RESUMO
Human T-cell leukemia virus type 1 (HTLV-1) has two late domain (LD) motifs, PPPY and PTAP, which are important for viral budding. Mutations in the PPPY motif are more deleterious for viral release than changes in the PTAP motif. Several reports have shown that the interaction of PPPY with the WW domains of a Nedd4 (neuronal precursor cell-expressed developmentally down-regulated-4) family ubiquitin ligase (UL) is a critical event in virus release. We tested nine members of the Nedd4 family ULs and found that ITCH is the main contributor to HTLV-1 budding. ITCH overexpression strongly inhibited release and infectivity of wild-type (wt) HTLV-1, but rescued the release of infectious virions with certain mutations in the PPPY motif. Electron microscopy showed either fewer or misshapen virus particles when wt HTLV-1 was produced in the presence of overexpressed ITCH, whereas mutants with changes in the PPPY motif yielded normal looking particles at wt level. The other ULs had significantly weaker or no effects on HTLV-1 release and infectivity except for SMURF-1, which caused enhanced release of wt and all PPPY(-) mutant particles. These particles were poorly infectious and showed abnormal morphology by electron microscopy. Budding and infectivity defects due to overexpression of ITCH and SMURF-1 were correlated with higher than normal ubiquitination of Gag. Only silencing of ITCH, but not of WWP1, WWP2, and Nedd4, resulted in a reduction of HTLV-1 budding from 293T cells. The binding efficiencies between the HTLV-1 LD and WW domains of different ULs as measured by mammalian two-hybrid interaction did not correlate with the strength of their effect on HTLV-1 budding.
Assuntos
Vírus Linfotrópico T Tipo 1 Humano/metabolismo , Proteínas Repressoras/fisiologia , Ubiquitina-Proteína Ligases/fisiologia , Liberação de Vírus , Motivos de Aminoácidos/genética , Linhagem Celular , Complexos Endossomais de Distribuição Requeridos para Transporte , Produtos do Gene gag/metabolismo , Vírus Linfotrópico T Tipo 1 Humano/genética , Humanos , Microscopia Eletrônica , Ubiquitina-Proteína Ligases Nedd4 , UbiquitinaçãoRESUMO
Human T-cell leukemia virus type 1 (HTLV-1) infection and transformation are associated with an incremental switch in the expression of the Src-related protein tyrosine kinases Lck and Lyn. We examined the physical and functional interactions of Lyn with receptors and signal transduction proteins in HTLV-1-infected T cells. Lyn coimmunoprecipitates with the interleukin-2 beta receptor (IL-2Rß) and JAK3 proteins; however, the association of Lyn with the IL-2Rß and Lyn kinase activity was independent of IL-2 stimulation. Phosphorylation of Janus kinase 3 (JAK3) and signal transducers and activator of transcription 5 (STAT5) proteins was reduced by treatment of cells with the Src kinase inhibitor PP2 or by ectopic expression of a dominant negative Lyn kinase protein.
