RESUMO
HIV-1 infection of human macrophages leads to the downmodulation of human mannose receptor 1 (hMRC1), a cell-surface glycoprotein that is involved in the host innate immune response. We previously reported that downmodulation of hMRC1 involves the transactivator of transcription (Tat)-dependent transcriptional silencing of the hMRC1 promoter. However, the inhibitory effect of Tat on hMRC1 transcription was indirect and involved inhibition of the transcriptional activator PU.1, which normally upregulates hMRC1 expression in macrophages and other myeloid cells. We cloned a 284-bp fragment of the hMRC1 promoter, and within it, we identified four PU.1 box elements. We assessed the relative contribution of each of the four PU.1 boxes to PU.1-dependent transcriptional regulation and, surprisingly, found that only one of the four PU.1 boxes [PU.1(b)] was critically required for PU.1-mediated upregulation of luciferase expression. Transfer of this PU.1 box to a heterologous promoter conferred PU.1 responsiveness to an otherwise PU.1 insensitive promoter. Electrophoretic mobility shift assays identified this PU.1 box as a direct binding site for PU.1 both in the context of the hMRC1 promoter and the heterologous promoter. Furthermore, mutational analysis of the PU.1 protein identified the C-terminal DNA-binding domain in PU.1 as the region responsible for interaction with the PU.1 box. Recombinant HIV-1 Tat protein did not bind to the hMRC1 promoter element but efficiently interfered with the binding of PU.1 protein to the hMRC1 promoter. Thus, Tat is likely to inhibit the formation of active PU.1 transcription complexes, presumably by binding to and depleting common transcriptional cofactors.IMPORTANCEHIV-1 infection of cells results in the modulation of cellular gene expression by virus-encoded proteins in a manner that benefits the virus. We reported that HIV-1 transactivator of transcription (Tat) dysregulates the expression of the human mannose receptor 1 (hMRC1). hMRC1 is involved in the innate immune response of macrophages to foreign pathogens. Tat does not act directly on the hMRC1 promoter but instead inhibits PU.1, a cellular transcription factor regulating hMRC1 gene expression. Here, we characterize the PU.1-dependent regulation of hMRC1 expression. We identified four potential PU.1 binding sites in the hMRC1 promoter region but found that only one, PU.1(b), functioned as a true binding site for PU.1. Transfer of the PU.1(b) box to a heterologous promoter did not activate this promoter per se but rendered it responsive to PU.1. Our results support the view that PU.1 acts as a transcriptional co-factor whose activity can be regulated by HIV-1 Tat.
Assuntos
HIV-1 , Receptor de Manose , Proteínas Proto-Oncogênicas , Transativadores , Humanos , HIV-1/fisiologia , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Transativadores/genética , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica , Ativação TranscricionalRESUMO
Human mannose receptor 1 (MRC1) is a cell surface receptor expressed in macrophages and other myeloid cells that inhibits human immunodeficiency virus type 1 (HIV-1) particle release by tethering virions to producer cell membranes. HIV-1 counteracts MRC1 expression by inhibiting mrc1 transcription. Here, we investigated the mechanism of MRC1 downregulation in HIV-1-infected macrophages. We identified the myeloid cell-specific transcription factor PU.1 as critical for regulating MRC1 expression. In the course of our study, we recognized a complex interplay between HIV-1 Tat and PU.1 transcription factors: Tat upregulated HIV-1 gene expression but inhibited mrc1 transcription, whereas PU.1 inhibited HIV-1 transcription but activated MRC1 expression. Disturbing this equilibrium by silencing PU.1 resulted in increased HIV-1 gene expression and reduced MRC1 promoter activity. Our study identified PU.1 as a central player in transcriptional control, regulating a complex interplay between viral and host gene expression in HIV-infected macrophages. IMPORTANCE HIV-1 replication in primary human cells depends on the activity of virus-encoded proteins but also involves cellular factors that can either promote (viral dependency factors) or inhibit (host restriction factors) virus replication. In previous work, we identified human MRC1 as a macrophage-specific host restriction factor that inhibits the detachment of viral particles from infected cells. Here, we report that HIV-1 counteracts this effect of MRC1 by imposing a transcriptional block on cellular MRC1 gene expression. The transcriptional inhibition of the MRC1 gene is accomplished by Tat, an HIV-1 factor whose best-described function actually is the enhancement of HIV-1 gene expression. Thus, HIV-1 has evolved to use the same protein for (i) activation of its own gene expression while (ii) inhibiting expression of MRC1 and other host factors.
