RESUMO
In chronic hepatitis C virus (HCV) infection, exhausted HCV-specific CD8+ T cells comprise memory-like and terminally exhausted subsets. However, little is known about the molecular profile and fate of these two subsets after the elimination of chronic antigen stimulation by direct-acting antiviral (DAA) therapy. Here, we report a progenitor-progeny relationship between memory-like and terminally exhausted HCV-specific CD8+ T cells via an intermediate subset. Single-cell transcriptomics implicated that memory-like cells are maintained and terminally exhausted cells are lost after DAA-mediated cure, resulting in a memory polarization of the overall HCV-specific CD8+ T cell response. However, an exhausted core signature of memory-like CD8+ T cells was still detectable, including, to a smaller extent, in HCV-specific CD8+ T cells targeting variant epitopes. These results identify a molecular signature of T cell exhaustion that is maintained as a chronic scar in HCV-specific CD8+ T cells even after the cessation of chronic antigen stimulation.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Hepacivirus/imunologia , Hepatite C Crônica/imunologia , Memória Imunológica/genética , Transcriptoma , Antígenos Virais/imunologia , Antivirais/uso terapêutico , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/virologia , Perfilação da Expressão Gênica , Redes Reguladoras de Genes , Hepacivirus/efeitos dos fármacos , Hepatite C Crônica/tratamento farmacológico , Hepatite C Crônica/genética , Hepatite C Crônica/virologia , Interações Hospedeiro-Patógeno , Humanos , Fenótipo , Indução de Remissão , Análise de Célula Única , Resultado do TratamentoRESUMO
Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) remodels the endoplasmic reticulum (ER) to form replication organelles, leading to ER stress and unfolded protein response (UPR). However, the role of specific UPR pathways in infection remains unclear. Here, we found that SARS-CoV-2 infection causes marginal activation of signaling sensor IRE1α leading to its phosphorylation, clustering in the form of dense ER-membrane rearrangements with embedded membrane openings, and XBP1 splicing. By investigating the factors regulated by IRE1α-XBP1 during SARS-CoV-2 infection, we identified stress-activated kinase NUAK2 as a novel host-dependency factor for SARS-CoV-2, HCoV-229E, and MERS-CoV entry. Reducing NUAK2 abundance or kinase activity impaired SARS-CoV-2 particle binding and internalization by decreasing cell surface levels of viral receptors and viral trafficking likely by modulating the actin cytoskeleton. IRE1α-dependent NUAK2 levels were elevated in SARS-CoV-2-infected and bystander non-infected cells, promoting viral spread by maintaining ACE2 cell surface levels and facilitating virion binding to bystander cells.
Assuntos
Proteínas Serina-Treonina Quinases , SARS-CoV-2 , Internalização do Vírus , Humanos , Quinases Proteína-Quinases Ativadas por AMP , Proteínas Quinases Ativadas por AMP/metabolismo , COVID-19/metabolismo , COVID-19/patologia , COVID-19/virologia , Endorribonucleases/genética , Endorribonucleases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , SARS-CoV-2/fisiologia , Resposta a Proteínas não DobradasRESUMO
Dengue is a major health threat and the number of symptomatic infections caused by the four dengue serotypes is estimated to be 96 million1 with annually around 10,000 deaths2. However, no antiviral drugs are available for the treatment or prophylaxis of dengue. We recently described the interaction between non-structural proteins NS3 and NS4B as a promising target for the development of pan-serotype dengue virus (DENV) inhibitors3. Here we present JNJ-1802-a highly potent DENV inhibitor that blocks the NS3-NS4B interaction within the viral replication complex. JNJ-1802 exerts picomolar to low nanomolar in vitro antiviral activity, a high barrier to resistance and potent in vivo efficacy in mice against infection with any of the four DENV serotypes. Finally, we demonstrate that the small-molecule inhibitor JNJ-1802 is highly effective against viral infection with DENV-1 or DENV-2 in non-human primates. JNJ-1802 has successfully completed a phase I first-in-human clinical study in healthy volunteers and was found to be safe and well tolerated4. These findings support the further clinical development of JNJ-1802, a first-in-class antiviral agent against dengue, which is now progressing in clinical studies for the prevention and treatment of dengue.