Assuntos
Transformação Celular Viral , Vírus Linfotrópico T Tipo 1 Humano/patogenicidade , Subunidade beta de Receptor de Interleucina-2/metabolismo , Janus Quinase 3/metabolismo , Fatores de Transcrição STAT/metabolismo , Linfócitos T/virologia , Quinases da Família src/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Imunoprecipitação , Fosforilação , Processamento de Proteína Pós-TraducionalRESUMO
The human immunodeficiency virus type 1 (HIV-1) Gag matrix (MA) domain facilitates Gag targeting and binding to the plasma membrane (PM) during virus assembly. Interaction with a PM phospholipid, phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P(2)], plays a key role in these MA functions. Previous studies showed that overexpression of polyphosphoinositide 5-phosphatase IV (5ptaseIV), which depletes cellular PI(4,5)P(2), mislocalizes HIV-1 Gag to the cytosol and greatly reduces HIV-1 release efficiency. In this study, we sought to determine the role of the MA-PI(4,5)P(2) interaction in Gag localization and membrane binding of a deltaretrovirus, human T-lymphotropic virus type 1 (HTLV-1). We compared the chimeric HIV-1 Gag (HTMA), in which MA was replaced with HTLV-1 MA, with wild-type HIV-1 and HTLV-1 Gag for PI(4,5)P(2) dependence. Our results demonstrate that, unlike HIV-1 Gag, subcellular localization of and VLP release by HTLV-1 and HTMA Gag were minimally sensitive to 5ptaseIV overexpression. These results suggest that the interaction of HTLV-1 MA with PI(4,5)P(2) is not essential for HTLV-1 particle assembly. Furthermore, liposome-binding analyses showed that both HTLV-1 and HTMA Gag can bind membrane efficiently even in the absence of PI(4,5)P(2). Efficient HTLV-1 Gag binding to liposomes was largely driven by electrostatic interaction, unlike that of HIV-1 Gag, which required specific interaction with PI(4,5)P(2). Furthermore, membrane binding of HTLV-1 Gag in vitro was not suppressed by RNA, in contrast to HIV-1 Gag. Altogether, our data suggest that Gag targeting and membrane binding mediated by HTLV-1 MA does not require PI(4,5)P(2) and that distinct mechanisms regulate HIV-1 and HTLV-1 Gag membrane binding.
Assuntos
Produtos do Gene gag/metabolismo , Vírus Linfotrópico T Tipo 1 Humano/fisiologia , Fosfatidilinositol 4,5-Difosfato/metabolismo , Proteínas da Matriz Viral/metabolismo , Liberação de Vírus , Membrana Celular/metabolismo , HIV-1/genética , Vírus Linfotrópico T Tipo 1 Humano/genética , Humanos , Recombinação GenéticaRESUMO
Human APOBEC3B (A3B) has been described as a potent inhibitor of retroviral infection and retrotransposition. However, we found that the predominantly nuclear A3B only weakly restricted infection by HIV-1, HIV-1Δvif, and human T-cell leukemia virus type 1 (HTLV-1), while significantly inhibiting LINE-1 retrotransposition. The chimeric construct A3G/B, in which the first 60 amino acids of A3B were replaced with those of A3G, restricted HIV-1, HIV-1Δvif, and HTLV-1 infection, as well as LINE-1 retrotransposition. In contrast to the exclusively cytoplasmic A3G, which is inactive against LINE-1 retrotransposition, the A3G/B protein, while localized mainly to the cytoplasm, was also present in the nucleus. Further mutational analysis revealed that residues 18, 19, 22, and 24 in A3B were the major determinants for nuclear versus cytoplasmic localization and antiretroviral activity. HIV-1Δvif packages A3G, A3B, and A3G/B into particles with close-to-equal efficiencies. Mutation E68Q or E255Q in the active centers of A3G/B resulted in loss of the inhibitory activity against HIV-1Δvif, while not affecting activity against LINE-1 retrotransposition. The low inhibition of HIV-1Δvif by A3B correlated with a low rate of G-to-A hypermutation. In contrast, viruses that had been exposed to A3G/B showed a high number of G-to-A transitions. The mutation pattern was similar to that previously reported for A3B, with a preference for the GA context. In summary, these observations suggest that changing 4 residues in the amino terminus of A3B not only retargets the protein from the nucleus to the cytoplasm but also enhances its ability to restrict HIV while retaining inhibition of retrotransposition.
