein that antagonizes host antiviral responses, modulating virus pathogenesis. As such, it serves as a good target for research and diagnostic assay development. In this study, we have generated a novel monoclonal antibody (mAb) 19H9 and epitope mapping revealed that two residues, P85 and Y89, of NS1 are essential for interacting with this mAb. Furthermore, residues P85 and Y89 are found to be highly conserved across different IAV subtypes, namely seasonal H1N1 and H3N2, as well as the highly pathogenic H5N1 and H5N6 avian strains. Indeed, mAb 19H9 exhibits broad cross-reactivity with IAV strains of different subtypes. The binding of mAb 19H9 to residue Y89 was further confirmed by the abrogation of interaction between NS1 and p85β. Additionally, mAb 19H9 also detected NS1 proteins expressed in IAV-infected cells, showing NS1 intracellular localization in the cytoplasm and nucleolus. To our knowledge, mAb 19H9 is the first murine mAb to bind at the juxtaposition between the N-terminal RNA-binding domain and C-terminal effector domain of NS1. It could serve as a useful research tool for studying the conformational plasticity and dynamic changes in NS1.

Our previous work has shown that Saffold virus (SAFV) induced several rodent and primate cell lines to undergo apoptosis (Xu et al. in Emerg Microb Infect 3:1–8, 2014), but the essential viral proteins of SAFV involved in apoptotic activity lack study. In this study, we individually transfected the viral proteins of SAFV into HEp-2 and Vero cells to assess their ability to induce apoptosis, and found that the 2B and 3C proteins are proapoptotic. Further investigation indicated the transmembrane domain of the 2B protein is essential for the apoptotic activity and tetramer formation of the 2B protein. Our research provides clues for the possible mechanisms of apoptosis induced by SAFV in different cell lines. It also opens up new directions to study viral proteins (the 2B, 3C protein), and sets the stage for future exploration of any possible link between SAFV, inclusive of its related uncultivable genotypes, and multiple sclerosis.

The highly pathogenic avian influenza A (H5N6) virus has caused sporadic human infections with a high case fatality rate. Due to the continuous evolution of this virus subtype and its ability to transmit to humans, there is an urgent need to develop effective antiviral therapeutics. In this study, a murine monoclonal antibody 9F4 was shown to display broad binding affinity against H5Nx viruses. Furthermore, 9F4 can neutralize H5N6 pseudotyped particles and prevent entry into host cells. Additionally, ADCC/ADCP deficient L234A, L235A (LALA) and CDC deficient K322A mutants were generated and displayed comparable binding affinity and neutralizing activity as wild type 9F4 (9F4-WT). Notably, 9F4-WT, 9F4-LALA and 9F4-K322A exhibit <i>in vivo</i> protective efficacies against H5N6 infections in that they were able to reduce viral loads in mice. However, only 9F4-WT and 9F4-K322A but not 9F4-LALA were able to reduce viral pathogenesis in H5N6 challenged mice. Furthermore, depletion of phagocytic cells in mice lungs nullifies 9F4-WT's protection against H5N6 infections, suggesting a crucial role of the host's immune cells in 9F4 antiviral activity. Collectively, these findings reveal the importance of ADCC/ADCP function for 9F4-WT protection against HPAIV H5N6 and demonstrate the potential of 9F4 to confer protection against the reassortant H5-subtype HPAIVs.