RESUMO
Hepatitis B virus (HBV) is a small double-stranded DNA virus that chronically infects 296 million people. Over half of its compact genome encodes proteins in two overlapping reading frames, and during evolution, multiple selective pressures can act on shared nucleotides. This study combines an RNA-based HBV cell culture system with deep mutational scanning (DMS) to uncouple cis- and trans-acting sequence requirements in the HBV genome. The results support a leaky ribosome scanning model for polymerase translation, provide a fitness map of the HBV polymerase at single-nucleotide resolution, and identify conserved prolines adjacent to the HBV polymerase termination codon that stall ribosomes. Further experiments indicated that stalled ribosomes tether the nascent polymerase to its template RNA, ensuring cis-preferential RNA packaging and reverse transcription of the HBV genome.
Assuntos
Vírus da Hepatite B , Transcrição Reversa , Humanos , Genoma Viral/genética , Vírus da Hepatite B/genética , Mutação , Ribossomos/metabolismo , RNA Viral/genética , RNA Viral/metabolismo , Linhagem CelularRESUMO
Interferon-stimulated gene (ISG) products take on a number of diverse roles. Collectively, they are highly effective at resisting and controlling pathogens. In this review, we begin by introducing interferon (IFN) and the JAK-STAT signaling pathway to highlight features that impact ISG production. Next, we describe ways in which ISGs both enhance innate pathogen-sensing capabilities and negatively regulate signaling through the JAK-STAT pathway. Several ISGs that directly inhibit virus infection are described with an emphasis on those that impact early and late stages of the virus life cycle. Finally, we describe ongoing efforts to identify and characterize antiviral ISGs, and we provide a forward-looking perspective on the ISG landscape.
Assuntos
Regulação da Expressão Gênica , Imunidade/fisiologia , Interferons/metabolismo , Animais , Humanos , Janus Quinases/metabolismo , Receptores de Interferon/metabolismo , Fatores de Transcrição STAT/metabolismo , Transdução de SinaisRESUMO
Flaviviruses pose a constant threat to human health. These RNA viruses are transmitted by the bite of infected mosquitoes and ticks and regularly cause outbreaks. To identify host factors required for flavivirus infection, we performed full-genome loss of function CRISPR-Cas9 screens. Based on these results, we focused our efforts on characterizing the roles that TMEM41B and VMP1 play in the virus replication cycle. Our mechanistic studies on TMEM41B revealed that all members of the Flaviviridae family that we tested require TMEM41B. We tested 12 additional virus families and found that SARS-CoV-2 of the Coronaviridae also required TMEM41B for infection. Remarkably, single nucleotide polymorphisms present at nearly 20% in East Asian populations reduce flavivirus infection. Based on our mechanistic studies, we propose that TMEM41B is recruited to flavivirus RNA replication complexes to facilitate membrane curvature, which creates a protected environment for viral genome replication.
Assuntos
Infecções por Flavivirus/genética , Flavivirus/fisiologia , Proteínas de Membrana/metabolismo , Animais , Povo Asiático/genética , Autofagia , COVID-19/genética , COVID-19/metabolismo , COVID-19/virologia , Sistemas CRISPR-Cas , Linhagem Celular , Infecções por Flavivirus/imunologia , Infecções por Flavivirus/metabolismo , Infecções por Flavivirus/virologia , Técnicas de Inativação de Genes , Estudo de Associação Genômica Ampla , Interações Hospedeiro-Patógeno , Humanos , Imunidade Inata , Proteínas de Membrana/genética , Polimorfismo de Nucleotídeo Único , SARS-CoV-2/fisiologia , Replicação Viral , Vírus da Febre Amarela/fisiologia , Zika virus/fisiologiaRESUMO
The coronavirus disease 2019 (COVID-19) pandemic has claimed the lives of over one million people worldwide. The causative agent, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a member of the Coronaviridae family of viruses that can cause respiratory infections of varying severity. The cellular host factors and pathways co-opted during SARS-CoV-2 and related coronavirus life cycles remain ill defined. To address this gap, we performed genome-scale CRISPR knockout screens during infection by SARS-CoV-2 and three seasonal coronaviruses (HCoV-OC43, HCoV-NL63, and HCoV-229E). These screens uncovered host factors and pathways with pan-coronavirus and virus-specific functional roles, including major dependency on glycosaminoglycan biosynthesis, sterol regulatory element-binding protein (SREBP) signaling, bone morphogenetic protein (BMP) signaling, and glycosylphosphatidylinositol biosynthesis, as well as a requirement for several poorly characterized proteins. We identified an absolute requirement for the VMP1, TMEM41, and TMEM64 (VTT) domain-containing protein transmembrane protein 41B (TMEM41B) for infection by SARS-CoV-2 and three seasonal coronaviruses. This human coronavirus host factor compendium represents a rich resource to develop new therapeutic strategies for acute COVID-19 and potential future coronavirus pandemics.