Assuntos
Antivirais/imunologia , Citidina Desaminase/imunologia , HIV-1/imunologia , Vírus Linfotrópico T Tipo 1 Humano/imunologia , Substituição de Aminoácidos/genética , Antivirais/metabolismo , Linhagem Celular , Núcleo Celular/química , Citidina Desaminase/genética , Citidina Desaminase/metabolismo , Citoplasma/química , HIV-1/crescimento & desenvolvimento , Vírus Linfotrópico T Tipo 1 Humano/crescimento & desenvolvimento , Humanos , Antígenos de Histocompatibilidade Menor , Proteínas Mutantes/metabolismoRESUMO
We have developed an efficient method to quantify cell-to-cell infection with single-cycle, replication dependent reporter vectors. This system was used to examine the mechanisms of infection with HTLV-1 and HIV-1 vectors in lymphocyte cell lines. Effector cells transfected with reporter vector, packaging vector, and Env expression plasmid produced virus-like particles that transduced reporter gene activity into cocultured target cells with zero background. Reporter gene expression was detected exclusively in target cells and required an Env-expression plasmid and a viral packaging vector, which provided essential structural and enzymatic proteins for virus replication. Cell-cell fusion did not contribute to infection, as reporter protein was rarely detected in syncytia. Coculture of transfected Jurkat T cells and target Raji/CD4 B cells enhanced HIV-1 infection two fold and HTLV-1 infection ten thousand fold in comparison with cell-free infection of Raji/CD4 cells. Agents that interfere with actin and tubulin polymerization strongly inhibited HTLV-1 and modestly decreased HIV-1 cell-to-cell infection, an indication that cytoskeletal remodeling was more important for HTLV-1 transmission. Time course studies showed that HTLV-1 transmission occurred very rapidly after cell mixing, whereas slower kinetics of HIV-1 coculture infection implies a different mechanism of infectious transmission. HTLV-1 Tax was demonstrated to play an important role in altering cell-cell interactions that enhance virus infection and replication. Interestingly, superantigen-induced synapses between Jurkat cells and Raji/CD4 cells did not enhance infection for either HTLV-1 or HIV-1. In general, the dependence on cell-to-cell infection was determined by the virus, the effector and target cell types, and by the nature of the cell-cell interaction.
Assuntos
Comunicação Celular/fisiologia , Infecções por Deltaretrovirus/transmissão , Vetores Genéticos , Infecções por HIV/transmissão , Linfócitos/virologia , Vírion/fisiologia , Separação Celular , Técnicas de Cocultura , Citometria de Fluxo , Genes Reporter , HIV-1/fisiologia , Vírus Linfotrópico T Tipo 1 Humano/fisiologia , Humanos , Células Jurkat , Transfecção , Replicação ViralRESUMO
We have investigated the underlying mechanism by which direct cell-cell contact enhances the efficiency of cell-to-cell transmission of retroviruses. Applying 4D imaging to a model retrovirus, the murine leukemia virus, we directly monitor and quantify sequential assembly, release, and transmission events for individual viral particles as they happen in living cells. We demonstrate that de novo assembly is highly polarized towards zones of cell-cell contact. Viruses assembled approximately 10-fold more frequently at zones of cell contact with no change in assembly kinetics. Gag proteins were drawn to adhesive zones formed by viral Env glycoprotein and its cognate receptor to promote virus assembly at cell-cell contact. This process was dependent on the cytoplasmic tail of viral Env. Env lacking the cytoplasmic tail while still allowing for contact formation, failed to direct virus assembly towards contact sites. Our data describe a novel role for the viral Env glycoprotein in establishing cell-cell adhesion and polarization of assembly prior to becoming a fusion protein to allow virus entry into cells.
Assuntos
Vírus da Leucemia Murina/metabolismo , Animais , Células COS , Adesão Celular , Células Cultivadas , Chlorocebus aethiops , Citoplasma/metabolismo , Produtos do Gene gag/metabolismo , Humanos , Cinética , Vírus da Leucemia Murina/patogenicidade , Proteínas do Envelope Viral/metabolismo , Ligação ViralRESUMO
Human T-lymphotropic virus type 1 (HTLV-1) envelope (Env) glycoprotein mediates binding of the virus to its receptor on the surface of target cells and subsequent fusion of virus and cell membranes. To better understand the mechanisms that control HTLV-1 Env trafficking and activity, we have examined two protein-protein interaction motifs in the cytoplasmic domain of Env. One is the sequence YSLI, which matches the consensus YXXPhi motifs that are known to interact with various adaptor protein complexes; the other is the sequence ESSL at the C terminus of Env, which matches the consensus PDZ-binding motif. We show here that mutations that destroy the YXXPhi motif increased Env expression on the cell surface and increased cell-cell fusion activity. In contrast, mutation of the PDZ-binding motif greatly diminished Env expression in cells, which could be restored to wild-type levels either by mutating the YXXPhi motif or by silencing AP2 and AP3, suggesting that interactions with PDZ proteins oppose an Env degradation pathway mediated by AP2 and AP3. Silencing of the PDZ protein hDlg1 did not affect Env expression, suggesting that hDlg1 is not a binding partner for Env. Substitution of the YSLI sequence in HTLV-1 Env with YXXPhi elements from other cell or virus membrane-spanning proteins resulted in alterations in Env accumulation in cells, incorporation into virions, and virion infectivity. Env variants containing YXXPhi motifs that are predicted to have high-affinity interaction with AP2 accumulated to lower steady-state levels. Interestingly, mutations that destroy the YXXPhi motif resulted in viruses that were not infectious by cell-free or cell-associated routes of infection. Unlike YXXPhi, the function of the PDZ-binding motif manifests itself only in the producer cells; AP2 silencing restored the incorporation of PDZ-deficient Env into virus-like particles (VLPs) and the infectivity of these VLPs to wild-type levels.