Assuntos
Antivirais , Vírus da Dengue , Dengue , Primatas , Proteínas não Estruturais Virais , Animais , Humanos , Camundongos , Antivirais/efeitos adversos , Antivirais/farmacologia , Antivirais/uso terapêutico , Ensaios Clínicos Fase I como Assunto , Dengue/tratamento farmacológico , Dengue/prevenção & controle , Dengue/virologia , Vírus da Dengue/classificação , Vírus da Dengue/efeitos dos fármacos , Relação Dose-Resposta a Droga , Farmacorresistência Viral , Técnicas In Vitro , Terapia de Alvo Molecular , Primatas/virologia , Ligação Proteica/efeitos dos fármacos , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas não Estruturais Virais/metabolismo , Replicação ViralRESUMO
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.
Assuntos
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
Mosquito-borne flaviviruses including Zika virus (ZIKV) represent a public health problem in some parts of the world. Although ZIKV infection is predominantly asymptomatic or associated with mild symptoms, it can lead to neurological complications. ZIKV infection can also cause antibody-dependent enhancement (ADE) of infection with similar viruses, warranting further studies of virion assembly and the function of envelope (E) protein-specific antibodies. Although extracellular vesicles (EVs) from flavivirus-infected cells have been reported to transmit infection, this interpretation is challenged by difficulties in separating EVs from flavivirions due to their similar biochemical composition and biophysical properties. In the present study, a rigorous EV-virion separation method combining sequential ultracentrifugation and affinity capture was developed to study EVs from ZIKV-infected cells. We find that these EVs do not transmit infection, but EVs display abundant E proteins which have an antigenic landscape similar to that of virions carrying E. ZIKV E-coated EVs attenuate antibody-dependent enhancement mediated by ZIKV E-specific and DENV-cross-reactive antibodies in both cell culture and mouse models. We thus report an alternative route for Flavivirus E protein secretion. These results suggest that modulation of E protein release via virions and EVs may present a new approach to regulating flavivirus-host interactions.
Assuntos
Vírus da Dengue , Dengue , Vesículas Extracelulares , Infecção por Zika virus , Zika virus , Animais , Camundongos , Infecção por Zika virus/prevenção & controle , Proteínas Virais , Anticorpos Neutralizantes , Anticorpos Antivirais , Dengue/prevenção & controleRESUMO
Dengue virus causes approximately 96 million symptomatic infections annually, manifesting as dengue fever or occasionally as severe dengue1,2. There are no antiviral agents available to prevent or treat dengue. Here, we describe a highly potent dengue virus inhibitor (JNJ-A07) that exerts nanomolar to picomolar activity against a panel of 21 clinical isolates that represent the natural genetic diversity of known genotypes and serotypes. The molecule has a high barrier to resistance and prevents the formation of the viral replication complex by blocking the interaction between two viral proteins (NS3 and NS4B), thus revealing a previously undescribed mechanism of antiviral action. JNJ-A07 has a favourable pharmacokinetic profile that results in outstanding efficacy against dengue virus infection in mouse infection models. Delaying start of treatment until peak viraemia results in a rapid and significant reduction in viral load. An analogue is currently in further development.