Assuntos
Infecções por HIV , Repetição Terminal Longa de HIV , Receptor de Manose , Regulação para Cima , Regulação Viral da Expressão Gênica , Infecções por HIV/fisiopatologia , Infecções por HIV/virologia , HIV-1/fisiologia , Humanos , Macrófagos/virologia , Receptor de Manose/genética , Regiões Promotoras Genéticas , Ativação TranscricionalRESUMO
Vif is a lentiviral accessory protein that counteracts the antiviral activity of cellular APOBEC3 (A3) cytidine deaminases in infected cells. The exact contribution of each member of the A3 family for the restriction of HIV-2 is still unclear. Thus, the aim of this work was to identify the A3s with anti-HIV-2 activity and compare their restriction potential for HIV-2 and HIV-1. We found that A3G is a strong restriction factor of both types of viruses and A3C restricts neither HIV-1 nor HIV-2. Importantly, A3B exhibited potent antiviral activity against HIV-2, but its effect was negligible against HIV-1. Whereas A3B is packaged with similar efficiency into both viruses in the absence of Vif, HIV-2 and HIV-1 differ in their sensitivity to A3B. HIV-2 Vif targets A3B by reducing its cellular levels and inhibiting its packaging into virions, whereas HIV-1 Vif did not evolve to antagonize A3B. Our observations support the hypothesis that during wild-type HIV-1 and HIV-2 infections, both viruses are able to replicate in host cells expressing A3B but using different mechanisms, probably resulting from a Vif functional adaptation over evolutionary time. Our findings provide new insights into the differences between Vif protein and their cellular partners in the two human viruses. Of note, A3B is highly expressed in some cancer cells and may cause deamination-induced mutations in these cancers. Thus, A3B may represent an important therapeutic target. As such, the ability of HIV-2 Vif to induce A3B degradation could be an effective tool for cancer therapy. IMPORTANCE Primate lentiviruses encode a series of accessory genes that facilitate virus adaptation to its host. Among those, the vif-encoded protein functions primarily by targeting the APOBEC3 (A3) family of cytidine deaminases. All lentiviral Vif proteins have the ability to antagonize A3G; however, antagonizing other members of the A3 family is variable. Here, we report that HIV-2 Vif, unlike HIV-1 Vif, can induce degradation of A3B. Consequently, HIV-2 Vif but not HIV-1 Vif can inhibit the packaging of A3B. Interestingly, while A3B is packaged efficiently into the core of both HIV-1 and HIV-2 virions in the absence of Vif, it only affects the infectivity of HIV-2 particles. Thus, HIV-1 and HIV-2 have evolved two distinct mechanisms to antagonize the antiviral activity of A3B. Aside from its antiviral activity, A3B has been associated with mutations in some cancers. Degradation of A3B by HIV-2 Vif may be useful for cancer therapies.
Assuntos
Citidina Desaminase/metabolismo , Produtos do Gene vif/metabolismo , HIV-1/metabolismo , HIV-2/metabolismo , Antígenos de Histocompatibilidade Menor/metabolismo , Produtos do Gene vif do Vírus da Imunodeficiência Humana/metabolismo , Animais , Citidina Desaminase/genética , Células HEK293 , Infecções por HIV , Humanos , Antígenos de Histocompatibilidade Menor/genética , Receptor EphB2RESUMO
Vif counteracts the host restriction factor APOBEC3G (A3G) and other APOBEC3s by preventing the incorporation of A3G into progeny virions. We previously identified Vif mutants with a dominant-negative (D/N) phenotype that interfered with the function of wild-type Vif, inhibited the degradation of A3G, and reduced the infectivity of viral particles by increased packaging of A3G. However, the mechanism of interference remained unclear, in particular since all D/N Vif mutants were unable to bind Cul5 and some mutants additionally failed to bind A3G, ruling out competitive binding to A3G or the E3 ubiquitin ligase complex as the sole mechanism. The goal of the current study was to revisit the mechanism of D/N interference by Vif mutants and analyze the possible involvement of core binding factor beta (CBFß) in this process. We found a clear correlation of D/N properties of Vif mutants with their ability to engage CBFß. Only mutants that retained the ability to bind CBFß exhibited the D/N phenotype. Competition studies revealed that D/N Vif mutants directly interfered with the association of CBFß and wild-type Vif. Furthermore, overexpression of CBFß counteracted the interference of D/N Vif mutants with A3G degradation by wild-type Vif. Finally, overexpression of Runx1 mimicked the effect of D/N Vif mutants and inhibited the degradation of A3G by wild-type Vif. Taken together, we identified CBFß as the key player involved in D/N interference by Vif.IMPORTANCE Of all the accessory proteins encoded by HIV-1 and other primate lentiviruses, Vif has arguably the strongest potential as a target for antiviral therapy. This conclusion is based on the observation that replication of HIV-1 in vivo is critically dependent on Vif. Thus, inhibiting the function of Vif via small-molecule inhibitors or other approaches has significant therapeutic potential. We previously identified dominant-negative (D/N) Vif variants whose expression interferes with the function of virus-encoded wild-type Vif. We now show that D/N interference involves competitive binding of D/N Vif variants to the transcriptional cofactor core binding factor beta (CBFß), which is expressed in cells in limiting quantities. Overexpression of CBFß neutralized the D/N phenotype of Vif. In contrast, overexpression of Runx1, a cellular binding partner of CBFß, phenocopied the D/N Vif phenotype by sequestering endogenous CBFß. Thus, our results provide proof of principle that D/N Vif variants could have therapeutic potential.