Assuntos
Infecções por Coronavirus/genética , Estudo de Associação Genômica Ampla , SARS-CoV-2/fisiologia , Células A549 , Linhagem Celular , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Coronavirus Humano 229E/fisiologia , Infecções por Coronavirus/virologia , Coronavirus Humano NL63/fisiologia , Coronavirus Humano OC43/fisiologia , Técnicas de Inativação de Genes , Células HEK293 , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Humanos , Proteínas de Membrana/metabolismo , Redes e Vias Metabólicas/efeitos dos fármacos , Mapeamento de Interação de ProteínasRESUMO
Stem cells are highly resistant to viral infection compared to their differentiated progeny; however, the mechanism is mysterious. Here, we analyzed gene expression in mammalian stem cells and cells at various stages of differentiation. We find that, conserved across species, stem cells express a subset of genes previously classified as interferon (IFN) stimulated genes (ISGs) but that expression is intrinsic, as stem cells are refractory to interferon. This intrinsic ISG expression varies in a cell-type-specific manner, and many ISGs decrease upon differentiation, at which time cells become IFN responsive, allowing induction of a broad spectrum of ISGs by IFN signaling. Importantly, we show that intrinsically expressed ISGs protect stem cells against viral infection. We demonstrate the in vivo importance of intrinsic ISG expression for protecting stem cells and their differentiation potential during viral infection. These findings have intriguing implications for understanding stem cell biology and the evolution of pathogen resistance.
Assuntos
Imunidade Inata , Células-Tronco Pluripotentes/imunologia , Viroses/imunologia , Animais , Células Cultivadas , Feminino , Células HEK293 , Humanos , Interferons/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos NOD , Células-Tronco Pluripotentes/virologia , Especificidade da EspécieRESUMO
Interferon-stimulated genes (ISGs) form the backbone of the innate immune system and are important for limiting intra- and intercellular viral replication and spread. We conducted a mass-spectrometry-based survey to understand the fundamental organization of the innate immune system and to explore the molecular functions of individual ISGs. We identified interactions between 104 ISGs and 1,401 cellular binding partners engaging in 2,734 high-confidence interactions. 90% of these interactions are unreported so far, and our survey therefore illuminates a far wider activity spectrum of ISGs than is currently known. Integration of the resulting ISG-interaction network with published datasets and functional studies allowed us to identify regulators of immunity and processes related to the immune system. Given the extraordinary robustness of the innate immune system, this ISG network may serve as a blueprint for therapeutic targeting of cellular systems to efficiently fight viral infections.