Assuntos
Motivos de Aminoácidos/genética , Produtos do Gene env/metabolismo , Vírus Linfotrópico T Tipo 1 Humano , Domínios PDZ , Tirosina/genética , Complexo 2 de Proteínas Adaptadoras/genética , Complexo 2 de Proteínas Adaptadoras/metabolismo , Complexo 3 de Proteínas Adaptadoras/genética , Complexo 3 de Proteínas Adaptadoras/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Fusão Celular , Linhagem Celular , Proteína 1 Homóloga a Discs-Large , Produtos do Gene env/genética , Vírus Linfotrópico T Tipo 1 Humano/genética , Vírus Linfotrópico T Tipo 1 Humano/metabolismo , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , Domínios PDZ/genética , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Tirosina/metabolismoRESUMO
Human T-cell leukemia/lymphoma virus type 1 (HTLV-1) persists despite a vigorous virus-specific host immune response, and causes adult T-cell leukemia and lymphoma in approximately 2% of infected individuals. Here we report that HTLV-1 has evolved a genetic function to restrict its own replication by a novel post-transcriptional mechanism. The HTLV-1-encoded p30(II) is a nuclear-resident protein that binds to, and retains in the nucleus, the doubly spliced mRNA encoding the Tax and Rex proteins. Because Tex and Rex are positive regulators of viral gene expression, their inhibition by p30(II) reduces virion production. p30(II) inhibits virus expression by reducing Tax and Rex protein expression.
Assuntos
Regulação Viral da Expressão Gênica , Vírus Linfotrópico T Tipo 1 Humano/fisiologia , Proteínas dos Retroviridae/metabolismo , Replicação Viral/fisiologia , Linhagem Celular , Produtos do Gene rex/genética , Produtos do Gene rex/metabolismo , Genes Reporter , Genes pX , Vírus Linfotrópico T Tipo 1 Humano/genética , Humanos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas dos Retroviridae/genética , Transcrição GênicaRESUMO
Human T-cell leukemia virus (HTLV-1) Env carries a typical disulfide isomerization motif, C(225)XXC, in the C-terminal domain SU. Here we have tested whether this motif is used for isomerization of the intersubunit disulfide of Env and whether this rearrangement is required for membrane fusion. We introduced the C225A and C228A mutations into Env and found that the former but not the latter mutant matured into covalently linked SU-TM complexes in transfected cells. Next, we constructed a secreted Env ectodomain and showed that it underwent incubation-dependent intersubunit disulfide isomerization on target cells. However, the rearrangement was blocked by the C225A mutation, suggesting that C(225) carried the isomerization-active thiol. Still, it was possible to reduce the intersubunit disulfide of the native C225A ectodomain mutant with dithiothreitol (DTT). The importance of the CXXC-mediated disulfide isomerization for infection was studied using murine leukemia virus vectors pseudotyped with wild-type or C225A HTLV-1 Env. We found that the mutant Env blocked infection, but this could be rescued with DTT. The fusion activity was tested in a fusion-from-within assay using a coculture of rat XC target and transfected BHK-21 effector cells. We found that the mutation blocked polykaryon formation, but this could be reversed with DTT. Similar DTT-reversible inhibition of infection and fusion was observed when a membrane-impermeable alkylator was present during the infection/fusion incubation. We conclude that the fusion activity of HTLV-1 Env is controlled by an SU CXXC-mediated isomerization of the intersubunit disulfide. Thus, this extends the applicability of the isomerization model from gammaretroviruses to deltaretroviruses.