Assuntos
Desaminase APOBEC-3G/metabolismo , Subunidade beta de Fator de Ligação ao Core/metabolismo , Produtos do Gene vif do Vírus da Imunodeficiência Humana/metabolismo , Ligação Competitiva , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Proteínas Culina/metabolismo , Elonguina/metabolismo , Genes Dominantes , Células HEK293 , HIV-1/fisiologia , Humanos , Leucócitos Mononucleares/metabolismo , Mutação , Fenótipo , VírionRESUMO
Apolipoprotein E (ApoE) belongs to a class of cellular proteins involved in lipid metabolism. ApoE is a polymorphic protein produced primarily in macrophages and astrocytes. Different isoforms of ApoE have been associated with susceptibility to various diseases including Alzheimer's and cardiovascular diseases. ApoE expression has also been found to affect susceptibility to several viral diseases, including Hepatitis C and E, but its effect on the life cycle of HIV-1 remains obscure. In this study, we initially found that HIV-1 infection selectively up-regulated ApoE in human monocyte-derived macrophages (MDMs). Interestingly, ApoE knockdown in MDMs enhanced the production and infectivity of HIV-1, and was associated with increased localization of viral envelope (Env) proteins to the cell surface. Consistent with this, ApoE over-expression in 293T cells suppressed Env expression and viral infectivity, which was also observed with HIV-2 Env, but not with VSV-G Env. Mechanistic studies revealed that the C-terminal region of ApoE was required for its inhibitory effect on HIV-1 Env expression. Moreover, we found that ApoE and Env co-localized in the cells, and ApoE associated with gp160, the precursor form of Env, and that the suppression of Env expression by ApoE was cancelled by the treatment with lysosomal inhibitors. Overall, our study revealed that ApoE is an HIV-1-inducible inhibitor of viral production and infectivity in macrophages that exerts its anti-HIV-1 activity through association with gp160 Env via the C-terminal region, which results in subsequent degradation of gp160 Env in the lysosomes.
Assuntos
Apolipoproteínas E/fisiologia , Infecções por HIV/metabolismo , Macrófagos/metabolismo , Adulto , Apolipoproteínas/metabolismo , Apolipoproteínas E/metabolismo , Linfócitos T CD4-Positivos/metabolismo , Regulação da Expressão Gênica/genética , Células HEK293 , Proteína gp120 do Envelope de HIV/metabolismo , Proteína gp41 do Envelope de HIV/metabolismo , Infecções por HIV/prevenção & controle , HIV-1/metabolismo , Humanos , Macrófagos/virologia , Masculino , Regulação para Cima , Replicação Viral/genética , Replicação Viral/fisiologia , Produtos do Gene env do Vírus da Imunodeficiência Humana/metabolismoRESUMO
For nearly 20 years, the principal biological function of the HIV-2/SIV Vpx gene has been thought to be required for optimal virus replication in myeloid cells. Mechanistically, this Vpx activity was recently reported to involve the degradation of Sterile Alpha Motif and HD domain-containing protein 1 (SAMHD1) in this cell lineage. Here we show that when macaques were inoculated with either the T cell tropic SIVmac239 or the macrophage tropic SIVmac316 carrying a Vpx point mutation that abrogates the recruitment of DCAF1 and the ensuing degradation of endogenous SAMHD1 in cultured CD4+ T cells, virus acquisition, progeny virion production in memory CD4+ T cells during acute infection, and the maintenance of set-point viremia were greatly attenuated. Revertant viruses emerging in two animals exhibited an augmented replication phenotype in memory CD4+ T lymphocytes both in vitro and in vivo, which was associated with reduced levels of endogenous SAMHD1. These results indicate that a critical role of Vpx in vivo is to promote the degradation of SAMHD1 in memory CD4+ T lymphocytes, thereby generating high levels of plasma viremia and the induction of immunodeficiency.
Assuntos
Linfócitos T CD4-Positivos/metabolismo , Interações Hospedeiro-Patógeno , Proteínas Monoméricas de Ligação ao GTP/antagonistas & inibidores , Síndrome de Imunodeficiência Adquirida dos Símios/metabolismo , Vírus da Imunodeficiência Símia/metabolismo , Proteínas Virais Reguladoras e Acessórias/metabolismo , Substituição de Aminoácidos , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/virologia , Células Cultivadas , Deleção de Genes , Células HEK293 , Humanos , Memória Imunológica , Macaca mulatta , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Fragmentos de Peptídeos , Fosforilação , Mutação Puntual , Processamento de Proteína Pós-Traducional , Proteólise , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteína 1 com Domínio SAM e Domínio HD , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Vírus da Imunodeficiência Símia/imunologia , Proteínas Virais Reguladoras e Acessórias/química , Proteínas Virais Reguladoras e Acessórias/genética , Viremia/imunologia , Viremia/metabolismo , Viremia/virologiaRESUMO
UNLABELLED: Vif is a lentiviral accessory protein that regulates viral infectivity in part by inducing proteasomal degradation of APOBEC3G (A3G). Recently, CBFß was found to facilitate Vif-dependent degradation of A3G. However, the exact role of CBFß remains unclear. Several studies noted reduced Vif expression in CBFß knockdown cells while others saw no significant impact of CBFß on Vif stability. Here, we confirmed that CBFß increases Vif steady-state levels. CBFß affected expression of neither viral Gag nor Vpu protein, indicating that CBFß regulates Vif expression posttranscriptionally. Kinetic studies revealed effects of CBFß on both metabolic stability and the rate of Vif biosynthesis. These effects were dependent on the ability of CBFß to interact with Vif. Importantly, at comparable Vif levels, CBFß further enhanced A3G degradation, suggesting that CBFß facilitates A3G degradation by increasing the levels of Vif and by independently augmenting the ability of Vif to target A3G for degradation. CBFß also increased expression of RUNX1 by enhancing RUNX1 biosynthesis. Unlike Vif, however, CBFß had no detectable effect on RUNX1 metabolic stability. We propose that CBFß acts as a chaperone to stabilize Vif during and after synthesis and to facilitate interaction of Vif with cellular cofactors required for the efficient degradation of A3G. IMPORTANCE: In this study, we show that CBFß has a profound effect on the expression of the HIV-1 infectivity factor Vif and the cellular transcription factor RUNX1, two proteins that physically interact with CBFß. Kinetic studies revealed that CBFß increases the rate of Vif and RUNX1 biosynthesis at the level of translation. Mutants of Vif unable to physically interact with CBFß were nonresponsive to CBFß. Our data suggest that CBFß exerts a chaperone-like activity (i) to minimize the production of defective ribosomal products (DRiPs) by binding to nascent protein to prevent premature termination and (ii) to stabilize mature protein conformation to ensure proper function of Vif and RUNX1. Thus, we identified a novel mechanism of protein regulation that affects both viral and cellular factors and thus has broad implications beyond the immediate HIV field.