Assuntos
Antivirais/farmacologia , Vírus da Dengue/classificação , Vírus da Dengue/efeitos dos fármacos , Dengue/virologia , Proteínas de Membrana/metabolismo , Proteínas não Estruturais Virais/metabolismo , Animais , Antivirais/farmacocinética , Antivirais/uso terapêutico , Dengue/tratamento farmacológico , Vírus da Dengue/genética , Vírus da Dengue/metabolismo , Modelos Animais de Doenças , Feminino , Masculino , Proteínas de Membrana/antagonistas & inibidores , Camundongos , RNA Helicases/antagonistas & inibidores , RNA Helicases/metabolismo , Serina Endopeptidases/metabolismo , Carga Viral/efeitos dos fármacos , Proteínas não Estruturais Virais/antagonistas & inibidores , Viremia/tratamento farmacológico , Viremia/virologia , Replicação Viral/efeitos dos fármacosRESUMO
Virus discovery by genomics and metagenomics empowered studies of viromes, facilitated characterization of pathogen epidemiology, and redefined our understanding of the natural genetic diversity of viruses with profound functional and structural implications. Here we employed a data-driven virus discovery approach that directly queries unprocessed sequencing data in a highly parallelized way and involves a targeted viral genome assembly strategy in a wide range of sequence similarity. By screening more than 269,000 datasets of numerous authors from the Sequence Read Archive and using two metrics that quantitatively assess assembly quality, we discovered 40 nidoviruses from six virus families whose members infect vertebrate hosts. They form 13 and 32 putative viral subfamilies and genera, respectively, and include 11 coronaviruses with bisegmented genomes from fishes and amphibians, a giant 36.1 kilobase coronavirus genome with a duplicated spike glycoprotein (S) gene, 11 tobaniviruses and 17 additional corona-, arteri-, cremega-, nanhypo- and nangoshaviruses. Genome segmentation emerged in a single evolutionary event in the monophyletic lineage encompassing the subfamily Pitovirinae. We recovered the bisegmented genome sequences of two coronaviruses from RNA samples of 69 infected fishes and validated the presence of poly(A) tails at both segments using 3'RACE PCR and subsequent Sanger sequencing. We report a genetic linkage between accessory and structural proteins whose phylogenetic relationships and evolutionary distances are incongruent with the phylogeny of replicase proteins. We rationalize these observations in a model of inter-family S recombination involving at least five ancestral corona- and tobaniviruses of aquatic hosts. In support of this model, we describe an individual fish co-infected with members from the families Coronaviridae and Tobaniviridae. Our results expand the scale of the known extraordinary evolutionary plasticity in nidoviral genome architecture and call for revisiting fundamentals of genome expression, virus particle biology, host range and ecology of vertebrate nidoviruses.
Assuntos
Coronavirus , Genoma Viral , Nidovirales , Filogenia , Animais , Nidovirales/genética , Coronavirus/genética , Coronavirus/classificação , Vertebrados/virologia , Vertebrados/genética , Peixes/virologia , Evolução Molecular , Mineração de Dados , Infecções por Nidovirales/virologia , Infecções por Nidovirales/genéticaRESUMO
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virions are surrounded by a lipid bilayer from which spike (S) protein trimers protrude1. Heavily glycosylated S trimers bind to the angiotensin-converting enzyme 2 receptor and mediate entry of virions into target cells2-6. S exhibits extensive conformational flexibility: it modulates exposure of its receptor-binding site and subsequently undergoes complete structural rearrangement to drive fusion of viral and cellular membranes2,7,8. The structures and conformations of soluble, overexpressed, purified S proteins have been studied in detail using cryo-electron microscopy2,7,9-12, but the structure and distribution of S on the virion surface remain unknown. Here we applied cryo-electron microscopy and tomography to image intact SARS-CoV-2 virions and determine the high-resolution structure, conformational flexibility and distribution of S trimers in situ on the virion surface. These results reveal the conformations of S on the virion, and provide a basis from which to understand interactions between S and neutralizing antibodies during infection or vaccination.
