RESUMO
The Coronaviridae are a family of viruses that cause disease in humans ranging from mild respiratory infection to potentially lethal acute respiratory distress syndrome. Finding host factors common to multiple coronaviruses could facilitate the development of therapies to combat current and future coronavirus pandemics. Here, we conducted genome-wide CRISPR screens in cells infected by SARS-CoV-2 as well as two seasonally circulating common cold coronaviruses, OC43 and 229E. This approach correctly identified the distinct viral entry factors ACE2 (for SARS-CoV-2), aminopeptidase N (for 229E), and glycosaminoglycans (for OC43). Additionally, we identified phosphatidylinositol phosphate biosynthesis and cholesterol homeostasis as critical host pathways supporting infection by all three coronaviruses. By contrast, the lysosomal protein TMEM106B appeared unique to SARS-CoV-2 infection. Pharmacological inhibition of phosphatidylinositol kinases and cholesterol homeostasis reduced replication of all three coronaviruses. These findings offer important insights for the understanding of the coronavirus life cycle and the development of host-directed therapies.
Assuntos
COVID-19/genética , Infecções por Coronavirus/genética , Coronavirus/fisiologia , Estudo de Associação Genômica Ampla , Interações Hospedeiro-Patógeno , SARS-CoV-2/fisiologia , Células A549 , Animais , Vias Biossintéticas/efeitos dos fármacos , COVID-19/virologia , Linhagem Celular , Chlorocebus aethiops , Colesterol/biossíntese , Colesterol/metabolismo , Análise por Conglomerados , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Resfriado Comum/genética , Resfriado Comum/virologia , Coronavirus/classificação , Infecções por Coronavirus/virologia , Técnicas de Inativação de Genes , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Humanos , Camundongos , Fosfatidilinositóis/biossíntese , Células Vero , Internalização do Vírus/efeitos dos fármacos , Replicação ViralRESUMO
My scientific journeys began at Oxford nearly 50 years ago. My paths have taken me from magnetic resonance through enzyme systems to antibodies, which led directly to glycobiology. Oxford University's first industrial grant helped the development of the technology for isolating and sequencing oligosaccharides from glycoproteins. This technology was disseminated through a spin-off company, Oxford GlycoSystems, and by the establishment of the Glycobiology Institute. The technology gave rise to the concept of glycoforms, which allow diversification of a protein's properties. Iminosugars, which are glucosidase inhibitors, can interfere with the initial steps of glycan processing on proteins and inhibit three-dimensional folding of glycoproteins. Glucosidase targets for therapy include viral envelope glycoproteins. Clinical trials of an iminosugar as an antiviral for dengue virus are under way. Another iminosugar activity, inhibition of glycolipid synthesis, resulted in a drug for Gaucher disease, which was approved worldwide in 2002. The success of the company and the institute allowed me to undertake several initiatives, in the United Kingdom and abroad, that might help the paths of future generations of scientists.
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Glicômica/história , Alergia e Imunologia/história , Animais , Antígenos , Pesquisa Biomédica/história , Desenho de Fármacos , Inglaterra , Glucosidases/química , História do Século XX , História do Século XXI , Humanos , IsraelRESUMO
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19). SARS-CoV-2 relies on cellular RNA-binding proteins (RBPs) to replicate and spread, although which RBPs control its life cycle remains largely unknown. Here, we employ a multi-omic approach to identify systematically and comprehensively the cellular and viral RBPs that are involved in SARS-CoV-2 infection. We reveal that SARS-CoV-2 infection profoundly remodels the cellular RNA-bound proteome, which includes wide-ranging effects on RNA metabolic pathways, non-canonical RBPs, and antiviral factors. Moreover, we apply a new method to identify the proteins that directly interact with viral RNA, uncovering dozens of cellular RBPs and six viral proteins. Among them are several components of the tRNA ligase complex, which we show regulate SARS-CoV-2 infection. Furthermore, we discover that available drugs targeting host RBPs that interact with SARS-CoV-2 RNA inhibit infection. Collectively, our results uncover a new universe of host-virus interactions with potential for new antiviral therapies against COVID-19.