Assuntos
Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Subunidade beta de Fator de Ligação ao Core/metabolismo , Citidina Desaminase/metabolismo , Produtos do Gene vif do Vírus da Imunodeficiência Humana/metabolismo , Desaminase APOBEC-3G , Células HeLa , Humanos , Chaperonas Moleculares/metabolismo , Biossíntese de Proteínas , ProteóliseRESUMO
In humans, sterile alpha motif (SAM) domain- and histidine-aspartic acid (HD) domain-containing protein 1 (SAMHD1) is a dNTPase enzyme that prevents HIV-1 infection in non-cycling cells, such as differentiated THP-1 cells and human primary macrophages. Although phosphorylation of threonine 592 (T592) in SAMHD1 is recognized as the primary regulator of the ability to prevent HIV-1 infection, the contributions of SAMHD1 acetylation to this ability remain unknown. Mass spectrometry analysis of SAMHD1 proteins derived from cycling and non-cycling THP-1 cells, primary cycling B cells, and primary macrophages revealed that SAMHD1 is preferentially acetylated at lysine residues 354, 494, and 580 (K354, K494, and K580). In non-cycling cells, SAMHD1 is preferentially acetylated at K580, suggesting that this post-translational modification may contribute to the ability of SAMHD1 to block HIV-1 infection. Consistent with this finding, we found that mutations in K580 disrupted the ability of SAMHD1 to block HIV-1 infection without affecting the ability of SAMHD1 to deplete cellular dNTP levels. Gene editing of SAMHD1 in macrophage-like cells revealed that an intact K580 is required for HIV-1 restriction. This finding suggests that K580 acetylation in SAMHD1 is essential for blocking HIV-1 infection. More importantly, we found that a larger proportion of SAMHD1 featuring K580 acetylation could be detected in human primary macrophages when compared to human primary monocytes. In agreement, we found that SAMHD1 is acetylated during the monocyte-to-macrophage differentiation process. This finding agrees with the idea that the blockade of HIV-1 infection in macrophages requires SAMHD1 acetylation.IMPORTANCEThe natural inhibitor of HIV-1, sterile alpha motif (SAM) domain- and histidine-aspartic acid (HD) domain-containing protein 1 (SAMHD1), plays a pivotal role in preventing HIV-1 infection of macrophages and dendritic cells, which are vital components of the immune system. This study unveils that SAMHD1 undergoes post-translational modifications, specifically acetylation at lysines 354, 494, and 580. Our research underscores the significance of these modifications, demonstrating that acetylation at residue K580 is indispensable for SAMHD1's efficacy in blocking HIV-1 infection. Notably, K580 is found in a critical regulatory domain of SAMHD1, highlighting acetylation as a novel layer of SAMHD1 regulation for HIV-1 restriction in humans. A comprehensive understanding of the regulation mechanisms governing this anti-HIV-1 protein is crucial for leveraging nature's defense mechanisms against HIV-1 and could pave the way for innovative therapeutic strategies.