Assuntos
Imunidade Inata , Interferons/fisiologia , Mapeamento de Interação de Proteínas , Antígenos de Neoplasias/metabolismo , Biomarcadores Tumorais/metabolismo , Proteínas de Transporte/metabolismo , Linhagem Celular , Expressão Gênica , Glicoproteínas/metabolismo , Células HEK293 , Células HeLa , Humanos , Imunidade Inata/genética , Espectrometria de Massas , Receptores CCR4/metabolismo , Receptores de Peptídeos/metabolismo , Ribonucleoproteínas/metabolismo , Proteínas Virais/metabolismoRESUMO
Interferons (IFNs) play a crucial role in the regulation and evolution of host-virus interactions. Here, we conducted a genome-wide arrayed CRISPR knockout screen in the presence and absence of IFN to identify human genes that influence Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection. We then performed an integrated analysis of genes interacting with SARS-CoV-2, drawing from a selection of 67 large-scale studies, including our own. We identified 28 genes of high relevance in both human genetic studies of Coronavirus Disease 2019 (COVID-19) patients and functional genetic screens in cell culture, with many related to the IFN pathway. Among these was the IFN-stimulated gene PLSCR1. PLSCR1 did not require IFN induction to restrict SARS-CoV-2 and did not contribute to IFN signaling. Instead, PLSCR1 specifically restricted spike-mediated SARS-CoV-2 entry. The PLSCR1-mediated restriction was alleviated by TMPRSS2 overexpression, suggesting that PLSCR1 primarily restricts the endocytic entry route. In addition, recent SARS-CoV-2 variants have adapted to circumvent the PLSCR1 barrier via currently undetermined mechanisms. Finally, we investigate the functional effects of PLSCR1 variants present in humans and discuss an association between PLSCR1 and severe COVID-19 reported recently.
Assuntos
COVID-19 , SARS-CoV-2 , Internalização do Vírus , Humanos , SARS-CoV-2/genética , COVID-19/virologia , COVID-19/genética , Células HEK293 , Sistemas CRISPR-Cas/genética , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismo , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Interferons/metabolismo , Interferons/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Antígenos de DiferenciaçãoRESUMO
Type III interferons (IFN-λs) signal through a heterodimeric receptor complex composed of the IFN-λR1 subunit, specific for IFN-λs, and interleukin-10Rß (IL-10Rß), which is shared by multiple cytokines in the IL-10 superfamily. Low affinity of IL-10Rß for cytokines has impeded efforts aimed at crystallizing cytokine-receptor complexes. We used yeast surface display to engineer a higher-affinity IFN-λ variant, H11, which enabled crystallization of the ternary complex. The structure revealed that IL-10Rß uses a network of tyrosine residues as hydrophobic anchor points to engage IL-10 family cytokines that present complementary hydrophobic binding patches, explaining its role as both a cross-reactive but cytokine-specific receptor. H11 elicited increased anti-proliferative and antiviral activities in vitro and in vivo. In contrast, engineered higher-affinity type I IFNs did not increase antiviral potency over wild-type type I IFNs. Our findings provide insight into cytokine recognition by the IL-10R family and highlight the plasticity of type III interferon signaling and its therapeutic potential.
Assuntos
Interferons/imunologia , Receptores de Interferon/imunologia , Receptores de Interleucina-10/imunologia , Animais , Linhagem Celular , Cristalografia por Raios X , Citometria de Fluxo , Humanos , Camundongos , Reação em Cadeia da Polimerase , Ressonância de Plasmônio de SuperfícieRESUMO
Co-opting host cell protein synthesis is a hallmark of many virus infections. In response, certain host defense proteins limit mRNA translation globally, albeit at the cost of the host cell's own protein synthesis. Here, we describe an interferon-stimulated helicase, DDX60, that decreases translation from viral internal ribosome entry sites (IRESs). DDX60 acts selectively on type II IRESs of encephalomyocarditis virus (EMCV) and foot and mouth disease virus (FMDV), but not by other IRES types or by 5' cap. Correspondingly, DDX60 reduces EMCV and FMDV (type II IRES) replication, but not that of poliovirus or bovine enterovirus 1 (BEV-1; type I IRES). Furthermore, replacing the IRES of poliovirus with a type II IRES is sufficient for DDX60 to inhibit viral replication. Finally, DDX60 selectively modulates the amount of translating ribosomes on viral and in vitro transcribed type II IRES mRNAs, but not 5' capped mRNA. Our study identifies a novel facet in the repertoire of interferon-stimulated effector genes, the selective downregulation of translation from viral type II IRES elements.