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COVID-19/metabolismo , Proteoma/metabolismo , RNA Viral/metabolismo , Proteínas de Ligação a RNA/metabolismo , SARS-CoV-2/fisiologia , Proteínas Virais/metabolismo , Replicação Viral/fisiologia , Células A549 , COVID-19/genética , Humanos , Proteoma/genética , RNA Viral/genética , Proteínas de Ligação a RNA/genética , Proteínas Virais/genéticaRESUMO
Human transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), causative pathogen of the COVID-19 pandemic, exerts a massive health and socioeconomic crisis. The virus infects alveolar epithelial type 2 cells (AT2s), leading to lung injury and impaired gas exchange, but the mechanisms driving infection and pathology are unclear. We performed a quantitative phosphoproteomic survey of induced pluripotent stem cell-derived AT2s (iAT2s) infected with SARS-CoV-2 at air-liquid interface (ALI). Time course analysis revealed rapid remodeling of diverse host systems, including signaling, RNA processing, translation, metabolism, nuclear integrity, protein trafficking, and cytoskeletal-microtubule organization, leading to cell cycle arrest, genotoxic stress, and innate immunity. Comparison to analogous data from transformed cell lines revealed respiratory-specific processes hijacked by SARS-CoV-2, highlighting potential novel therapeutic avenues that were validated by a high hit rate in a targeted small molecule screen in our iAT2 ALI system.
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Células Epiteliais Alveolares/metabolismo , COVID-19/metabolismo , Fosfoproteínas/metabolismo , Proteoma/metabolismo , SARS-CoV-2/metabolismo , Células Epiteliais Alveolares/patologia , Células Epiteliais Alveolares/virologia , Animais , Antivirais , COVID-19/genética , COVID-19/patologia , Chlorocebus aethiops , Efeito Citopatogênico Viral , Citoesqueleto , Avaliação Pré-Clínica de Medicamentos , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/patologia , Células-Tronco Pluripotentes Induzidas/virologia , Fosfoproteínas/genética , Transporte Proteico , Proteoma/genética , SARS-CoV-2/genética , Transdução de Sinais , Células Vero , Tratamento Farmacológico da COVID-19RESUMO
The pulmonary surfactant system of the lung is a lipid and protein complex, which regulates the biophysical properties of the alveoli to prevent lung collapse and the innate immune system in the lung. Pulmonary surfactant is a lipoprotein complex consisting of 90% phospholipids and 10% protein, by weight. Two minor components of pulmonary surfactant phospholipids, phosphatidylglycerol (PG) and phosphatidylinositol (PI), exist at very high concentrations in the extracellular alveolar compartments. We have reported that one of the most dominant molecular species of PG, palmitoyl-oleoyl-phosphatidylglycerol (POPG) and PI inhibit inflammatory responses induced by multiple toll-like receptors (TLR2/1, TLR3, TLR4, and TLR2/6) by interacting with subsets of multiprotein receptor components. These lipids also exert potent antiviral effects against RSV and influenza A, in vitro, by inhibiting virus binding to host cells. POPG and PI inhibit these viral infections in vivo, in multiple animal models. Especially noteworthy, these lipids markedly attenuate SARS-CoV-2 infection including its variants. These lipids are natural compounds that already exist in the lung and, thus, are less likely to cause adverse immune responses by hosts. Collectively, these data demonstrate that POPG and PI have strong potential as novel therapeutics for applications as anti-inflammatory compounds and preventatives, as treatments for broad ranges of RNA respiratory viruses.