RESUMO
Gelsolin (GSN) is a structural actin-binding protein that is known to affect actin dynamics in the cell. Using mass spectrometry, we identified GSN as a novel Vpr-interacting protein. Endogenous GSN protein was expressed at detectable levels in monocyte-derived macrophages (MDM) and in THP-1 cells, but it was undetectable at the protein level in other cell lines tested. The HIV-1 infection of MDM was associated with a reduction in GSN steady-state levels, presumably due to the Vpr-induced degradation of GSN. Indeed, the coexpression of GSN and Viral protein R (Vpr) in transiently transfected HEK293T cells resulted in the Vpr-dependent proteasomal degradation of GSN. This effect was observed for Vprs from multiple virus isolates. The overexpression of GSN in HEK293T cells had no effect on Gag expression or particle release, but it reduced the expression and packaging of the HIV-1 envelope (Env) glycoprotein and reduced viral infectivity. An analysis of the HIV-1 splicing patterns did not reveal any GSN-dependent differences, suggesting that the effect of GSN on Env expression was regulated at a posttranscriptional level. Indeed, the treatment of transfected cells with lysosomal inhibitors reversed the effect of GSN on Env stability, suggesting that GSN reduced Env expression via enhanced lysosomal degradation. Our data identify GSN as a macrophage-specific host antiviral factor that reduces the expression of HIV-1 Env. IMPORTANCE Despite dramatic progress in drug therapies, HIV-1 infection remains an incurable disease that affects millions of people worldwide. The virus establishes long-lasting reservoirs that are resistant to currently available drug treatments and allow the virus to rebound whenever drug therapy is interrupted. Macrophages are long-lived cells that are relatively insensitive to HIV-1-induced cytopathicity and thus could contribute to the viral reservoir. Here, we identified a novel host factor, gelsolin, that is expressed at high levels in macrophages and inhibits viral infectivity by modulating the expression of the HIV-1 Env glycoprotein, which is critical in the spread of an HIV-1 infection. Importantly, the viral protein Vpr induces the degradation of gelsolin and thus counteracts its antiviral activity. Our study provides significant and novel insights into HIV-1 virus-host interactions and furthers our understanding of the importance of Vpr in HIV-1 infection and pathogenesis.
Assuntos
Infecções por HIV , HIV-1 , Humanos , Produtos do Gene vpr do Vírus da Imunodeficiência Humana/genética , Produtos do Gene vpr do Vírus da Imunodeficiência Humana/metabolismo , Gelsolina/metabolismo , Produtos do Gene env/metabolismo , Células HEK293 , Células Mieloides/metabolismo , Antivirais/metabolismoRESUMO
BACKGROUND: Sterile Alpha Motif and HD domain-containing protein 1 (SAMHD1) is a recently identified host factor that restricts HIV-1 replication in dendritic and myeloid cells. SAMHD1 is a dNTPase that presumably reduces the cellular dNTP levels to levels too low for retroviral reverse transcription to occur. However, HIV-2 and SIV encoded Vpx counteracts the antiviral effects of SAMHD1 by targeting the protein for proteasomal degradation. SAMHD1 is encoded by a multiply spliced mRNA and consists of 16 coding exons. RESULTS: Here, we identified two naturally occurring splice variants lacking exons 8-9 and 14, respectively. Like wildtype SAMHD1, both splice variants localize primarily to the nucleus, interact with Vpx, and retain some sensitivity to Vpx-dependent degradation. However, the splice variants differ from full-length SAMHD1 in their metabolic stability and catalytic activity. While full-length SAMHD1 is metabolically stable in uninfected cells, both splice variants were inherently metabolically unstable and were rapidly degraded even in the absence of Vpx. Vpx strongly increased the rate of degradation of full-length SAMHD1 and further accelerated the degradation of the splice variants. However, the effect of Vpx on the splice variants was more modest due to the inherent instability of these proteins. Analysis of dNTPase activity indicates that neither splice variant is catalytically active. CONCLUSIONS: The identification of SAMHD1 splice variants exposes a potential regulatory mechanism that could enable the cell to control its dNTPase activity on a post-transcriptional level.
Assuntos
Variação Genética , Isoenzimas/genética , Isoenzimas/metabolismo , Proteínas Monoméricas de Ligação ao GTP/genética , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Linhagem Celular , Núcleo Celular/química , Éxons , Humanos , Modelos Moleculares , Estabilidade Proteica , Proteína 1 com Domínio SAM e Domínio HDRESUMO
BST-2/CD317/tetherin is a host factor that inhibits the release of HIV-1 and other unrelated viruses. A current model proposes that BST-2 physically tethers virions to the surface of virus-producing cells. The HIV-1-encoded Vpu protein effectively antagonizes the activity of BST-2. How Vpu accomplishes this task remains unclear; however, it is known that Vpu has the ability to down-modulate BST-2 from the cell surface. Here we analyzed the effects of Vpu on BST-2 by performing a series of kinetic studies with HeLa, 293T, and CEMx174 cells. Our results indicate that the surface downregulation of BST-2 is not due to an accelerated internalization or reduced recycling of internalized BST-2 but instead is caused by interference with the resupply of newly synthesized BST-2 from within the cell. While our data confirm previous reports that the high-level expression of Vpu can cause the endoplasmic reticulum (ER)-associated degradation of BST-2, we found no evidence that Vpu targets endogenous BST-2 in the ER in the course of a viral infection. Instead, we found that Vpu acts in a post-ER compartment and increases the turnover of newly synthesized mature BST-2. Our observation that Vpu does not affect the recycling of BST-2 suggests that Vpu does not act directly at the cell surface but may interfere with the trafficking of newly synthesized BST-2 to the cell surface, resulting in the accelerated targeting of BST-2 to the lysosomal compartment for degradation.