Assuntos
Interferons , Sítios Internos de Entrada RibossomalRESUMO
Human adenosine deaminase acting on RNA 1 (ADAR1) catalyzes adenosine-to-inosine deamination reactions on double-stranded RNA molecules to regulate cellular responses to endogenous and exogenous RNA. Defective ADAR1 editing leads to disorders such as Aicardi-Goutières syndrome, an autoinflammatory disease that manifests in the brain and skin, and dyschromatosis symmetrica hereditaria, a skin pigmentation disorder. Two ADAR1 protein isoforms, p150 (150 kDa) and p110 (110 kDa), are expressed and can edit RNA, but the contribution of each isoform to the editing landscape remains unclear, largely because of the challenges in expressing p150 without p110. In this study, we demonstrate that p110 is coexpressed with p150 from the canonical p150-encoding mRNA due to leaky ribosome scanning downstream of the p150 start codon. The presence of a strong Kozak consensus context surrounding the p110 start codon suggests the p150 mRNA is optimized to leak p110 alongside expression of p150. To reduce leaky scanning and translation initiation at the p110 start codon, we introduced synonymous mutations in the coding region between the p150 and p110 start codons. Cells expressing p150 constructs with these mutations produced significantly reduced levels of p110. Editing analysis of total RNA from ADAR1 knockout cells reconstituted separately with modified p150 and p110 revealed that more than half of the A-to-I edit sites are selectively edited by p150, and the other half are edited by either p150 or p110. This method of isoform-selective editing analysis, making use of the modified p150, has the potential to be adapted for other cellular contexts.
Assuntos
Adenosina Desaminase/genética , Regulação da Expressão Gênica , Isoformas de Proteínas/genética , Edição de RNA , Proteínas de Ligação a RNA/genética , Doenças Autoimunes do Sistema Nervoso/genética , Suscetibilidade a Doenças , Técnicas de Inativação de Genes , Predisposição Genética para Doença , Humanos , Malformações do Sistema Nervoso/genética , Transtornos da Pigmentação/congênito , Transtornos da Pigmentação/genéticaRESUMO
Primary human hepatocytes (PHHs) are an essential tool for modeling drug metabolism and liver disease. However, variable plating efficiencies, short lifespan in culture, and resistance to genetic manipulation have limited their use. Here, we show that the pyrrolizidine alkaloid retrorsine improves PHH repopulation of chimeric mice on average 10-fold and rescues the ability of even poorly plateable donor hepatocytes to provide cells for subsequent ex vivo cultures. These mouse-passaged (mp) PHH cultures overcome the marked donor-to-donor variability of cryopreserved PHH and remain functional for months as demonstrated by metabolic assays and infection with hepatitis B virus and Plasmodium falciparum mpPHH can be efficiently genetically modified in culture, mobilized, and then recultured as spheroids or retransplanted to create highly humanized mice that carry a genetically altered hepatocyte graft. Together, these advances provide flexible tools for the study of human liver disease and evaluation of hepatocyte-targeted gene therapy approaches.