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COVID-19 , Surfactantes Pulmonares , Animais , Humanos , Fosfolipídeos/metabolismo , Surfactantes Pulmonares/uso terapêutico , Surfactantes Pulmonares/química , Surfactantes Pulmonares/metabolismo , Antivirais/farmacologia , Antivirais/uso terapêutico , Receptor 2 Toll-Like , SARS-CoV-2 , Pulmão/metabolismo , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/uso terapêutico , Fosfatidilgliceróis/uso terapêutico , Fosfatidilgliceróis/farmacologiaRESUMO
The SARS-CoV-2 infection cycle is a multistage process that relies on functional interactions between the host and the pathogen. Here, we repurposed antiviral drugs against both viral and host enzymes to pharmaceutically block methylation of the viral RNA 2'-O-ribose cap needed for viral immune escape. We find that the host cap 2'-O-ribose methyltransferase MTr1 can compensate for loss of viral NSP16 methyltransferase in facilitating virus replication. Concomitant inhibition of MTr1 and NSP16 efficiently suppresses SARS-CoV-2 replication. Using in silico target-based drug screening, we identify a bispecific MTr1/NSP16 inhibitor with anti-SARS-CoV-2 activity in vitro and in vivo but with unfavorable side effects. We further show antiviral activity of inhibitors that target independent stages of the host SAM cycle providing the methyltransferase co-substrate. In particular, the adenosylhomocysteinase (AHCY) inhibitor DZNep is antiviral in in vitro, in ex vivo, and in a mouse infection model and synergizes with existing COVID-19 treatments. Moreover, DZNep exhibits a strong immunomodulatory effect curbing infection-induced hyperinflammation and reduces lung fibrosis markers ex vivo. Thus, multispecific and metabolic MTase inhibitors constitute yet unexplored treatment options against COVID-19.
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Tratamento Farmacológico da COVID-19 , SARS-CoV-2 , Animais , Antivirais/farmacologia , Inflamação/tratamento farmacológico , Metiltransferases/metabolismo , Camundongos , Capuzes de RNA/metabolismo , RNA Viral/genética , Ribose , Proteínas não Estruturais Virais/genéticaRESUMO
The treatment for COVID-19 has evolved rapidly since the start of the pandemic and now consists mainly of antiviral and immunomodulatory agents. Antivirals, such as remdesivir and nirmatrelvir-ritonavir, have proved to be most useful earlier in illness (e.g., as outpatient therapy) and for less severe disease. Immunomodulatory therapies, such as dexamethasone and interleukin-6 or Janus kinase inhibitors, are most useful in severe disease or critical illness. The role of anti-SARS-CoV-2 monoclonal antibodies has diminished because of the emergence of viral variants that are not anticipated to be susceptible to these treatments, and there still is not a consensus on the use of convalescent plasma. COVID-19 has been associated with increased rates of venous thromboembolism, but the role of antithrombotic therapy is limited. Multiple investigational agents continue to be studied, which will alter current treatment paradigms as new data are released.
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COVID-19 , Inibidores de Janus Quinases , Humanos , Soroterapia para COVID-19 , Imunomodulação , Interleucina-6 , Inibidores de Janus Quinases/uso terapêuticoRESUMO
The COVID-19 pandemic reminded us of the urgent need for new antivirals to control emerging infectious diseases and potential future pandemics. Immunotherapy has revolutionized oncology and could complement the use of antivirals, but its application to infectious diseases remains largely unexplored. Nucleoside analogs are a class of agents widely used as antiviral and anti-neoplastic drugs. Their antiviral activity is generally based on interference with viral nucleic acid replication or transcription. Based on our previous work and computer modeling, we hypothesize that antiviral adenosine analogs, like remdesivir, have previously unrecognized immunomodulatory properties which contribute to their therapeutic activity. In the case of remdesivir, we here show that these properties are due to its metabolite, GS-441524, acting as an Adenosine A2A Receptor antagonist. Our findings support a new rationale for the design of next-generation antiviral agents with dual - immunomodulatory and intrinsic - antiviral properties. These compounds could represent game-changing therapies to control emerging viral diseases and future pandemics.