Assuntos
Dissulfetos/metabolismo , Produtos do Gene env/metabolismo , Vírus Linfotrópico T Tipo 1 Humano/fisiologia , Internalização do Vírus , Substituição de Aminoácidos/genética , Animais , Linhagem Celular , Cricetinae , Ditiotreitol/farmacologia , Produtos do Gene env/genética , Humanos , Mutação de Sentido Incorreto , Ratos , Substâncias Redutoras/farmacologiaRESUMO
APOBEC3 proteins are cytidine deaminases which help defend cells against retroviral infections. One antiviral mechanism involves deaminating dC residues in minus-strand DNA during reverse transcription, resulting in G-to-A mutations in the coding strand. We investigated the effects of mouse APOBEC3 (mA3) and human APOBEC3G (hA3G) upon Moloney murine leukemia virus (MLV). We find that mA3 inactivates MLV but is significantly less effective against MLV than is hA3G. In contrast, mA3 is as potent against human immunodeficiency virus type 1 (HIV-1, lacking the protective Vif protein) as is hA3G. The two APOBEC3 proteins are packaged to similar extents in MLV particles. Dose-response profiles imply that a single APOBEC3 molecule (or oligomer) is sufficient to inactivate an MLV particle. The inactivation of MLV by mA3 and hA3G is accompanied by relatively small reductions in the amount of viral DNA in infected cells. Although hA3G induces significant levels of G-to-A mutations in both MLV and HIV DNAs, and mA3 induces these mutations in HIV DNA, no such mutations were detected in DNA synthesized by MLV inactivated by mA3. Thus, MLV has apparently evolved to partially resist the antiviral effects of mA3 and to totally resist the ability of mA3 to induce G-to-A mutation in viral DNA. Unlike the resistance of HIV-1 and human T-cell leukemia virus type 1 to hA3G, the resistance of MLV to mA3 is not mediated by the exclusion of APOBEC from the virus particle. The nature of its resistance and the mechanism of inactivation of MLV by mA3 are completely unknown.
Assuntos
Citidina Desaminase/metabolismo , Citosina Desaminase/metabolismo , DNA Viral/genética , Vírus da Leucemia Murina de Moloney/metabolismo , Inativação de Vírus , Desaminases APOBEC , Animais , Sequência de Bases , Linhagem Celular , Citidina Desaminase/genética , Citosina Desaminase/genética , Primers do DNA/genética , Humanos , Immunoblotting , Camundongos , Dados de Sequência Molecular , Vírus da Leucemia Murina de Moloney/genética , Mutação/genética , Análise de Sequência de DNARESUMO
Retrovirus infection proceeds by attachment of the envelope glycoprotein to a cell surface receptor, followed by fusion of the viral and cellular membranes. Once in the cell, the viral enzymes and structural proteins form a replication complex that converts the single-stranded viral genomic RNA into a double-stranded DNA, which is then integrated into the host cell chromosome. For HTLV-1, these events are not well characterized. We have developed cell culture systems, infectious molecular clones, and viral vectors that can be used to characterize the mechanisms of HTLV-1 infection and replication. Infection with cell-free HTLV-1 virions is orders of magnitude less efficient compared with other retroviruses. This inefficiency is the result of a block in the replication process after the virion is bound to the cell surface. We are determining whether this block is conferred by the viral replication enzymes, results from the actions of cellular restriction factors, reflects the need for cell-cell contact, or is caused by a combination of these factors.
