RESUMO
The Human immunodeficiency virus-1 (HIV-1) accessory protein Vpu modulates numerous proteins, including the host proteins CD4 and BST-2/tetherin. Vpu interacts with the Skp, Cullin, F-Box (SCF) ubiquitin ligase through interactions with the F-Box protein ßTrCP (1 and/or 2). This interaction is dependent on phosphorylation of S52,56 in Vpu. Mutation of S52,56, or inhibition of the SCF, abolishes most Vpu activity against CD4 and partly reduces activity against BST-2/tetherin. Recently, Vpu has also been reported to interact with the clathrin adapter proteins AP-1 and AP-2, and these interactions were also found to be required for BST-2/tetherin antagonism in an S52,56 -dependent manner. In assays where HIV-1 is pseudotyped with gibbon ape leukemia virus (GaLV Env), Vpu has also been found to prevent GaLV Env from being incorporated into viral particles, but the mechanism for this antagonism is not fully understood. To clarify the role of the ßTrCPs in Vpu function we used CRISPR/Cas9 to generate a clonal cell line lacking both ßTrCP-1 and -2. Vpu activity against CD4 and GaLV Env was abolished in this cell line, and activity against BST-2/tetherin reduced significantly. Mutation of the S52,56 residues no longer affected Vpu activity against BST-2/tetherin in this cell line. These data suggest that the primary role of the S52,56 residues in antagonism of CD4, GaLV Env, and BST-2/tetherin is to recruit the SCF/ßTrCP ubiquitin ligase.
Assuntos
Antígenos CD/metabolismo , Antígenos CD4/metabolismo , Infecções por HIV/metabolismo , HIV-1/metabolismo , Proteínas do Vírus da Imunodeficiência Humana/metabolismo , Proteínas do Envelope Viral/metabolismo , Proteínas Virais Reguladoras e Acessórias/metabolismo , Proteínas Contendo Repetições de beta-Transducina/metabolismo , Antígenos CD/genética , Antígenos CD4/genética , Linhagem Celular , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/metabolismo , Infecções por HIV/genética , Infecções por HIV/virologia , HIV-1/genética , Interações Hospedeiro-Patógeno , Proteínas do Vírus da Imunodeficiência Humana/genética , Humanos , Vírus da Leucemia do Macaco Gibão/genética , Vírus da Leucemia do Macaco Gibão/metabolismo , Ligação Proteica , Proteínas do Envelope Viral/genética , Proteínas Virais Reguladoras e Acessórias/genética , Proteínas Contendo Repetições de beta-Transducina/genéticaRESUMO
Type I interferon (IFN)-stimulated genes (ISGs) have critical roles in inhibiting virus replication and dissemination. Despite advances in understanding the molecular basis of ISG restriction, the antiviral mechanisms of many remain unclear. The 20-kDa ISG ISG20 is a nuclear 3'-5' exonuclease with preference for single-stranded RNA (ssRNA) and has been implicated in the IFN-mediated restriction of several RNA viruses. Although the exonuclease activity of ISG20 has been shown to degrade viral RNA in vitro, evidence has yet to be presented that virus inhibition in cells requires this activity. Here, we utilized a combination of an inducible, ectopic expression system and newly generated Isg20-/- mice to investigate mechanisms and consequences of ISG20-mediated restriction. Ectopically expressed ISG20 localized primarily to Cajal bodies in the nucleus and restricted replication of chikungunya and Venezuelan equine encephalitis viruses. Although restriction by ISG20 was associated with inhibition of translation of infecting genomic RNA, degradation of viral RNAs was not observed. Instead, translation inhibition of viral RNA was associated with ISG20-induced upregulation of over 100 other genes, many of which encode known antiviral effectors. ISG20 modulated the production of IFIT1, an ISG that suppresses translation of alphavirus RNAs. Consistent with this observation, the pathogenicity of IFIT1-sensitive alphaviruses was increased in Isg20-/- mice compared to that of wild-type viruses but not in cells ectopically expressing ISG20. Our findings establish an indirect role for ISG20 in the early restriction of RNA virus replication by regulating expression of other ISGs that inhibit translation and possibly other activities in the replication cycle.IMPORTANCE The host immune responses to infection lead to the production of type I interferon (IFN), and the upregulation of interferon-stimulated genes (ISGs) reduces virus replication and virus dissemination within a host. Ectopic expression of the interferon-induced 20-kDa exonuclease ISG20 suppressed replication of chikungunya virus and Venezuelan equine encephalitis virus, two mosquito-vectored RNA alphaviruses. Since the replication of alphavirus genomes occurs exclusively in the cytoplasm, the mechanism of nucleus-localized ISG20 inhibition of replication is unclear. In this study, we determined that ISG20 acts as a master regulator of over 100 genes, many of which are ISGs. Specifically, ISG20 upregulated IFIT1 genes and inhibited translation of the alphavirus genome. Furthermore, IFIT1-sensitive alphavirus replication was increased in Isg20-/- mice compared to the replication of wild-type viruses but not in cells ectopically expressing ISG20. We propose that ISG20 acts as an indirect regulator of RNA virus replication in the cytoplasm through the upregulation of many other ISGs.