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Monofosfato de Adenosina , Adenosina , Alanina , Antivirais , COVID-19 , SARS-CoV-2 , Antivirais/farmacologia , Antivirais/química , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/imunologia , Adenosina/análogos & derivados , Adenosina/farmacologia , Adenosina/química , Humanos , Monofosfato de Adenosina/análogos & derivados , Monofosfato de Adenosina/farmacologia , Alanina/análogos & derivados , Alanina/farmacologia , Alanina/química , COVID-19/imunologia , COVID-19/virologia , Animais , Agentes de Imunomodulação/farmacologia , Agentes de Imunomodulação/química , Antagonistas do Receptor A2 de Adenosina/farmacologia , Antagonistas do Receptor A2 de Adenosina/química , Antagonistas do Receptor A2 de Adenosina/uso terapêutico , Pandemias , Tratamento Farmacológico da COVID-19 , Chlorocebus aethiops , Replicação Viral/efeitos dos fármacos , Células Vero , Betacoronavirus/efeitos dos fármacos , Betacoronavirus/imunologia , Receptor A2A de Adenosina/metabolismo , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/virologiaRESUMO
The ongoing COVID-19 pandemic has had great societal and health consequences. Despite the availability of vaccines, infection rates remain high due to immune evasive Omicron sublineages. Broad-spectrum antivirals are needed to safeguard against emerging variants and future pandemics. We used messenger RNA (mRNA) display under a reprogrammed genetic code to find a spike-targeting macrocyclic peptide that inhibits SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) Wuhan strain infection and pseudoviruses containing spike proteins of SARS-CoV-2 variants or related sarbecoviruses. Structural and bioinformatic analyses reveal a conserved binding pocket between the receptor-binding domain, N-terminal domain, and S2 region, distal to the angiotensin-converting enzyme 2 receptor-interaction site. Our data reveal a hitherto unexplored site of vulnerability in sarbecoviruses that peptides and potentially other drug-like molecules can target.
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COVID-19 , SARS-CoV-2 , Humanos , SARS-CoV-2/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo , Pandemias/prevenção & controle , Peptídeos/farmacologiaRESUMO
SUMMARYSince the emergence of COVID-19 in 2020, an unprecedented range of therapeutic options has been studied and deployed. Healthcare providers have multiple treatment approaches to choose from, but efficacy of those approaches often remains controversial or compromised by viral evolution. Uncertainties still persist regarding the best therapies for high-risk patients, and the drug pipeline is suffering fatigue and shortage of funding. In this article, we review the antiviral activity, mechanism of action, pharmacokinetics, and safety of COVID-19 antiviral therapies. Additionally, we summarize the evidence from randomized controlled trials on efficacy and safety of the various COVID-19 antivirals and discuss unmet needs which should be addressed.