Assuntos
Infecções por HTLV-I/transmissão , Vírus Linfotrópico T Tipo 1 Humano/metabolismo , Vírus Linfotrópico T Tipo 1 Humano/fisiologia , Replicação Viral , Animais , Antivirais/farmacologia , Regulação Viral da Expressão Gênica , Humanos , Leucemia de Células T/virologia , DNA Polimerase Dirigida por RNA/metabolismoRESUMO
Tetherin is part of the cellular innate immunity and impedes cell-free transmission of viruses that bud from the plasma membrane by retaining them on the cell surface. Some viruses have evolved activities in different proteins such as Vpu (HIV-1), K-protein (KSHV), Nef (SIV) or Env (HIV-2) to downregulate tetherin and overcome its restriction. We found that chronically HTLV-1 infected T-cell lines express eightfold more tetherin than uninfected transformed T-cell lines suggesting that tetherin expression is not inhibited by the virus. We observed that even small amounts of exogenous tetherin caused the retention of HTLV-1 on the cell surface and severely reduced cell-free infectivity of HTLV-1, but that cell-cell transmission, which is more relevant for HTLV-1, was significantly less decreased. However, knock-down of tetherin expresssion resulted in a slight increase in cell-cell infection indicating that the protein does not enhance this route of transmission.
Assuntos
Antígenos CD/metabolismo , Linfócitos T CD4-Positivos/virologia , Vírus Linfotrópico T Tipo 1 Humano/fisiologia , Linfócitos T/virologia , Liberação de Vírus , Antígenos CD/genética , Linfócitos T CD4-Positivos/imunologia , Comunicação Celular , Linhagem Celular , Linhagem Celular Transformada , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/metabolismo , Vírus Linfotrópico T Tipo 1 Humano/crescimento & desenvolvimento , Humanos , Imunidade Inata , Interferência de RNA , RNA Interferente Pequeno , Linfócitos T/imunologiaRESUMO
Virus transmission can occur either by a cell-free mode through the extracellular space or by cell-to-cell transmission involving direct cell-to-cell contact. The factors that determine whether a virus spreads by either pathway are poorly understood. Here, we assessed the relative contribution of cell-free and cell-to-cell transmission to the spreading of the human immunodeficiency virus (HIV). We demonstrate that HIV can spread by a cell-free pathway if all the steps of the viral replication cycle are efficiently supported in highly permissive cells. However, when the cell-free path was systematically hindered at various steps, HIV transmission became contact-dependent. Cell-to-cell transmission overcame barriers introduced in the donor cell at the level of gene expression and surface retention by the restriction factor tetherin. Moreover, neutralizing antibodies that efficiently inhibit cell-free HIV were less effective against cell-to-cell transmitted virus. HIV cell-to-cell transmission also efficiently infected target T cells that were relatively poorly susceptible to cell-free HIV. Importantly, we demonstrate that the donor and target cell types influence critically the extent by which cell-to-cell transmission can overcome each barrier. Mechanistically, cell-to-cell transmission promoted HIV spread to more cells and infected target cells with a higher proviral content than observed for cell-free virus. Our data demonstrate that the frequently observed contact-dependent spread of HIV is the result of specific features in donor and target cell types, thus offering an explanation for conflicting reports on the extent of cell-to-cell transmission of HIV.
Assuntos
Comunicação Celular , Infecções por HIV/transmissão , HIV/patogenicidade , Anticorpos Neutralizantes/imunologia , Células Cultivadas , Técnicas de Cocultura , Regulação Viral da Expressão Gênica , Células HEK293 , HIV/fisiologia , Infecções por HIV/imunologia , Infecções por HIV/virologia , Humanos , Células Jurkat , Linfócitos T/virologia , Internalização do VírusRESUMO
The tetraspanin superfamily proteins play important roles in organizing membrane protein complexes, modulating integrin function, and controlling T cell adhesion. Tetraspanins such as CD82 contain two extracellular loops with its N terminus, C terminus, and inner loop exposed to the cytoplasm. The matrix (MA) domain of human T cell lymphotrophic virus, type 1 (HTLV-1), Gag interacts with the cytoplasmic face of the plasma membrane and is concentrated at tetraspanin-enriched microdomains. To understand the basis of this association, we generated site-directed mutations in the various domains of CD82 and used coimmunoprecipitation and colocalization approaches to examine interactions with HTLV-1 MA. The large extracellular loop of CD82, which is important for interactions with integrins, was not required for the association with HTLV-1 MA. The cytoplasmic N terminus and C terminus of CD82 were also dispensable for CD82-MA interactions. In contrast, mutations of conserved amino acids in the inner loop of CD82 or of palmitoylated cysteines that flank the inner loop diminished CD82 association with MA. HTLV-1 MA also interacted with the inner loop of CD81. Thus, association of HTLV-1 Gag with tetraspanin-enriched microdomains is mediated by the inner loops of CD81 and CD82.