Assuntos
Exonucleases/genética , Exorribonucleases/genética , Interferon Tipo I/genética , Replicação Viral , Proteínas Adaptadoras de Transdução de Sinal , Animais , Proteínas de Transporte/genética , Linhagem Celular , Vírus Chikungunya/fisiologia , Vírus da Encefalite Equina Venezuelana/fisiologia , Feminino , Interações Hospedeiro-Patógeno , Imunidade Inata , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia de Fluorescência , RNA Viral/análise , Proteínas de Ligação a RNA , Regulação para CimaRESUMO
Although the outcome of flavivirus infection can vary from asymptomatic to lethal, environmental factors modulating disease severity are poorly defined. Here, we observed increased susceptibility of mice to severe West Nile (WNV), Dengue, and Zika virus infections after treatment with oral antibiotics (Abx) that depleted the gut microbiota. Abx treatment impaired the development of optimal T cell responses, with decreased levels of WNV-specific CD8+ T cells associated with increased infection and immunopathology. Abx treatments that resulted in enhanced WNV susceptibility generated changes in the overall structure of the gut bacterial community and in the abundance of specific bacterial taxa. As little as 3 days of treatment with ampicillin was sufficient to alter host immunity and WNV outcome. Our results identify oral Abx therapy as a potential environmental determinant of systemic viral disease, and they raise the possibility that perturbation of the gut microbiota may have deleterious consequences for subsequent flavivirus infections.
Assuntos
Antibacterianos/efeitos adversos , Flavivirus/isolamento & purificação , Infecção por Zika virus/tratamento farmacológico , Administração Oral , Aedes , Ampicilina/efeitos adversos , Ampicilina/farmacologia , Animais , Antibacterianos/farmacologia , Ceco/efeitos dos fármacos , Ceco/microbiologia , Chlorocebus aethiops , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microbiota/efeitos dos fármacos , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Células Vero , Infecção por Zika virus/imunologia , Infecção por Zika virus/microbiologia , Infecção por Zika virus/patologiaRESUMO
Innate and adaptive immune cells modulate heart failure pathogenesis during viral myocarditis, yet their identities and functions remain poorly defined. We utilized a combination of genetic fate mapping, parabiotic, transcriptional, and functional analyses and demonstrated that the heart contained two major conventional dendritic cell (cDC) subsets, CD103+ and CD11b+, which differentially relied on local proliferation and precursor recruitment to maintain their tissue residency. Following viral infection of the myocardium, cDCs accumulated in the heart coincident with monocyte infiltration and loss of resident reparative embryonic-derived cardiac macrophages. cDC depletion abrogated antigen-specific CD8+ T cell proliferative expansion, transforming subclinical cardiac injury to overt heart failure. These effects were mediated by CD103+ cDCs, which are dependent on the transcription factor BATF3 for their development. Collectively, our findings identified resident cardiac cDC subsets, defined their origins, and revealed an essential role for CD103+ cDCs in antigen-specific T cell responses during subclinical viral myocarditis.