Assuntos
Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Hepatopatias/genética , Alcaloides de Pirrolizidina/farmacologia , Animais , Transplante de Células , Quimera , Modelos Animais de Doenças , Feminino , Terapia Genética , Hepatite B , Vírus da Hepatite B , Hepatócitos/transplante , Proteínas de Homeodomínio/genética , Humanos , Hidrolases/genética , Subunidade gama Comum de Receptores de Interleucina/genética , Fígado/patologia , Hepatopatias/patologia , Malária , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos Knockout , Plasmodium falciparumRESUMO
Given the unprecedented scale of the recent Ebola and Zika viral epidemics, it is crucial to understand the biology of host factors with broad antiviral action in order to develop novel therapeutic approaches. Here, we look into one such factor: zinc finger antiviral protein (ZAP) inhibits a variety of RNA and DNA viruses. Alternative splicing results in two isoforms that differ at their C termini: ZAPL (long) encodes a poly(ADP-ribose) polymerase (PARP)-like domain that is missing in ZAPS (short). Previously, it has been shown that ZAPL is more antiviral than ZAPS, while the latter is more induced by interferon (IFN). In this study, we discovered and confirmed the expression of two additional splice variants of human ZAP: ZAPXL (extralong) and ZAPM (medium). We also found two haplotypes of human ZAP. Since ZAPL and ZAPS have differential activities, we hypothesize that all four ZAP isoforms have evolved to mediate distinct antiviral and/or cellular functions. By taking a gene-knockout-and-reconstitution approach, we have characterized the antiviral, translational inhibition, and IFN activation activities of individual ZAP isoforms. Our work demonstrates that ZAPL and ZAPXL are more active against alphaviruses and hepatitis B virus (HBV) than ZAPS and ZAPM and elucidates the effects of splice variants on the action of a broad-spectrum antiviral factor.IMPORTANCE ZAP is an IFN-induced host factor that can inhibit a wide range of viruses, and there is great interest in fully characterizing its antiviral mechanism. This is the first study that defines the antiviral capacities of individual ZAP isoforms in the absence of endogenous ZAP expression and, hence, cross talk with other isoforms. Our data demonstrate that ZAP is expressed as four different forms: ZAPS, ZAPM, ZAPL, and ZAPXL. The longer ZAP isoforms better inhibit alphaviruses and HBV, while all isoforms equally inhibit Ebola virus transcription and replication. In addition, there is no difference in the abilities of ZAP isoforms to enhance the induction of type I IFN expression. Our results show that the full spectrum of ZAP activities can change depending on the virus target and the relative levels of basal expression and induction by IFN or infection.
Assuntos
Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Células A549 , Alphavirus/genética , Processamento Alternativo , Linhagem Celular , Células HEK293 , Haplótipos , Células HeLa , Vírus da Hepatite B/genética , Humanos , Isoformas de Proteínas , Splicing de RNA/genética , RNA Viral/genética , Replicação Viral/efeitos dos fármacos , Dedos de ZincoRESUMO
The host factor and interferon (IFN)-stimulated gene (ISG) product, zinc-finger antiviral protein (ZAP), inhibits a number of diverse viruses by usurping and intersecting with multiple cellular pathways. To elucidate its antiviral mechanism, we perform a loss-of-function genome-wide RNAi screen to identify cellular cofactors required for ZAP antiviral activity against the prototype alphavirus, Sindbis virus (SINV). In order to exclude off-target effects, we carry out stringent confirmatory assays to verify the top hits. Important ZAP-liaising partners identified include proteins involved in membrane ion permeability, type I IFN signaling, and post-translational protein modification. The factor contributing most to the antiviral function of ZAP is TRIM25, an E3 ubiquitin and ISG15 ligase. We demonstrate here that TRIM25 interacts with ZAP through the SPRY domain, and TRIM25 mutants lacking the RING or coiled coil domain fail to stimulate ZAP's antiviral activity, suggesting that both TRIM25 ligase activity and its ability to form oligomers are critical for its cofactor function. TRIM25 increases the modification of both the short and long ZAP isoforms by K48- and K63-linked polyubiquitin, although ubiquitination of ZAP does not directly affect its antiviral activity. However, TRIM25 is critical for ZAP's ability to inhibit translation of the incoming SINV genome. Taken together, these data uncover TRIM25 as a bona fide ZAP cofactor that leads to increased ZAP modification enhancing its translational inhibition activity.