Assuntos
Microscopia Crioeletrônica , SARS-CoV-2/metabolismo , SARS-CoV-2/ultraestrutura , Glicoproteína da Espícula de Coronavírus/análise , Glicoproteína da Espícula de Coronavírus/ultraestrutura , Vírion/química , Vírion/ultraestrutura , Anticorpos Neutralizantes/imunologia , COVID-19/imunologia , Vacinas contra COVID-19/imunologia , Linhagem Celular Tumoral , Humanos , Modelos Moleculares , Maleabilidade , Conformação Proteica , Multimerização Proteica , SARS-CoV-2/química , SARS-CoV-2/isolamento & purificação , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/isolamento & purificação , Vírion/isolamento & purificação , Vírion/metabolismoRESUMO
BACKGROUND & AIMS: High expression of phosphatidylinositol 4-kinase III alpha (PI4KIIIα) correlates with poor survival rates in patients with hepatocellular carcinoma. In addition, hepatitis C virus (HCV) infections activate PI4KIIIα and contribute to hepatocellular carcinoma progression. We aimed at mechanistically understanding the impact of PI4KIIIα on the progression of liver cancer and the potential contribution of HCV in this process. METHODS: Several hepatic cell culture and mouse models were used to study the functional importance of PI4KIIIα on liver pathogenesis. Antibody arrays, gene silencing, and PI4KIIIα-specific inhibitor were applied to identify the involved signaling pathways. The contribution of HCV was examined by using HCV infection or overexpression of its nonstructural protein. RESULTS: High PI4KIIIα expression and/or activity induced cytoskeletal rearrangements via increased phosphorylation of paxillin and cofilin. This led to morphologic alterations and higher migratory and invasive properties of liver cancer cells. We further identified the liver-specific lipid kinase phosphatidylinositol 3-kinase C2 domain-containing subunit gamma (PIK3C2γ) working downstream of PI4KIIIα in regulation of the cytoskeleton. PIK3C2γ generates plasma membrane phosphatidylinositol 3,4-bisphosphate-enriched, invadopodia-like structures that regulate cytoskeletal reorganization by promoting Akt2 phosphorylation. CONCLUSIONS: PI4KIIIα regulates cytoskeleton organization via PIK3C2γ/Akt2/paxillin-cofilin to favor migration and invasion of liver cancer cells. These findings provide mechanistic insight into the contribution of PI4KIIIα and HCV to the progression of liver cancer and identify promising targets for therapeutic intervention.
Assuntos
Fatores de Despolimerização de Actina , Carcinoma Hepatocelular , Movimento Celular , Citoesqueleto , Neoplasias Hepáticas , Invasividade Neoplásica , Paxilina , Transdução de Sinais , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/genética , Humanos , Animais , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/genética , Citoesqueleto/metabolismo , Citoesqueleto/patologia , Paxilina/metabolismo , Camundongos , Fatores de Despolimerização de Actina/metabolismo , Fatores de Despolimerização de Actina/genética , Fosforilação , Hepacivirus , Linhagem Celular Tumoral , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Células Hep G2 , Hepatite C/patologia , Hepatite C/metabolismo , Hepatite C/virologia , Interferência de RNARESUMO
Liver-generated plasma Apolipoprotein E (ApoE)-containing lipoproteins (LPs) (ApoE-LPs) play central roles in lipid transport and metabolism. Perturbations of ApoE can result in several metabolic disorders and ApoE genotypes have been associated with multiple diseases. ApoE is synthesized at the endoplasmic reticulum and transported to the Golgi apparatus for LP assembly; however, the ApoE-LPs transport pathway from there to the plasma membrane is largely unknown. Here, we established an integrative imaging approach based on a fully functional fluorescently tagged ApoE. We found that newly synthesized ApoE-LPs accumulate in CD63-positive endosomes of hepatocytes. In addition, we observed the co-egress of ApoE-LPs and CD63-positive intraluminal vesicles (ILVs), which are precursors of extracellular vesicles (EVs), along the late endosomal trafficking route in a microtubule-dependent manner. A fraction of ApoE-LPs associated with CD63-positive EVs appears to be co-transmitted from cell to cell. Given the important role of ApoE in viral infections, we employed as well-studied model the hepatitis C virus (HCV) and found that the viral replicase component nonstructural protein 5A (NS5A) is enriched in ApoE-containing ILVs. Interaction between NS5A and ApoE is required for the efficient release of ILVs containing HCV RNA. These vesicles are transported along the endosomal ApoE egress pathway. Taken together, our data argue for endosomal egress and transmission of hepatic ApoE-LPs, a pathway that is hijacked by HCV. Given the more general role of EV-mediated cell-to-cell communication, these insights provide new starting points for research into the pathophysiology of ApoE-related metabolic and infection-related disorders.