Assuntos
Antígenos CD/análise , Infecções por Cardiovirus/complicações , Células Dendríticas/imunologia , Vírus da Encefalomiocardite , Insuficiência Cardíaca/prevenção & controle , Cadeias alfa de Integrinas/análise , Miocardite/complicações , Animais , Antígeno CD11b/análise , Linfócitos T CD8-Positivos/imunologia , Infecções por Cardiovirus/imunologia , Movimento Celular , Feminino , Hematopoese , Memória Imunológica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Miocardite/imunologia , Receptores CCR2/fisiologiaRESUMO
UNLABELLED: The mammalian host responds to viral infections by inducing expression of hundreds of interferon-stimulated genes (ISGs). While the functional significance of many ISGs has yet to be determined, their cell type and temporal nature of expression suggest unique activities against specific pathogens. Using a combination of ectopic expression and gene silencing approaches in cell culture, we previously identified Ifi27l2a as a candidate antiviral ISG within neuronal subsets of the central nervous system (CNS) that restricts infection by West Nile virus (WNV), an encephalitic flavivirus of global concern. To investigate the physiological relevance of Ifi27l2a in the context of viral infection, we generated Ifi27l2a(-/-) mice. Although adult mice lacking Ifi27l2a were more vulnerable to lethal WNV infection, the viral burden was greater only within the CNS, particularly in the brain stem, cerebellum, and spinal cord. Within neurons of the cerebellum and brain stem, in the context of WNV infection, a deficiency of Ifi27l2a was associated with less cell death, which likely contributed to sustained viral replication and higher titers in these regions. Infection studies in a primary cell culture revealed that Ifi27l2a(-/-) cerebellar granule cell neurons and macrophages but not cerebral cortical neurons, embryonic fibroblasts, or dendritic cells sustained higher levels of WNV infection than wild-type cells and that this difference was greater under conditions of beta interferon (IFN-ß) pretreatment. Collectively, these findings suggest that Ifi27l2a has an antiviral phenotype in subsets of cells and that at least some ISGs have specific inhibitory functions in restricted tissues. IMPORTANCE: The interferon-stimulated Ifi27l2a gene is expressed differentially within the central nervous system upon interferon stimulation or viral infection. Prior studies in cell culture suggested an antiviral role for Ifi27l2a during infection by West Nile virus (WNV). To characterize its antiviral activity in vivo, we generated mice with a targeted gene deletion of Ifi27l2a. Based on extensive virological analyses, we determined that Ifi27l2a protects mice from WNV-induced mortality by contributing to the control of infection of the hindbrain and spinal cord, possibly by regulating cell death of neurons. This antiviral activity was validated in granule cell neurons derived from the cerebellum and in macrophages but was not observed in other cell types. Collectively, these data suggest that Ifi27l2a contributes to innate immune restriction of WNV in a cell-type- and tissue-specific manner.
Assuntos
Imunidade Inata , Interferons/metabolismo , Proteínas/metabolismo , Vírus do Nilo Ocidental/imunologia , Animais , Encéfalo/patologia , Encéfalo/virologia , Células Cultivadas , Modelos Animais de Doenças , Feminino , Macrófagos/fisiologia , Macrófagos/virologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios/fisiologia , Neurônios/virologia , Proteínas/genética , Medula Espinal/patologia , Medula Espinal/virologia , Febre do Nilo Ocidental/patologia , Febre do Nilo Ocidental/virologiaRESUMO
BACKGROUND: Retroviruses can acquire not only their own glycoproteins as they bud from the cellular membrane, but also some cellular and foreign viral glycoproteins. Many of these non-native glycoproteins are actively recruited to budding virions, particularly other viral glycoproteins. This observation suggests that there may be a conserved mechanism underlying the recruitment of glycoproteins into viruses. If a conserved mechanism is used, diverse glycoproteins should localize to a single budding retroviral particle. On the other hand, if viral glycoproteins have divergent mechanisms for recruitment, the different glycoproteins could segregate into different particles. RESULTS: To determine if co-packaging occurs among different glycoproteins, we designed an assay that combines virion antibody capture and a determination of infectivity based on a luciferase reporter. Virions were bound to a plate with an antibody against one glycoprotein, and then the infectivity was measured with cells that allow entry only with a second glycoprotein. We tested pairings of glycoproteins from HIV, murine leukemia virus (MLV), Rous sarcoma virus (RSV), vesicular stomatitis virus (VSV), and Ebola virus. The results showed that glycoproteins that were actively recruited into virions were co-packaged efficiently with each other. We also tested cellular proteins and found CD4 also had a similar correlation between active recruitment and efficient co-packaging, but other cellular proteins did not. CONCLUSION: Glycoproteins that are actively incorporated into HIV-1 virions are efficiently co-packaged into the same virus particles, suggesting that the same general mechanism for recruitment may act in many viruses.