Assuntos
Infecções por Alphavirus/prevenção & controle , Antivirais/metabolismo , Proteínas de Ligação a RNA/metabolismo , Sindbis virus/crescimento & desenvolvimento , Fatores de Transcrição/metabolismo , Proteínas com Motivo Tripartido/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Linhagem Celular , Cricetinae , Células HEK293 , Humanos , Interferon Tipo I/metabolismo , Domínios Proteicos , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Interferência de RNA , RNA Interferente Pequeno/genética , Proteínas de Ligação a RNA/genética , Fatores de Transcrição/genética , Proteínas com Motivo Tripartido/genética , Ubiquitina-Proteína Ligases/genética , UbiquitinaçãoRESUMO
Over half a century has passed since interferons (IFNs) were discovered and shown to inhibit virus infection in cultured cells. Since then, researchers have steadily brought to light the molecular details of IFN signaling, catalogued their pleiotropic effects on cells, and harnessed their therapeutic potential for a variety of maladies. While advances have been plentiful, several fundamental questions have yet to be answered and much complexity remains to be unraveled. We explore the current knowledge surrounding four main questions: are type I IFN subtypes differentially produced in response to distinct pathogens? How are IFN subtypes distinguished by cells? What are the mechanisms and consequences of viral antagonism? Lastly, how can the IFN response be harnessed to improve vaccine efficacy?
Assuntos
Imunidade Inata , Interferon Tipo I/imunologia , Interleucinas/imunologia , Linfócitos/imunologia , Vírus/imunologia , Imunidade Adaptativa , Evolução Biológica , Linhagem da Célula/imunologia , Regulação da Expressão Gênica/imunologia , Humanos , Evasão da Resposta Imune , Interferon Tipo I/classificação , Interferon Tipo I/genética , Interleucinas/classificação , Interleucinas/genética , Linfócitos/virologia , Filogenia , Receptores de Reconhecimento de Padrão/genética , Receptores de Reconhecimento de Padrão/imunologia , Transdução de Sinais , Vírus/patogenicidadeRESUMO
BACKGROUND & AIMS: As important virological markers, serum hepatitis B surface antigen (HBsAg) and hepatitis B virus (HBV) DNA levels show large fluctuations among chronic hepatitis B patients. The aim of this study was to reveal the potential impact and mechanisms of amino acid substitutions in small hepatitis B surface proteins (SHBs) on serum HBsAg and HBV DNA levels. METHODS: Serum samples from 230 untreated chronic hepatitis B patients with genotype C HBV were analyzed in terms of HBV DNA levels, serological markers of HBV infection and SHBs sequences. In vitro functional analysis of the identified SHBs mutants was performed. RESULTS: Among 230 SHBs sequences, there were 39 (16.96%) sequences with no mutation detected (wild-type) and 191 (83.04%) with single or multiple mutations. SHBs consist of 226 amino acids, of which 104 (46.02%) had mutations in our study. Some mutations (e.g., sE2G, sL21S, sR24K, sT47A/K, sC69stop (sC69∗), sL95W, sL98V, and sG145R) negatively correlated with serum HBsAg levels. HBsAg and HBV DNA levels from this group of patients had a positive correlation (r=0.61, p<0.001). In vitro analysis showed that these mutations reduced extracellular HBsAg and HBV DNA levels by restricting virion secretion and antibody binding capacity. Virion secretion could be rescued for sE2G, sC69∗, and sG145R by co-expression of wild-type HBsAg. CONCLUSION: The serum HBsAg levels were lower in untreated CHB patients with novel SHBs mutations outside the major antigenic region than those without mutations. Underlying mechanisms include impairment of virion secretion and lower binding affinity to antibodies used for HBsAg measurements. LAY SUMMARY: The hepatitis B surface antigen (HBsAg) is a major viral protein of the hepatitis B virus (HBV) secreted into patient blood serum and its quantification value serves as an important marker for the evaluation of chronic HBV infection and antiviral response. We found a few new amino acid substitutions in HBsAg associated with lower serum HBsAg and HBV DNA levels. These different substitutions might impair virion secretion, change the ability of HBsAg to bind to antibodies, or impact HBV replication. These could all result in decreased detectable levels of serum HBsAg. The factors affecting circulating HBsAg level and HBsAg detection are varied and caution is needed when interpreting clinical significance of serum HBsAg levels. Clinical trial number: NCT01088009.