Assuntos
Hepacivirus , Hepatite C , Humanos , Hepacivirus/fisiologia , Lipopolissacarídeos/metabolismo , Montagem de Vírus/fisiologia , Endossomos/metabolismo , Apolipoproteínas E/metabolismoRESUMO
Cell-autonomous induction of type I interferon must be stringently regulated. Rapid induction is key to control virus infection, whereas proper limitation of signaling is essential to prevent immunopathology and autoimmune disease. Using unbiased kinome-wide RNAi screening followed by thorough validation, we identified 22 factors that regulate RIG-I/IRF3 signaling activity. We describe a negative-feedback mechanism targeting RIG-I activity, which is mediated by death associated protein kinase 1 (DAPK1). RIG-I signaling triggers DAPK1 kinase activation, and active DAPK1 potently inhibits RIG-I stimulated IRF3 activity and interferon-beta production. DAPK1 phosphorylates RIG-I in vitro at previously reported as well as other sites that limit 5'ppp-dsRNA sensing and virtually abrogate RIG-I activation.
Assuntos
Proteínas Quinases Associadas com Morte Celular/metabolismo , RNA Interferente Pequeno/genética , Receptores do Ácido Retinoico/metabolismo , Células A549 , Animais , Células Cultivadas , Retroalimentação Fisiológica , Células HEK293 , Humanos , Camundongos , Fosforilação , Proteínas Quinases/metabolismo , Transdução de SinaisRESUMO
IMPORTANCE: Remodeling of the cellular endomembrane system by viruses allows for efficient and coordinated replication of the viral genome in distinct subcellular compartments termed replication organelles. As a critical step in the viral life cycle, replication organelle formation is an attractive target for therapeutic intervention, but factors central to this process are only partially understood. In this study, we corroborate that two viral proteins, nsp3 and nsp4, are the major drivers of membrane remodeling in SARS-CoV-2 infection. We further report a number of host cell factors interacting with these viral proteins and supporting the viral replication cycle, some of them by contributing to the formation of the SARS-CoV-2 replication organelle.
Assuntos
COVID-19 , SARS-CoV-2 , Proteínas não Estruturais Virais , Replicação Viral , Humanos , Organelas/metabolismo , Proteômica , SARS-CoV-2/fisiologia , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismoRESUMO
The hepatitis C virus (HCV) life cycle is highly regulated and characterized by a step-wise succession of interactions between viral and host cell proteins resulting in the assembly of macromolecular complexes, which catalyse genome replication and/or virus production. Non-structural (NS) protein 3, comprising a protease and a helicase domain, is involved in orchestrating these processes by undergoing protein interactions in a temporal fashion. Recently, we identified a multifunctional NS3 protease surface patch promoting pivotal protein-protein interactions required for early steps of the HCV life cycle, including NS3-mediated NS2 protease activation and interactions required for replicase assembly. In this work, we extend this knowledge by identifying further NS3 surface determinants important for NS5A hyperphosphorylation, replicase assembly or virion morphogenesis, which map to protease and helicase domain and form a contiguous NS3 surface area. Functional interrogation led to the identification of phylogenetically conserved amino acid positions exerting a critical function in virion production without affecting RNA replication. These findings illustrate that NS3 uses a multipurpose protein surface to orchestrate the step-wise assembly of functionally distinct multiprotein complexes. Taken together, our data provide a basis to dissect the temporal formation of viral multiprotein complexes required for the individual steps of the HCV life cycle.
Assuntos
Hepacivirus , Hepatite C , Humanos , Hepacivirus/fisiologia , Proteínas não Estruturais Virais/metabolismo , Montagem de Vírus/genética , Replicação Viral/fisiologia , Linhagem Celular , Vírion/metabolismo , Hepatite C/metabolismo , Morfogênese , Peptídeo Hidrolases/metabolismoRESUMO
Hepatitis C virus (HCV) is highly diverse and grouped into eight genotypes (gts). Infectious cell culture models are limited to a few subtypes and isolates, hampering the development of prophylactic vaccines. A consensus gt1b genome (termed GLT1) was generated from an HCV infected liver-transplanted patient. GLT1 replicated to an outstanding efficiency in Huh7 cells upon SEC14L2 expression, by use of replication enhancing mutations or with a previously developed inhibitor-based regimen. RNA replication levels almost reached JFH-1, but full-length genomes failed to produce detectable amounts of infectious virus. Long-term passaging led to the adaptation of a genome carrying 21 mutations and concomitant production of high levels of transmissible infectivity (GLT1cc). During the adaptation, GLT1 spread in the culture even in absence of detectable amounts of free virus, likely due to cell-to-cell transmission, which appeared to substantially contribute to spreading of other isolates as well. Mechanistically, genome replication and particle production efficiency were enhanced by adaptation, while cell entry competence of HCV pseudoparticles was not affected. Furthermore, GLT1cc retained the ability to replicate in human liver chimeric mice, which was critically dependent on a mutation in domain 3 of nonstructural protein NS5A. Over the course of infection, only one mutation in the surface glycoprotein E2 consistently reverted to wildtype, facilitating assembly in cell culture but potentially affecting CD81 interaction in vivo. Overall, GLT1cc is an efficient gt1b infectious cell culture model, paving the road to a rationale-based establishment of new infectious HCV isolates and represents an important novel tool for the development of prophylactic HCV vaccines.