Assuntos
Antígenos CD4/metabolismo , Glicoproteínas/metabolismo , HIV-1/fisiologia , Proteínas Virais/metabolismo , Montagem de Vírus , Linhagem Celular , HumanosRESUMO
During human immunodeficiency virus-1 (HIV-1) assembly, the host proteins CD4 (the HIV-1 receptor) and tetherin (an interferon stimulated anti-viral protein) both reduce viral fitness. The HIV-1 accessory gene Vpu counteracts both of these proteins, but it is thought to do so through two distinct mechanisms. Modulation of CD4 likely occurs through proteasomal degradation from the endoplasmic reticulum. The exact mechanism of tetherin modulation is less clear, with possible roles for degradation and alteration of protein transport to the plasma membrane. Most investigations of Vpu function have used different assays for CD4 and tetherin. In addition, many of these investigations used exogenously expressed Vpu, which could result in variable expression levels. Thus, few studies have investigated these two Vpu functions in parallel assays, making direct comparisons difficult. Here, we present results from a rapid assay used to simultaneously investigate Vpu-targeting of both tetherin and a viral glycoprotein, gibbon ape leukemia virus envelope (GaLV Env). We previously reported that Vpu modulates GaLV Env and prevents its incorporation into HIV-1 particles through a recognition motif similar to that found in CD4. Using this assay, we performed a comprehensive mutagenic scan of Vpu in its native proviral context to identify features required for both types of activity. We observed considerable overlap in the Vpu sequences required to modulate tetherin and GaLV Env. We found that features in the cytoplasmic tail of Vpu, specifically within the cytoplasmic tail hinge region, were required for modulation of both tetherin and GaLV Env. Interestingly, these same regions features have been determined to be critical for CD4 downmodulation. We also observed a role for the transmembrane domain in the restriction of tetherin, as previously reported, but not of GaLV Env. We propose that Vpu may target both proteins in a mechanistically similar manner, albeit in different cellular locations.
Assuntos
Antígenos CD/metabolismo , HIV-1/crescimento & desenvolvimento , Proteínas do Vírus da Imunodeficiência Humana/metabolismo , Vírus da Leucemia do Macaco Gibão/crescimento & desenvolvimento , Proteínas Mutantes/metabolismo , Domínios e Motivos de Interação entre Proteínas/fisiologia , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Virais Reguladoras e Acessórias/metabolismo , Sequência de Aminoácidos , Antígenos CD/genética , Citoplasma/metabolismo , Citometria de Fluxo , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/metabolismo , Células HEK293/metabolismo , Células HEK293/virologia , Proteínas do Vírus da Imunodeficiência Humana/genética , Humanos , Vírus da Leucemia do Macaco Gibão/genética , Dados de Sequência Molecular , Proteínas Mutantes/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Virais Reguladoras e Acessórias/genética , Vírion/metabolismo , Montagem de Vírus , Replicação ViralRESUMO
HIV-1 efficiently forms pseudotyped particles with many gammaretrovirus glycoproteins, such as Friend murine leukemia virus (F-MLV) Env, but not with the related gibbon ape leukemia virus (GaLV) Env or with a chimeric F-MLV Env with a GaLV cytoplasmic tail domain (CTD). This incompatibility is modulated by the HIV-1 accessory protein Vpu. Because the GaLV Env CTD does not resemble tetherin or CD4, the well-studied targets of Vpu, we sought to characterize the modular sequence in the GaLV Env CTD required for this restriction in the presence of Vpu. Using a systematic mutagenesis scan, we determined that the motif that makes GaLV Env sensitive to Vpu is INxxIxxVKxxVxRxK. This region in the CTD of GaLV Env is predicted to form a helix. Mutations in the CTD that would break this helix abolish sensitivity to Vpu. Although many of these positions can be replaced with amino acids with similar biophysical properties without disrupting the Vpu sensitivity, the final lysine residue is required. This Vpu sensitivity sequence appears to be modular, as the unrelated Rous sarcoma virus (RSV) Env can be made Vpu sensitive by replacing its CTD with the GaLV Env CTD. In addition, F-MLV Env can be made Vpu sensitive by mutating two amino acids in its cytoplasmic tail to make it resemble more closely the Vpu sensitivity motif. Surprisingly, the core components of this Vpu sensitivity sequence are also present in the host surface protein CD4, which is also targeted by Vpu through its CTD.