Assuntos
Antígenos CD/metabolismo , HIV-1/imunologia , HIV-1/patogenicidade , Proteínas do Vírus da Imunodeficiência Humana/metabolismo , Proteínas Virais Reguladoras e Acessórias/metabolismo , Antígenos CD/imunologia , Linhagem Celular , Regulação para Baixo , Proteínas Ligadas por GPI/imunologia , Proteínas Ligadas por GPI/metabolismo , Expressão Gênica , Proteínas do Vírus da Imunodeficiência Humana/imunologia , Humanos , Transporte Proteico , Proteínas Virais Reguladoras e Acessórias/imunologiaRESUMO
BST-2/CD317/HM1.24/tetherin is a B-cell antigen overexpressed on the surface of myeloma cell lines and on neoplastic plasma cells of patients with multiple myeloma. Antibodies to BST-2 are in clinical trial for the treatment of multiple myeloma and are considered for the treatment of solid tumors with high BST-2 antigen levels. Functionally, BST-2 restricts the secretion of retroviruses, including human immunodeficiency virus type 1, as well as members of the herpesvirus, filovirus, and arenavirus families, presumably by tethering nascent virions to the cell surface. Here we report that BST-2 antibody treatment facilitates virus release from BST-2(+) cells by interfering with the tethering activity of BST-2. BST-2 antibodies were unable to release already tethered virions and were most effective when added early during virus production. BST-2 antibody treatment did not affect BST-2 dimerization and did not reduce the cell surface expression of BST-2. Interestingly, BST-2 antibody treatment reduced the nonspecific shedding of BST-2 and limited the encapsidation of BST-2 into virions. Finally, flotation analyses indicate that BST-2 antibodies affect the distribution of BST-2 within membrane rafts. Our data suggest that BST-2 antibody treatment may enhance virus release by inducing a redistribution of BST-2 at the cell surface, thus preventing it from accumulating at the sites of virus budding.
Assuntos
Anticorpos/metabolismo , Antígenos CD/imunologia , HIV-1/fisiologia , HIV-2/fisiologia , Liberação de Vírus , Linhagem Celular , Proteínas Ligadas por GPI/antagonistas & inibidores , Proteínas Ligadas por GPI/imunologia , HIV-1/crescimento & desenvolvimento , HIV-2/crescimento & desenvolvimento , HumanosRESUMO
HIV-1 Vpu enhances the release of virions from infected cells. Recent work identified Bst-2/CD317/tetherin as a host factor whose inhibitory activity on viral release is counteracted by Vpu. A current working model proposes that Bst-2 inhibits virus release by tethering viral particles to the cell surface. Here, we analyzed endogenous Bst-2 with respect to its effect on virus release from HeLa cells, T cells, and macrophages. We noted significant cell type-dependent variation in Bst-2 expression. Vpu caused a reduction in Bst-2 expression in transfected HeLa cells and long-term infected macrophages. However, Vpu expression did not result in cell surface down-modulation of Bst-2 or a reduction in intracellular Bst-2 expression in CEMx174 or H9 cells, yet virus replication in these cells was Vpu-responsive. Surprisingly, Bst-2 was undetectable in cell-free virions that were recovered from the surface of HeLa cells by physical shearing, suggesting that a tethering model may not explain all of the functional properties of Bst-2. Taken together we conclude that enhancement of virus release by Vpu does not, at least in CEMx174 and H9 cells, require cell surface down-modulation or intracellular depletion of Bst-2, nor does it entail exclusion of Bst-2 from viral particles.
Assuntos
Antígenos CD/fisiologia , Regulação para Baixo , HIV-1/fisiologia , Proteínas do Vírus da Imunodeficiência Humana/fisiologia , Glicoproteínas de Membrana/fisiologia , Proteínas Virais Reguladoras e Acessórias/fisiologia , Antígenos CD/metabolismo , Western Blotting , Linhagem Celular , Membrana Celular/metabolismo , Citometria de Fluxo , Proteínas Ligadas por GPI , Humanos , Soros Imunes , Glicoproteínas de Membrana/metabolismo , Vírion , Montagem de VírusRESUMO
APOBEC3G (A3G) is a host cytidine deaminase that serves as a potent intrinsic inhibitor of retroviral replication. A3G is packaged into human immunodeficiency virus type 1 virions and deaminates deoxycytidine to deoxyuridine on nascent minus-strand retroviral cDNA, leading to hyper-deoxyguanine-to-deoxyadenine mutations on positive-strand cDNA and inhibition of viral replication. The antiviral activity of A3G is suppressed by Vif, a lentiviral accessory protein that prevents encapsidation of A3G. In this study, we identified dominant negative mutants of Vif that interfered with the ability of wild-type Vif to inhibit the encapsidation and antiviral activity of A3G. These mutants were nonfunctional due to mutations in the highly conserved HCCH and/or SOCS box motifs, which are required for assembly of a functional Cul5-E3 ubiquitin ligase complex. Similarly, mutation or deletion of a PPLP motif, which was previously reported to be important for Vif dimerization, induced a dominant negative phenotype. Expression of dominant negative Vif counteracted the Vif-induced reduction of intracellular A3G levels, presumably by preventing Vif-induced A3G degradation. Consequently, dominant negative Vif interfered with wild-type Vif's ability to exclude A3G from viral particles and reduced viral infectivity despite the presence of wild-type Vif. The identification of dominant negative mutants of Vif presents exciting possibilities for the design of novel antiviral strategies.