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Antivirais , Tratamento Farmacológico da COVID-19 , COVID-19 , SARS-CoV-2 , Humanos , Antivirais/uso terapêutico , SARS-CoV-2/efeitos dos fármacos , COVID-19/terapiaRESUMO
The globally reemerging respiratory pathogen enterovirus D68 (EV-D68) is implicated in outbreaks of severe respiratory illness and associated with acute flaccid myelitis. However, there remains a lack of effective treatments for EV-D68 infection. In this work, we found that the host Toll-like receptor 7 (TLR7) proteins, which function as powerful innate immune sensors, were selectively elevated in expression in response to EV-D68 infection. Subsequently, we investigated the impact of Vesatolimod (GS-9620), a Toll-like receptor 7 agonist, on EV-D68 replication. Our findings revealed that EV-D68 infection resulted in increased mRNA levels of TLR7. Treatment with Vesatolimod significantly inhibited EV-D68 replication [half maximal effective concentration (EC50) = 0.1427 µM] without inducing significant cytotoxicity at virucidal concentrations. Although Vesatolimod exhibited limited impact on EV-D68 attachment, it suppressed RNA replication and viral protein synthesis after virus entry. Vesatolimod broadly inhibited the replication of circulating isolated strains of EV-D68. Furthermore, our findings demonstrated that treatment with Vesatolimod conferred resistance to both respiratory and neural cells against EV-D68 infection. Overall, these results present a promising strategy for drug development by pharmacologically activating TLR7 to initiate an antiviral state in EV-D68-infected cells selectively.IMPORTANCEConventional strategies for antiviral drug development primarily focus on directly targeting viral proteases or key components, as well as host proteins involved in viral replication. In this study, based on our intriguing discovery that enterovirus D68 (EV-D68) infection specifically upregulates the expression of immune sensor Toll-like receptor 7 (TLR7) protein, which is either absent or expressed at low levels in respiratory cells, we propose a potential antiviral approach utilizing TLR7 agonists to activate EV-D68-infected cells into an anti-viral defense state. Notably, our findings demonstrate that pharmacological activation of TLR7 effectively suppresses EV-D68 replication in respiratory tract cells through a TLR7/MyD88-dependent mechanism. This study not only presents a promising drug candidate and target against EV-D68 dissemination but also highlights the potential to exploit unique alterations in cellular innate immune responses induced by viral infections, selectively inducing a defensive state in infected cells while safeguarding uninfected normal cells from potential adverse effects associated with therapeutic interventions.
Assuntos
Antivirais , Enterovirus Humano D , Receptor 7 Toll-Like , Replicação Viral , Receptor 7 Toll-Like/agonistas , Receptor 7 Toll-Like/metabolismo , Humanos , Replicação Viral/efeitos dos fármacos , Enterovirus Humano D/efeitos dos fármacos , Antivirais/farmacologia , Indóis/farmacologia , Infecções por Enterovirus/virologia , Imunidade Inata/efeitos dos fármacos , Linhagem Celular , Internalização do Vírus/efeitos dos fármacos , PteridinasRESUMO
Zika virus (ZIKV) is a re-emerging mosquito-borne flavivirus that has been associated with congenital neurological defects in fetuses born to infected mothers. At present, no vaccine or antiviral therapy is available to combat this devastating disease. Repurposing drugs that target essential host factors exploited by viruses is an attractive therapeutic approach. Here, we screened a panel of clinically approved small-molecule kinase inhibitors for their antiviral effects against a clinical isolate of ZIKV and thoroughly characterized their mechanisms of action. We found that the Raf kinase inhibitors Dabrafenib and Regorafenib potently impair the replication of ZIKV, but not that of its close relative dengue virus. Time-of-addition experiments showed that both inhibitors target ZIKV infection at post-entry steps. We found that Dabrafenib, but not Regorafenib, interfered with ZIKV genome replication by impairing both negative- and positive-strand RNA synthesis. Regorafenib, on the other hand, altered steady-state viral protein levels, viral egress, and blocked NS1 secretion. We also observed Regorafenib-induced ER fragmentation in ZIKV-infected cells, which might contribute to its antiviral effects. Because these inhibitors target different steps of the ZIKV infection cycle, their use in combination therapy may amplify their antiviral effects which could be further explored for future therapeutic strategies against ZIKV and possibly other flaviviruses. IMPORTANCE: There is an urgent need to develop effective therapeutics against re-emerging arboviruses associated with neurological disorders like Zika virus (ZIKV). We identified two FDA-approved kinase inhibitors, Dabrafenib and Regorafenib, as potent inhibitors of contemporary ZIKV strains at distinct stages of infection despite overlapping host targets. Both inhibitors reduced viral titers by ~1 to 2 log10 (~10-fold to 100-fold) with minimal cytotoxicity. Furthermore, we show that Dabrafenib inhibits ZIKV RNA replication whereas Regorafenib inhibits ZIKV translation and egress. Regorafenib has the added benefit of limiting NS1 secretion, which contributes to the pathogenesis and disease progression of several flaviviruses. Because these inhibitors affect distinct post-entry steps of ZIKV infection, their therapeutic potential may be amplified by combination therapy and likely does not require prophylactic administration. This study provides further insight into ZIKV-host interactions and has implications for the development of novel antivirals against ZIKV and possibly other flaviviruses.