Assuntos
Antígenos CD/química , Antígenos CD/fisiologia , Produtos do Gene gag/metabolismo , Vírus Linfotrópico T Tipo 1 Humano/química , Vírus Linfotrópico T Tipo 1 Humano/fisiologia , Proteína Kangai-1/química , Proteína Kangai-1/fisiologia , Sequência de Aminoácidos , Antígenos CD/genética , Linhagem Celular , Células HeLa , Vírus Linfotrópico T Tipo 1 Humano/genética , Humanos , Células Jurkat , Proteína Kangai-1/genética , Microdomínios da Membrana/química , Microdomínios da Membrana/genética , Microdomínios da Membrana/fisiologia , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Estrutura Terciária de Proteína/genética , Tetraspanina 28RESUMO
The PPPY motif in the matrix (MA) domain of human T-cell leukemia virus type 1 (HTLV-1) Gag associates with WWP1, a member of the HECT domain containing family of E3 ubiquitin ligases. Mutation of the PPPY motif arrests particle assembly at an early stage and abolishes ubiquitination of MA. Similar effects are seen when Gag is expressed in the presence of a truncated form of WWP1 that lacks the catalytically active HECT domain (C2WW). To understand the role of ubiquitination in budding, we mutated the four lysines in MA to arginines and identified lysine 74 as the unique site of ubiquitination. Virus-like particles produced by the K74R mutant did not contain ubiquitinated MA and showed a fourfold reduction in the release of infectious particles. Furthermore, the K74R mutation rendered assembly hypersensitive to C2WW inhibition; K74R Gag budding was inhibited at significantly lower levels of expression of C2WW compared with wild-type Gag. This finding indicates that the interaction between Gag and WWP1 is required for functions other than Gag ubiquitination. Additionally, we show that the PPPY(-) mutant Gag exerts a strong dominant-negative effect on the budding of wild-type Gag, further supporting the importance of recruitment of WWP1 to achieve particle assembly.
Assuntos
Produtos do Gene gag/metabolismo , Vírus Linfotrópico T Tipo 1 Humano/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina/metabolismo , Montagem de Vírus/fisiologia , Motivos de Aminoácidos/genética , Substituição de Aminoácidos , Produtos do Gene gag/genética , Células HeLa , Vírus Linfotrópico T Tipo 1 Humano/genética , Humanos , Mutação de Sentido Incorreto , Estrutura Terciária de Proteína/genética , Ubiquitina/genética , Ubiquitina-Proteína Ligases/genéticaRESUMO
Human T cell leukemia virus type 1 (HTLV-1) has evolved a remarkable strategy to thwart the antiviral effects of the cellular cytidine deaminase APOBEC3G (hA3G). HTLV-1 infects T lymphocytes in vivo, where, like HIV-1, it is likely to encounter hA3G. HIV-1 counteracts the innate antiviral activity of hA3G by producing an accessory protein, Vif, which hastens the degradation of hA3G. In contrast, HTLV-1 does not encode a Vif homologue; instead, HTLV-1 has evolved a cis-acting mechanism to prevent hA3G restriction. We demonstrate here that a peptide motif in the C terminus of the HTLV-1 nucleocapsid (NC) domain inhibits hA3G packaging into nascent virions. Mutation of amino acids within this region resulted in increased levels of hA3G incorporation into virions and increased susceptibility to hA3G restriction. Elements within the C-terminal extension of the NC domain are highly conserved among the primate T cell leukemia viruses, but this extension is absent in all other retroviral NC proteins.