Assuntos
Hepacivirus , Hepatite C , Animais , Técnicas de Cultura de Células , Genótipo , Humanos , Camundongos , Mutação , Proteínas não Estruturais Virais/metabolismo , Replicação ViralRESUMO
Plus-strand RNA viruses are the largest group of viruses. Many are human pathogens that inflict a socio-economic burden. Interestingly, plus-strand RNA viruses share remarkable similarities in their replication. A hallmark of plus-strand RNA viruses is the remodeling of intracellular membranes to establish replication organelles (so-called "replication factories"), which provide a protected environment for the replicase complex, consisting of the viral genome and proteins necessary for viral RNA synthesis. In the current study, we investigate pan-viral similarities and virus-specific differences in the life cycle of this highly relevant group of viruses. We first measured the kinetics of viral RNA, viral protein, and infectious virus particle production of hepatitis C virus (HCV), dengue virus (DENV), and coxsackievirus B3 (CVB3) in the immuno-compromised Huh7 cell line and thus without perturbations by an intrinsic immune response. Based on these measurements, we developed a detailed mathematical model of the replication of HCV, DENV, and CVB3 and showed that only small virus-specific changes in the model were necessary to describe the in vitro dynamics of the different viruses. Our model correctly predicted virus-specific mechanisms such as host cell translation shut off and different kinetics of replication organelles. Further, our model suggests that the ability to suppress or shut down host cell mRNA translation may be a key factor for in vitro replication efficiency, which may determine acute self-limited or chronic infection. We further analyzed potential broad-spectrum antiviral treatment options in silico and found that targeting viral RNA translation, such as polyprotein cleavage and viral RNA synthesis, may be the most promising drug targets for all plus-strand RNA viruses. Moreover, we found that targeting only the formation of replicase complexes did not stop the in vitro viral replication early in infection, while inhibiting intracellular trafficking processes may even lead to amplified viral growth.
Assuntos
Hepatite C , Vírus de RNA , Humanos , Antivirais/farmacologia , Replicação Viral/fisiologia , RNA Viral/genética , Modelos TeóricosRESUMO
SARS-CoV-2 is a human pathogenic virus responsible for the COVID-19 (coronavirus disease 2019) pandemic. The infection cycle of SARS-CoV-2 involves several related steps, including virus entry, gene expression, RNA replication, assembly of infectious virions and their egress. For all of these steps, the virus relies on and exploits host cell factors, cellular organelles, and processes such as endocytosis, nuclear transport, protein secretion, metabolite transport at membrane contact sites (MSC) and exocytotic pathways. To do this, SARS-CoV-2 has evolved multifunctional viral proteins that hijack cellular factors and modulate their function by unique strategies. In this Review, we highlight cellular trafficking factors, processes, and organelles of relevance to the SARS-CoV-2 infection cycle and how viral proteins make use of and perturb cellular transport during the viral infection cycle.