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Several viruses hijack various forms of endocytosis in order to infect host cells. Here, we report the discovery of a molecule with antiviral properties that we named virapinib, which limits viral entry by macropinocytosis. The identification of virapinib derives from a chemical screen using high-throughput microscopy, where we identified chemical entities capable of preventing infection with a pseudotype virus expressing the spike (S) protein from SARS-CoV-2. Subsequent experiments confirmed the capacity of virapinib to inhibit infection by SARS-CoV-2, as well as by additional viruses, such as mpox virus and TBEV. Mechanistic analyses revealed that the compound inhibited macropinocytosis, limiting this entry route for the viruses. Importantly, virapinib has no significant toxicity to host cells. In summary, we present the discovery of a molecule that inhibits macropinocytosis, thereby limiting the infectivity of viruses that use this entry route such as SARS-CoV2.
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Completion of the Lassa virus (LASV) life cycle critically depends on the activities of the virally encoded, RNA-dependent RNA polymerase in replication and transcription of the viral RNA genome in the cytoplasm of infected cells. The contribution of cellular proteins to these processes remains unclear. Here, we applied proximity proteomics to define the interactome of LASV polymerase in cells under conditions that recreate LASV RNA synthesis. We engineered a LASV polymerase-biotin ligase (TurboID) fusion protein that retained polymerase activity and successfully biotinylated the proximal proteome, which allowed the identification of 42 high-confidence LASV polymerase interactors. We subsequently performed a small interfering RNA (siRNA) screen to identify those interactors that have functional roles in authentic LASV infection. As proof of principle, we characterized eukaryotic peptide chain release factor subunit 3a (eRF3a/GSPT1), which we found to be a proviral factor that physically associates with LASV polymerase. Targeted degradation of GSPT1 by a small-molecule drug candidate, CC-90009, resulted in strong inhibition of LASV infection in cultured cells. Our work demonstrates the feasibility of using proximity proteomics to illuminate and characterize yet-to-be-defined host-pathogen interactome, which can reveal new biology and uncover novel targets for the development of antivirals against highly pathogenic RNA viruses.
Assuntos
Acetamidas , Antivirais , Isoindóis , Vírus Lassa , Fatores de Terminação de Peptídeos , Piperidonas , RNA Polimerase Dependente de RNA , Proteínas Virais , Acetamidas/farmacologia , Acetamidas/uso terapêutico , Antivirais/farmacologia , Antivirais/uso terapêutico , Linhagem Celular Tumoral , Humanos , Isoindóis/farmacologia , Isoindóis/uso terapêutico , Febre Lassa/tratamento farmacológico , Vírus Lassa/efeitos dos fármacos , Fatores de Terminação de Peptídeos/metabolismo , Piperidonas/metabolismo , Piperidonas/farmacologia , Piperidonas/uso terapêutico , Mapas de Interação de Proteínas/efeitos dos fármacos , Proteólise/efeitos dos fármacos , Proteoma , Proteômica , RNA Polimerase Dependente de RNA/metabolismo , Proteínas Virais/metabolismoRESUMO
Chronic viral hepatitis is caused by hepatitis B virus, hepatitis C virus or hepatitis D virus (HBV, HCV, and HDV). Despite different replication strategies, all these viruses rely on secretion through the host endoplasmic reticulum-Golgi pathway, providing potential host targets for antiviral therapy. Knockdown of transmembrane 6 superfamily member 2 (TM6SF2) in virus cell culture models reduced secretion of infectious HCV virions, HDV virions and HBV subviral particles. Moreover, in a cohort of people with hepatitis B a TM6SF2 polymorphism (rs58542926 CT/TT, which causes protein misfolding and reduced TM6SF2 in the liver) correlated with lower concentrations of subviral particles in blood, complementing our previous work showing decreased HCV viral load in people with this polymorphism. In conclusion, the host protein TM6SF2 plays a key role in secretion of HBV, HCV and HDV, providing the potential for novel pan-viral agents to treat people with chronic viral hepatitis.