Assuntos
HIV-1/crescimento & desenvolvimento , Proteínas do Vírus da Imunodeficiência Humana/metabolismo , Vírus da Leucemia do Macaco Gibão/crescimento & desenvolvimento , Proteínas do Envelope Viral/antagonistas & inibidores , Proteínas do Envelope Viral/metabolismo , Proteínas Virais Reguladoras e Acessórias/metabolismo , Análise Mutacional de DNA , Vírus da Leucemia Murina de Friend/genética , Vírus da Leucemia Murina de Friend/crescimento & desenvolvimento , Humanos , Vírus da Leucemia do Macaco Gibão/genética , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Recombinação Genética , Proteínas do Envelope Viral/genéticaRESUMO
The cytoplasmic tail domain (CTD) of retroviral envelope (Env) proteins has been implicated in modulating Env incorporation into viral particles. We generated a panel of murine leukemia virus (MLV) Env mutants and analyzed their ability to be recruited to human immunodeficiency virus-1 (HIV-1) assembly sites. Surprisingly, the entire CTD was dispensable for recruitment to assembly sites, but a mutation that disrupted the furin cleavage site in Env abolished recruitment. To determine if MLV Env can show selectivity for homologous assembly sites, cells were co-transfected with both HIV-1 and MLV assembly components along with each MLV Env construct and assayed for infectious particle production. MLV Env selectively formed infectious particles with the MLV components at the expense of infectious HIV-1 infectious particle production, but truncation of the CTD progressively reduced this selectivity. Collectively these data suggest that there are two separable mechanisms that govern MLV Env recruitment to viral assembly sites.
Assuntos
Vírus da Leucemia Murina/metabolismo , Proteínas do Envelope Viral/metabolismo , Vírion/metabolismo , Montagem de Vírus , Animais , Linhagem Celular , Citoplasma/metabolismo , Regulação Viral da Expressão Gênica , HIV-1/genética , HIV-1/metabolismo , Proteínas do Vírus da Imunodeficiência Humana/genética , Proteínas do Vírus da Imunodeficiência Humana/metabolismo , Humanos , Vírus da Leucemia Murina/genética , Mutação , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/genética , Vírion/genéticaRESUMO
The Env protein from gibbon ape leukemia virus (GaLV) has been shown to be incompatible with human immunodeficiency virus type 1 (HIV-1) in the production of infectious pseudotyped particles. This incompatibility has been mapped to the C-terminal cytoplasmic tail of GaLV Env. Surprisingly, we found that the HIV-1 accessory protein Vpu modulates this incompatibility. The infectivity of HIV-1 pseudotyped with murine leukemia virus (MLV) Env was not affected by Vpu. However, the infectivity of HIV-1 pseudotyped with an MLV Env with the cytoplasmic tail from GaLV Env (MLV/GaLV Env) was restricted 50- to 100-fold by Vpu. A Vpu mutant containing a scrambled membrane-spanning domain, Vpu(RD), was still able to restrict MLV/GaLV Env, but mutation of the serine residues at positions 52 and 56 completely alleviated the restriction. Loss of infectivity appeared to be caused by reduced MLV/GaLV Env incorporation into viral particles. The mechanism of this downmodulation appears to be distinct from Vpu-mediated CD4 downmodulation because Vpu-expressing cells that failed to produce infectious HIV-1 particles nonetheless continued to display robust surface MLV/GaLV Env expression. In addition, if MLV and HIV-1 were simultaneously introduced into the same cells, only the HIV-1 particle infectivity was restricted by Vpu. Collectively, these data suggest that Vpu modulates the cellular distribution of MLV/GaLV Env, preventing its recruitment to HIV-1 budding sites.