Assuntos
COVID-19 , Humanos , SARS-CoV-2/metabolismo , Proteínas Virais/metabolismo , Internalização do Vírus , Transporte ProteicoRESUMO
Zika virus (ZIKV) has recently emerged as a global health concern owing to its widespread diffusion and its association with severe neurological symptoms and microcephaly in newborns1. However, the molecular mechanisms that are responsible for the pathogenicity of ZIKV remain largely unknown. Here we use human neural progenitor cells and the neuronal cell line SK-N-BE2 in an integrated proteomics approach to characterize the cellular responses to viral infection at the proteome and phosphoproteome level, and use affinity proteomics to identify cellular targets of ZIKV proteins. Using this approach, we identify 386 ZIKV-interacting proteins, ZIKV-specific and pan-flaviviral activities as well as host factors with known functions in neuronal development, retinal defects and infertility. Moreover, our analysis identified 1,216 phosphorylation sites that are specifically up- or downregulated after ZIKV infection, indicating profound modulation of fundamental signalling pathways such as AKT, MAPK-ERK and ATM-ATR and thereby providing mechanistic insights into the proliferation arrest elicited by ZIKV infection. Functionally, our integrative study identifies ZIKV host-dependency factors and provides a comprehensive framework for a system-level understanding of ZIKV-induced perturbations at the levels of proteins and cellular pathways.
Assuntos
Interações Hospedeiro-Patógeno/fisiologia , Proteoma/análise , Proteômica , Zika virus/patogenicidade , Animais , Diferenciação Celular , Linhagem Celular , Chlorocebus aethiops , Interações Hospedeiro-Patógeno/genética , Humanos , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Células-Tronco Neurais/virologia , Fosfoproteínas/análise , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Mapas de Interação de Proteínas , Proteoma/genética , Proteoma/metabolismo , RNA Interferente Pequeno/genética , Proteínas Virais/genética , Proteínas Virais/metabolismo , Zika virus/genética , Zika virus/metabolismoRESUMO
Cell-penetrating peptides (CPPs) play a significant role in the delivery of cargos into human cells. We report the first CPPs based on peptide-bismuth bicycles, which can be readily obtained from commercially available peptide precursors, making them accessible for a wide range of applications. These CPPs enter human cells as demonstrated by live-cell confocal microscopy using fluorescently labelled peptides. We report efficient sequences that demonstrate increased cellular uptake compared to conventional CPPs like the TAT peptide (derived from the transactivating transcriptional activator of human immunodeficiency virus 1) or octaarginine (R8 ), despite requiring only three positive charges. Bicyclization triggered by the presence of bismuth(III) increases cellular uptake by more than one order of magnitude. Through the analysis of cell lysates using inductive coupled plasma mass spectrometry (ICP-MS), we have introduced an alternative approach to examine the cellular uptake of CPPs. This has allowed us to confirm the presence of bismuth in cells after exposure to our CPPs. Mechanistic studies indicated an energy-dependent endocytic cellular uptake sensitive to inhibition by rottlerin, most likely involving macropinocytosis.
Assuntos
Peptídeos Penetradores de Células , Humanos , Peptídeos Penetradores de Células/química , Endocitose/fisiologia , Bismuto , Ciclismo , PinocitoseRESUMO
BACKGROUND & AIMS: Retinoic acid inducible gene I (RIG-I)-like receptors (RLRs), including RIG-I, melanoma differentiation-associated protein 5 (MDA5), and laboratory of genetics and physiology 2 (LGP2), sense viral RNA to induce the antiviral interferon (IFN) response. LGP2, unable to activate the IFN response itself, modulates RIG-I and MDA5 signalling. HDV, a small RNA virus causing the most severe form of viral hepatitis, is sensed by MDA5. The mechanism underlying IFN induction and its effect on HDV replication is unclear. Here, we aimed to unveil the role of LGP2 and clinically relevant variants thereof in these processes. METHODS: RLRs were depleted in HDV susceptible HepaRGNTCP cells and primary human hepatocytes. Cells were reconstituted to express different LGP2 versions. HDV and IFN markers were quantified in a time-resolved manner. Interaction studies among LGP2, MDA5, and RNA were performed by pull-down assays. RESULTS: LGP2 is essential for the MDA5-mediated IFN response induced upon HDV infection. This induction requires both RNA binding and