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OBJECTIVE: To evaluate the impact of hepatitis C virus (HCV) infection and treatment status on COVID-19-related hospitalizations in Georgia. METHODS: We analyzed 2020-2021 Georgian health-registry data for COVID-19-positive individuals and categorized by HCV infection and treatment status. Logistic regression was used to assess the strengths of the associations. RESULTS: Treated HCV individuals had lower odds of COVID-19-related hospitalization compared to anti-HCV-negatives, while untreated HCV-viremic and anti-HCV-positive non-viremic individuals had higher odds. CONCLUSIONS: HCV treatment prior to COVID-19 infection was associated with lower odds of COVID-19-related hospitalization, highlighting the benefits of HCV management in the context of the pandemic.
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We present compelling evidence for the existence of an extended innate viperin-dependent pathway, which provides crucial evidence for an adaptive response to viral agents, such as SARS-CoV-2. We show the in vivo biosynthesis of a family of novel endogenous cytosine metabolites with potential antiviral activities. Two-dimensional nuclear magnetic resonance (NMR) spectroscopy revealed a characteristic spin-system motif, indicating the presence of an extended panel of urinary metabolites during the acute viral replication phase. Mass spectrometry additionally enabled the characterization and quantification of the most abundant serum metabolites, showing the potential diagnostic value of the compounds for viral infections. In total, we unveiled ten nucleoside (cytosine- and uracil-based) analogue structures, eight of which were previously unknown in humans allowing us to propose a new extended viperin pathway for the innate production of antiviral compounds. The molecular structures of the nucleoside analogues and their correlation with an array of serum cytokines, including IFN-α2, IFN-γ, and IL-10, suggest an association with the viperin enzyme contributing to an ancient endogenous innate immune defense mechanism against viral infection.
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COVID-19 , Humanos , Estrutura Molecular , SARS-CoV-2 , Imunidade Inata , Citosina , Redes e Vias Metabólicas , AntiviraisRESUMO
Feline infectious peritonitis (FIP) is a serious viral illness in cats, caused by feline coronavirus. Once a cat develops clinical FIP, the prognosis is poor. The effective treatment strategy for coronavirus infections with immunopathological complications such as SARS-CoV-2, MERS, and FIP is focused on antiviral and immunomodulatory agents to inhibit virus replication and enhance the protective immune response. In this article we report the binding and conformational alteration of feline alphacoronavirus (FCoV) nucleocapsid protein by a novel compound K31. K31 noncompetitively inhibited the interaction between the purified nucleocapsid protein and the synthetic 5' terminus of viral genomic RNA in vitro. K31 was well tolerated by cells and inhibited FCoV replication in cell culture with a selective index of 115. A single dose of K31inhibited FCoV replication to an undetectable level in 24 h post treatment. K31 did not affect the virus entry to the host cell but inhibited the postentry steps of virus replication. The nucleocapsid protein forms ribonucleocapsid in association with the viral genomic RNA that serves as a template for transcription and replication of the viral genome. Our results show that K31 treatment disrupted the structural integrity of ribonucleocapsid in virus-infected cells. After the COVID-19 pandemic, most of the antiviral drug development strategies have focused on RdRp and proteases encoded by the viral genome. Our results have shown that nucleocapsid protein is a druggable target for anticoronavirus drug discovery.