Assuntos
Citidina Desaminase/antagonistas & inibidores , HIV-1/imunologia , HIV-1/patogenicidade , Proteínas Mutantes/metabolismo , Mutação , Fatores de Virulência/metabolismo , Produtos do Gene vif do Vírus da Imunodeficiência Humana/metabolismo , Desaminase APOBEC-3G , Linhagem Celular , HIV-1/genética , Humanos , Viabilidade Microbiana , Proteínas Mutantes/genética , Fatores de Virulência/genética , Produtos do Gene vif do Vírus da Imunodeficiência Humana/genéticaRESUMO
APOBEC3F (A3F) is a member of the family of cytidine deaminases that is often coexpressed with APOBEC3G (A3G) in cells susceptible to HIV infection. A3F has been shown to have strong antiviral activity in transient-expression studies, and together with A3G, it is considered the most potent cytidine deaminase targeting HIV. Previous analyses suggested that the antiviral properties of A3F can be dissociated from its catalytic deaminase activity. We were able to confirm the deaminase-independent antiviral activity of exogenously expressed A3F; however, we also noted that exogenous expression was associated with very high A3F mRNA and protein levels. In analogy to our previous study of A3G, we produced stable HeLa cell lines constitutively expressing wild-type or deaminase-defective A3F at levels that were more in line with the levels of endogenous A3F in H9 cells. A3F expressed in stable HeLa cells was packaged into Vif-deficient viral particles with an efficiency similar to that of A3G and was properly targeted to the viral nucleoprotein complex. Surprisingly, however, neither wild-type nor deaminase-defective A3F inhibited HIV-1 infectivity. These results imply that the antiviral activity of endogenous A3F is negligible compared to that of A3G.
Assuntos
Citidina Desaminase/imunologia , Citosina Desaminase/imunologia , Desaminase APOBEC-3G , Citosina Desaminase/análise , Citosina Desaminase/genética , Infecções por HIV , HIV-1/patogenicidade , Células HeLa , Humanos , RNA Mensageiro/análise , Vírion , Produtos do Gene vif do Vírus da Imunodeficiência HumanaRESUMO
HIV-1 Vif counteracts the antiviral activity of APOBEC3G by inhibiting its encapsidation into virions. Here, we compared the relative sensitivity to Vif of APOBEC3G in stable HeLa cells containing APOBEC3G (HeLa-A3G cells) versus that of newly synthesized APOBEC3G. We observed that newly synthesized APOBEC3G was more sensitive to degradation than preexisting APOBEC3G. Nevertheless, preexisting and transiently expressed APOBEC3G were packaged with similar efficiencies into vif-deficient human immunodeficiency virus type 1 (HIV-1) virions, and Vif inhibited the encapsidation of both forms of APOBEC3G into HIV particles equally well. Our results suggest that HIV-1 Vif preferentially induces degradation of newly synthesized APOBEC3G but indiscriminately inhibits encapsidation of "old" and "new" APOBEC3G.
Assuntos
Citidina Desaminase/antagonistas & inibidores , Citidina Desaminase/metabolismo , Produtos do Gene vif do Vírus da Imunodeficiência Humana/metabolismo , Desaminase APOBEC-3G , HIV-1/fisiologia , Células HeLa , HumanosRESUMO
BACKGROUND: The Human Immunodeficiency virus type 1 (HIV-1) Vpu protein enhances virus release from infected cells and induces proteasomal degradation of CD4. Recent work identified BST-2/CD317 as a host factor that inhibits HIV-1 virus release in a Vpu sensitive manner. A current working model proposes that BST-2 inhibits virus release by tethering viral particles to the cell surface thereby triggering their subsequent endocytosis. RESULTS: Here we defined structural properties of BST-2 required for inhibition of virus release and for sensitivity to Vpu. We found that BST-2 is modified by N-linked glycosylation at two sites in the extracellular domain. However, N-linked glycosylation was not important for inhibition of HIV-1 virus release nor did it affect surface expression or sensitivity to Vpu. Rodent BST-2 was previously found to form cysteine-linked dimers. Analysis of single, double, or triple cysteine mutants revealed that any one of three cysteine residues present in the BST-2 extracellular domain was sufficient for BST-2 dimerization, for inhibition of virus release, and sensitivity to Vpu. In contrast, BST-2 lacking all three cysteines in its ectodomain was unable to inhibit release of wild type or Vpu-deficient HIV-1 virions. This defect was not caused by a gross defect in BST-2 trafficking as the mutant protein was expressed at the cell surface of transfected 293T cells and was down-modulated by Vpu similar to wild type BST-2. CONCLUSION: While BST-2 glycosylation was functionally irrelevant, formation of cysteine-linked dimers appeared to be important for inhibition of virus release. However lack of dimerization did not prevent surface expression or Vpu sensitivity of BST-2, suggesting Vpu sensitivity and inhibition of virus release are separable properties of BST-2.