ATPase activities of LGP2. The IFN response only moderately reduced HDV replication in resting cells but profoundly suppressed cell division-mediated HDV spread. An LGP2 variant (Q425R), predominating in Africans who develop less severe chronic hepatitis D, mediated detectably higher basal and faster HDV-induced IFN response as well as stronger HDV suppression. Mechanistically, LGP2 RNA binding was a prerequisite for the formation of stable MDA5-RNA complexes. MDA5 binding to RNA was enhanced by the Q425R LGP2 variant. CONCLUSIONS: LGP2 is essential to mount an antiviral IFN response induced by HDV and stabilises MDA5-RNA interaction required for downstream signalling. The natural Q425R LGP2 is a gain-of-function variant and might contribute to an attenuated course of hepatitis D. IMPACT AND IMPLICATIONS: HDV is the causative pathogen of chronic hepatitis D, a severe form of viral hepatitis that can lead to cirrhosis and hepatocellular carcinoma. Upon infection, the human immune system senses HDV and mounts an antiviral interferon (IFN) response. Here, we demonstrate that the immune sensor LGP2 cooperates with MDA5 to mount an IFN response that represses HDV replication. We mapped LGP2 determinants required for IFN system activation and characterised several natural genetic variants of LGP2. One of them reported to predominate in sub-Saharan Africans can accelerate HDV-induced IFN responses, arguing that genetic determinants, possibly including LGP2, might contribute to slower disease progression in this population. Our results will hopefully prompt further studies on genetic variations in LGP2 and other components of the innate immune sensing system, including assessments of their possible impact on the course of viral infection.
Assuntos
Hepatite D Crônica , Helicase IFIH1 Induzida por Interferon , Interferons , RNA Helicases , Humanos , Antivirais , Vírus Delta da Hepatite/genética , Imunidade Inata , Helicase IFIH1 Induzida por Interferon/genética , Helicase IFIH1 Induzida por Interferon/metabolismo , RNA Helicases/genética , RNA Helicases/metabolismo , RNA Viral/genética , RNA Viral/metabolismo , Replicação ViralRESUMO
BACKGROUND & AIMS: Hepatitis A virus (HAV) infections are considered not to trigger innate immunity in vivo, in contrast to hepatitis C virus (HCV). This lack of induction has been imputed to strong interference by HAV proteases 3CD and 3ABC. We aimed to elucidate the mechanisms of immune activation and counteraction by HAV and HCV in vivo and in vitro. METHODS: Albumin-urokinase-type plasminogen activator/severe combined immunodeficiency (Alb/uPA-SCID) mice with humanised livers were infected with HAV and HCV. Hepatic cell culture models were used to assess HAV and HCV sensing by Toll-like receptor 3 and retinoic acid-inducible gene I/melanoma differentiation-associated protein 5 (RIG-I/MDA5), respectively. Cleavage of the adaptor proteins TIR-domain-containing adapter-inducing interferon-ß (TRIF) and mitochondrial antiviral-signalling protein (MAVS) was analysed by transient and stable expression of HAV and HCV proteases and virus infection. RESULTS: We detected similar levels of interferon-stimulated gene induction in hepatocytes of HAV- and HCV-infected mice with humanised liver. In cell culture, HAV induced interferon-stimulated genes exclusively upon MDA5 sensing and depended on LGP2 (laboratory of genetics and physiology 2). TRIF and MAVS were only partially cleaved by HAV 3ABC and 3CD, not sufficiently to abrogate signalling. In contrast, HCV NS3-4A efficiently degraded MAVS, as previously reported, whereas TRIF cleavage was not detected. CONCLUSIONS: HAV induces an innate immune response in hepatocytes via MDA5/LGP2, with limited control of both pathways by proteolytic cleavage. HCV activates Toll-like receptor 3 and lacks TRIF cleavage, suggesting that this pathway mainly contributes to HCV-induced antiviral responses in hepatocytes. Our results shed new light on the induction of innate immunity and counteraction by HAV and HCV. IMPACT AND IMPLICATIONS: Understanding the mechanisms that determine the differential outcomes of HAV and HCV infections is crucial for the development of effective therapies. Our study provides insights into the interplay between these viruses and the host innate immune response in vitro and in vivo, shedding light on previously controversial or only partially investigated aspects. This knowledge could tailor the development of new strategies to combat HCV persistence, as well as improve our understanding of the factors underlying successful HAV clearance.