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Antivirais , Coronavirus Felino , Peritonite Infecciosa Felina , Proteínas do Nucleocapsídeo , Replicação Viral , Animais , Gatos , Antivirais/farmacologia , Antivirais/uso terapêutico , Técnicas de Cultura de Células , Coronavirus Felino/efeitos dos fármacos , Coronavirus Felino/fisiologia , Peritonite Infecciosa Felina/tratamento farmacológico , RNA Viral/genética , Replicação Viral/efeitos dos fármacosRESUMO
The fusion of haemagglutinin-neuraminidase (HN) protein of peste des petits ruminant (PPR) virus with signaling lymphocyte activation molecules (SLAM) host cell receptor consequences the virus entry and multiplication inside the host cell. The use of synthetic SLAM homologous peptides (i.e., molecular decoy for HN protein of PPR virus) may check PPR infection at the preliminary stage. Hence, the predicted SLAM homologous peptides using bioinformatics tools were synthesized by solid phase chemistry with standard Merrifield's 9-fluorenylmethoxycarbonyl (Fmoc) chemistry and were purified by reverse phase high performance liquid chromatography. The secondary structures of synthesized peptides were elucidated by circular dichroism spectroscopy. The in vitro interactions of these peptides were studied through indirect Enzyme Linked Immuno Sorbent Assay (ELISA) and visual surface plasmon UV-visible spectroscopy. The SLAM homologous peptides were able to interact with the peste des petits ruminant virus (PPRV) with varying binding efficiency. The interaction of SLAM homologous peptide with the PPR virus was ascertained by the change in the plasmon color from red wine to purple during visual detection and also by bathochromic shift in absorbance spectra under UV-visible spectrophotometry. The cytotoxic and anti-PPRV effect of these peptides were also evaluated in B95a cell line using PPR virus (Sungri/96). The cytotoxic concentration 50 (CC50 ) value of each peptide was greater than 1000 µg mL-1 . The anti-PPRV efficiency of SLAM-22 was relatively high among SLAM homologous peptides, SLAM-22 at 25 µg mL-1 concentration showed a reduction of more than log10 3 virus titer by priming of B95a cell line while the use of SLAM-15 and Muco-17 at the same concentration dropped virus titer from log10 4.8 to log10 2.5 and log10 3.1 respectively. The concentration of SLAM homologous peptide (25 µg mL-1 ) to exert its anti-PPRV effect was much less than its CC50 level (>1000 µg mL-1 ). Therefore, the synthetic SLAM homologous peptides may prove to be better agents to target PPRV.
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Peste dos Pequenos Ruminantes , Vírus da Peste dos Pequenos Ruminantes , Animais , Vírus da Peste dos Pequenos Ruminantes/metabolismo , Peste dos Pequenos Ruminantes/metabolismo , Linhagem Celular , Proteínas Virais/metabolismo , Peptídeos/farmacologia , Peptídeos/metabolismo , CabrasRESUMO
The goals of coronavirus disease 2019 (COVID-19) antiviral therapy early in the pandemic were to prevent severe disease, hospitalization, and death. As these outcomes have become infrequent in the age of widespread population immunity, the objectives have shifted. For the general population, COVID-19-directed antiviral therapy should decrease symptom severity and duration and minimize infectiousness, and for immunocompromised individuals, antiviral therapy should reduce severe outcomes and persistent infection. The increased recognition of virologic rebound following ritonavir-boosted nirmatrelvir (NMV/r) and the lack of randomized controlled trial data showing benefit of antiviral therapy for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection for standard-risk, vaccinated individuals remain major knowledge gaps. Here, we review data for selected antiviral agents and immunomodulators currently available or in late-stage clinical trials for use in outpatients. We do not review antibody products, convalescent plasma, systemic corticosteroids, IL-6 inhibitors, Janus kinase inhibitors, or agents that lack Food and Drug Administration approval or emergency use authorization or are not appropriate for outpatients.