Assuntos
DNA Viral , Antígenos de Superfície da Hepatite B , Vírus da Hepatite B , Hepatite B Crônica , Adulto , Substituição de Aminoácidos , DNA Viral/análise , DNA Viral/sangue , Feminino , Antígenos de Superfície da Hepatite B/sangue , Antígenos de Superfície da Hepatite B/genética , Vírus da Hepatite B/genética , Vírus da Hepatite B/isolamento & purificação , Hepatite B Crônica/diagnóstico , Hepatite B Crônica/virologia , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , Proteínas Virais/genética , Vírion/genética , Vírion/isolamento & purificação , Replicação ViralRESUMO
The hepatitis C virus (HCV) p7 protein is required for infectious virus production via its role in assembly and ion channel activity. Although NMR structures of p7 have been reported, the location of secondary structural elements and orientation of the p7 transmembrane domains differ among models. Furthermore, the p7 structure-function relationship remains unclear. Here, extensive mutagenesis, coupled with infectious virus production phenotyping and molecular modeling, demonstrates that the N-terminal helical region plays a previously underappreciated yet critical functional role, especially with respect to E2/p7 cleavage efficiency. Interrogation of specific N-terminal helix residues identified as having p7-specific defects and predicted to point toward the channel pore, in a context of independent E2/p7 cleavage, further supports p7 as a structurally plastic, minimalist ion channel. Together, our findings indicate that the p7 N-terminal helical region is critical for E2/p7 processing, protein-protein interactions, ion channel activity, and infectious HCV production.
Assuntos
Hepacivirus/metabolismo , Canais Iônicos/metabolismo , Proteínas do Envelope Viral/metabolismo , Proteínas Virais/metabolismo , Sequência de Aminoácidos , Linhagem Celular , Humanos , Modelos Moleculares , Montagem de Vírus , Replicação ViralRESUMO
Target-site selection by retroviral integrase (IN) proteins profoundly affects viral pathogenesis. We describe the solution nuclear magnetic resonance structure of the Moloney murine leukemia virus IN (M-MLV) C-terminal domain (CTD) and a structural homology model of the catalytic core domain (CCD). In solution, the isolated MLV IN CTD adopts an SH3 domain fold flanked by a C-terminal unstructured tail. We generated a concordant MLV IN CCD structural model using SWISS-MODEL, MMM-tree and I-TASSER. Using the X-ray crystal structure of the prototype foamy virus IN target capture complex together with our MLV domain structures, residues within the CCD α2 helical region and the CTD ß1-ß2 loop were predicted to bind target DNA. The role of these residues was analyzed in vivo through point mutants and motif interchanges. Viable viruses with substitutions at the IN CCD α2 helical region and the CTD ß1-ß2 loop were tested for effects on integration target site selection. Next-generation sequencing and analysis of integration target sequences indicate that the CCD α2 helical region, in particular P187, interacts with the sequences distal to the scissile bonds whereas the CTD ß1-ß2 loop binds to residues proximal to it. These findings validate our structural model and disclose IN-DNA interactions relevant to target site selection.
Assuntos
Integrases/química , Vírus da Leucemia Murina de Moloney/enzimologia , Proteínas Virais/química , Domínio Catalítico , DNA/química , DNA/metabolismo , Integrases/genética , Integrases/metabolismo , Modelos Moleculares , Mutação , Ligação Proteica , Estrutura Terciária de Proteína , Análise de Sequência de DNA , Proteínas Virais/metabolismoRESUMO
We report alterations to the murine leukemia virus (MLV) integrase (IN) protein that successfully result in decreasing its integration frequency at transcription start sites and CpG islands, thereby reducing the potential for insertional activation. The host bromo and extraterminal (BET) proteins Brd2, 3 and 4 interact with the MLV IN protein primarily through the BET protein ET domain. Using solution NMR, protein interaction studies, and next generation sequencing, we show that the C-terminal tail peptide region of MLV IN is important for the interaction with BET proteins and that disruption of this interaction through truncation mutations affects the global targeting profile of MLV vectors. The use of the unstructured tails of gammaretroviral INs to direct association with complexes at active promoters parallels that used by histones and RNA polymerase II. Viruses bearing MLV IN C-terminal truncations can provide new avenues to improve the safety profile of gammaretroviral vectors for human gene therapy.