Assuntos
Vírus Linfotrópico T Tipo 1 Humano/efeitos dos fármacos , Vírus Linfotrópico T Tipo 1 Humano/fisiologia , Nucleocapsídeo/química , Nucleocapsídeo/metabolismo , Nucleosídeo Desaminases/farmacologia , Proteínas Repressoras/farmacologia , Desaminase APOBEC-3G , Motivos de Aminoácidos , Sequência de Aminoácidos , Sequência Conservada , Citidina Desaminase , Produtos do Gene gag/química , Produtos do Gene gag/metabolismo , Produtos do Gene vif/química , Produtos do Gene vif/metabolismo , HIV-1 , Células HeLa , Humanos , Dados de Sequência Molecular , Proteínas Mutantes/metabolismo , Nucleocapsídeo/efeitos dos fármacos , Peptídeos/química , Estrutura Terciária de Proteína/efeitos dos fármacos , Montagem de Vírus/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Produtos do Gene vif do Vírus da Imunodeficiência HumanaRESUMO
The human pre-T-cell receptor alpha (TCRalpha; pTalpha) gene encodes a polypeptide which associates with the TCRbeta chain and CD3 molecules to form the pre-TCR complex. The surface expression of the pre-TCR is pTalpha dependent, and signaling through this complex triggers an early alphabeta T-cell developmental checkpoint inside the thymus, known as beta-selection. E2A transcription factors, which are involved at multiple stages of T-cell development, regulate the transcription of the pTalpha gene. Here we show that the regulatory protein Tax of the human T-cell leukemia virus type 1 (HTLV-1) efficiently suppresses the E47-mediated activation of the pTalpha promoter. Furthermore, we report that in Tax lentivirally transduced human MOLT-4 T cells, which constitutively express the pTalpha gene, the amount of pTalpha transcripts decreases. Such a decrease is not observed in MOLT-4 cells transduced by a vector encoding the Tax mutant K88A, which is unable to interact with p300. These data underline that Tax inhibits pTalpha transcription by recruiting this coactivator. Finally, we show that the expression of Tax in human immature thymocytes results in a decrease of pTalpha gene transcription but does not modify the level of E47 transcripts. These observations indicate that Tax, by silencing E proteins, down-regulates pTalpha gene transcription during early thymocyte development. They further provide evidence that Tax can interfere with an important checkpoint during T-cell differentiation in the thymus.
Assuntos
Regulação para Baixo , Produtos do Gene tax/metabolismo , Vírus Linfotrópico T Tipo 1 Humano , Glicoproteínas de Membrana/genética , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Linfócitos T/imunologia , Timo/imunologia , Diferenciação Celular/genética , Técnicas de Transferência de Genes , Genes Reporter , Humanos , Luciferases/análise , Regiões Promotoras Genéticas , RNA Mensageiro/metabolismo , Timo/citologiaRESUMO
It is well established that cell-free infection with human T-cell leukemia virus type 1 (HTLV-1) is less efficient than that with other retroviruses, though the specific infectivities of only a limited number of HTLV-1 isolates have been quantified. Earlier work indicated that a post-entry step in the infectious cycle accounted for the poor cell-free infectivity of HTLV-1. To determine whether variations in the pol gene sequence correlated with virus infectivity, we sequenced and phenotypically tested pol genes from a variety of HTLV-1 isolates derived from primary sources, transformed cell lines, and molecular clones. The pol genes and deduced amino acid sequences from 23 proviruses were sequenced and compared with 14 previously published sequences, revealing a limited number of amino acid variations among isolates. The variations appeared to be randomly dispersed among primary isolates and proviruses from cell lines and molecular clones. In addition, there was no correlation between reverse transcriptase sequence and the disease phenotype of the original source of the virus isolate. HTLV-1 pol gene fragments encoding reverse transcriptase were amplified from a variety of isolates and were subcloned into HTLV-1 vectors for both single-cycle infection and spreading-infection assays. Vectors carrying pol genes that matched the consensus sequence had the highest titers, and those with the largest number of variations from the consensus had the lowest titers. The molecular clone from CS-1 cells had four amino acid differences from the consensus sequence and yielded infectious titers that were approximately eight times lower than those of vectors encoding a consensus reverse transcriptase.