Assuntos
Antígenos CD/imunologia , HIV-1/imunologia , HIV-1/fisiologia , Proteínas do Vírus da Imunodeficiência Humana/fisiologia , Glicoproteínas de Membrana/imunologia , Proteínas Virais Reguladoras e Acessórias/fisiologia , Replicação Viral , Substituição de Aminoácidos/genética , Antígenos CD/metabolismo , Linhagem Celular , Membrana Celular/química , Cisteína/genética , Dimerização , Dissulfetos , Proteínas Ligadas por GPI , Glicosilação , Humanos , Glicoproteínas de Membrana/antagonistas & inibidores , Glicoproteínas de Membrana/metabolismo , Mutagênese Sítio-DirigidaRESUMO
BACKGROUND: The cellular cytidine deaminase APOBEC3G (A3G), when incorporated into the human immunodeficiency virus type 1 (HIV-1), renders viral particles non-infectious. We previously observed that mutation of a single cysteine residue of A3G (C100S) inhibited A3G packaging. In addition, several recent studies showed that mutation of tryptophan 127 (W127) and tyrosine 124 (Y124) inhibited A3G encapsidation suggesting that the N-terminal CDA constitutes a viral packaging signal in A3G. It was also reported that W127 and Y124 affect A3G oligomerization. RESULTS: Here we studied the mechanistic basis of the packaging defect of A3G W127A and Y124A mutants. Interestingly, cell fractionation studies revealed a strong correlation between encapsidation, lipid raft association, and genomic RNA binding of A3G. Surprisingly, the presence of a C-terminal epitope tag affected lipid raft association and encapsidation of the A3G W127A mutant but had no effect on wt A3G encapsidation, lipid raft association, and interaction with viral genomic RNA. Mutation of Y124 abolished A3G encapsidation irrespective of the presence or absence of an epitope tag. Contrasting a recent report, our co-immunoprecipitation studies failed to reveal a correlation between A3G oligomerization and A3G encapsidation. In fact, our W127A and Y124A mutants both retained the ability to oligomerize. CONCLUSION: Our results confirm that W127 and Y124 residues in A3G are important for encapsidation into HIV-1 virions and our data establish a novel correlation between genomic RNA binding, lipid raft association, and viral packaging of A3G. In contrast, we were unable to confirm a role of W127 and Y124 in A3G oligomerization and we thus failed to confirm a correlation between A3G oligomerization and virus encapsidation.
Assuntos
Citidina Desaminase/metabolismo , HIV-1/fisiologia , Microdomínios da Membrana/metabolismo , Montagem de Vírus , Desaminase APOBEC-3G , Substituição de Aminoácidos/genética , Fracionamento Celular , Citidina Desaminase/genética , Humanos , Imunoprecipitação , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutação de Sentido Incorreto , Ligação Proteica , RNA Viral/metabolismoRESUMO
APOBEC3G (APO3G) is a cellular cytidine deaminase with potent antiviral activity. Initial studies of the function of APO3G demonstrated extensive mutation of the viral genome, suggesting a model in which APO3G's antiviral activity is due to hypermutation of the viral genome. Recent studies, however, found that deaminase-defective APO3G mutants transiently expressed in virus-producing cells exhibited significant antiviral activity, suggesting that the antiviral activity of APO3G could be dissociated from its deaminase activity. To directly compare the antiviral activities of wild-type (wt) and deaminase-defective APO3G, we used two approaches: (i) we titrated wt and deaminase-defective APO3G in transient-transfection studies to achieve similar levels of virus-associated APO3G and (ii) we constructed stable cell lines and selected clones expressing comparable amounts of wt and deaminase-defective APO3G. Viruses produced under these conditions were tested for viral infectivity. The results from the two approaches were consistent and suggested that the antiviral activity of deaminase-defective APO3G was significantly lower than that of wt APO3G. We conclude that efficient inhibition of vif-defective human immunodeficiency virus type 1 requires catalytically active APO3G.
Assuntos
Antivirais/farmacologia , Citidina Desaminase/farmacologia , HIV-1/efeitos dos fármacos , HIV-1/patogenicidade , Desaminase APOBEC-3G , Animais , Antivirais/metabolismo , Linhagem Celular , Citidina Desaminase/química , Citidina Desaminase/genética , Citidina Desaminase/metabolismo , Genes vif , HIV-1/genética , Células HeLa , Humanos , Mutação , Coelhos , TransfecçãoRESUMO
Human mannose receptor 1 (hMRC1) is expressed on the surface of most tissue macrophages, dendritic cells, and select lymphatic or liver endothelial cells. HMRC1 contributes to the binding of HIV-1 to monocyte-derived macrophages (MDMs) and is involved in the endocytic uptake of HIV-1 into these cells. Here, we identify hMRC1 as an antiviral factor that inhibits virus release through a bone marrow stromal antigen 2 (BST-2)-like mechanism. Virions produced in the presence of hMRC1 accumulated in clusters at the cell surface but were fully infectious. HIV-1 counteracted the effect by transcriptional silencing of hMRC1. The effect of hMRC1 was not virus isolate specific. Surprisingly, deletion of the Env protein, which is known to interact with hMRC1, did not relieve the hMRC1 antiviral activity, suggesting the involvement of additional cellular factor(s) in the process. Our data reveal an antiviral mechanism that is active in primary human macrophages and is counteracted by HIV-1 through downregulation of hMRC1.