Assuntos
Integrases/química , Vírus da Leucemia Murina/genética , Proteínas de Ligação a RNA/química , Proteínas Virais/química , Integração Viral , Sequência de Aminoácidos , Sítios de Ligação , Ilhas de CpG , Células HEK293 , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Análise de Sequência de DNA , Deleção de Sequência , Fatores de Transcrição , Sítio de Iniciação de TranscriçãoRESUMO
The p12 protein of murine leukemia virus (MuLV) group-specific antigen (Gag) is associated with the preintegration complex, and mutants of p12 (PM14) show defects in nuclear entry or retention. Here we show that p12 proteins engineered to encode peptide sequences derived from known viral tethering proteins can direct chromatin binding during the early phase of viral replication and rescue a lethal p12-PM14 mutant. Peptides studied included segments of Kaposi sarcoma herpesvirus latency-associated nuclear antigen (LANA)(1-23), human papillomavirus 8 E2, and prototype foamy virus chromatin-binding sequences. Amino acid substitutions in Kaposi sarcoma herpesvirus LANA and prototype foamy virus chromatin-binding sequences that blocked nucleosome association failed to rescue MuLV p12-PM14. Rescue by a larger LANA peptide, LANA(1-32), required second-site mutations that are predicted to reduce peptide binding affinity to chromosomes, suggesting that excessively high binding affinity interfered with Gag/p12 function. This is supported by confocal microscopy of chimeric p12-GFP fusion constructs showing the reverted proteins had weaker association to condensed mitotic chromosomes. Analysis of the integration-site selection of these chimeric viruses showed no significant change in integration profile compared with wild-type MuLV, suggesting release of the tethered p12 post mitosis, before viral integration.
Assuntos
DNA Viral/genética , Produtos do Gene gag/genética , Vírus da Leucemia Murina/genética , Integração Viral/genética , Replicação Viral/genética , Animais , Antígenos Virais/metabolismo , Sequência de Bases , Western Blotting , Linhagem Celular , Cromatina/metabolismo , Clonagem Molecular , Cães , Recuperação de Fluorescência Após Fotodegradação , Proteínas de Fluorescência Verde/metabolismo , Humanos , Imunoprecipitação , Microscopia Confocal , Dados de Sequência Molecular , Mutação/genética , Proteínas Nucleares/metabolismo , Proteínas Oncogênicas Virais/metabolismo , Engenharia de Proteínas , Análise de Sequência de DNA , Transativadores/metabolismoRESUMO
Retrons are a retroelement class found in diverse prokaryotes that can be adapted to augment CRISPR-Cas9 genome engineering technology to efficiently rewrite short stretches of genetic information in bacteria and yeast; however, efficiency in human cells has been limited by unknown factors. We identified non-coding RNA (ncRNA) instability and impaired Cas9 activity as major contributors to poor retron editor efficiency. We re-engineered the Eco1 ncRNA to incorporate an exoribonuclease-resistant RNA pseudoknot from the Zika virus 3' UTR and devised an RNA processing strategy using Csy4 ribonuclease to liberate the sgRNA and ncRNA. These modifications yielded a ncRNA with 5'- and 3'-end protection and an sgRNA with minimal 5' extension. This strategy increased steady-state ncRNA levels and rescued Cas9 activity leading to enhanced efficiency of the Eco1 retron editor in human cells. The enhanced Eco1 retron editor enabled the insertion of missense mutations in human cells from a single integrated lentivirus, thereby ensuring genotype-phenotype linkage over multiple cell divisions. This work reveals a previously unappreciated role for ncRNA stability in retron editor efficiency in human cells. Here we present an enhanced Eco1 retron editor that enables efficient introduction of missense mutations in human cells from a single heritable genome copy.