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1.
Proc Natl Acad Sci U S A ; 121(33): e2407400121, 2024 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-39110735

RESUMO

HIV-1 transcript function is controlled in part by twinned transcriptional start site usage, where 5' capped RNAs beginning with a single guanosine (1G) are preferentially packaged into progeny virions as genomic RNA (gRNA) whereas those beginning with three sequential guanosines (3G) are retained in cells as mRNAs. In 3G transcripts, one of the additional guanosines base pairs with a cytosine located within a conserved 5' polyA element, resulting in formation of an extended 5' polyA structure as opposed to the hairpin structure formed in 1G RNAs. To understand how this remodeling influences overall transcript function, we applied in vitro biophysical studies with in-cell genome packaging and competitive translation assays to native and 5' polyA mutant transcripts generated with promoters that differentially produce 1G or 3G RNAs. We identified mutations that stabilize the 5' polyA hairpin structure in 3G RNAs, which promote RNA dimerization and Gag binding without sequestering the 5' cap. None of these 3G transcripts were competitively packaged, confirming that cap exposure is a dominant negative determinant of viral genome packaging. For all RNAs examined, conformations that favored 5' cap exposure were both poorly packaged and more efficiently translated than those that favored 5' cap sequestration. We propose that structural plasticity of 5' polyA and other conserved RNA elements place the 5' leader on a thermodynamic tipping point for low-energetic (~3 kcal/mol) control of global transcript structure and function.


Assuntos
Genoma Viral , HIV-1 , Conformação de Ácido Nucleico , Biossíntese de Proteínas , RNA Viral , HIV-1/genética , RNA Viral/genética , RNA Viral/metabolismo , RNA Viral/química , Humanos , Empacotamento do Genoma Viral , Mutação , Montagem de Vírus/genética , Capuzes de RNA/metabolismo , Capuzes de RNA/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
2.
PLoS Pathog ; 19(7): e1011492, 2023 07.
Artigo em Inglês | MEDLINE | ID: mdl-37459363

RESUMO

HIV-1 spreads efficiently through direct cell-to-cell transmission at virological synapses (VSs) formed by interactions between HIV-1 envelope proteins (Env) on the surface of infected cells and CD4 receptors on uninfected target cells. Env-CD4 interactions bring the infected and uninfected cellular membranes into close proximity and induce transport of viral and cellular factors to the VS for efficient virion assembly and HIV-1 transmission. Using novel, cell-specific stable isotope labeling and quantitative mass spectrometric proteomics, we identified extensive changes in the levels and phosphorylation states of proteins in HIV-1 infected producer cells upon mixing with CD4+ target cells under conditions inducing VS formation. These coculture-induced alterations involved multiple cellular pathways including transcription, TCR signaling and, unexpectedly, cell cycle regulation, and were dominated by Env-dependent responses. We confirmed the proteomic results using inhibitors targeting regulatory kinases and phosphatases in selected pathways identified by our proteomic analysis. Strikingly, inhibiting the key mitotic regulator Aurora kinase B (AURKB) in HIV-1 infected cells significantly increased HIV activity in cell-to-cell fusion and transmission but had little effect on cell-free infection. Consistent with this, we found that AURKB regulates the fusogenic activity of HIV-1 Env. In the Jurkat T cell line and primary T cells, HIV-1 Env:CD4 interaction also dramatically induced cell cycle-independent AURKB relocalization to the centromere, and this signaling required the long (150 aa) cytoplasmic C-terminal domain (CTD) of Env. These results imply that cytoplasmic/plasma membrane AURKB restricts HIV-1 envelope fusion, and that this restriction is overcome by Env CTD-induced AURKB relocalization. Taken together, our data reveal a new signaling pathway regulating HIV-1 cell-to-cell transmission and potential new avenues for therapeutic intervention through targeting the Env CTD and AURKB activity.


Assuntos
Infecções por HIV , HIV-1 , Humanos , HIV-1/fisiologia , Aurora Quinase B/metabolismo , Proteômica , Linfócitos T CD4-Positivos/metabolismo , Antígenos CD4/metabolismo , Infecções por HIV/metabolismo
3.
J Virol ; 96(1): e0134921, 2022 01 12.
Artigo em Inglês | MEDLINE | ID: mdl-34643428

RESUMO

HIV-1 virion production is driven by Gag and Gag-Pol (GP) proteins, with Gag forming the bulk of the capsid and driving budding, while GP binds Gag to deliver the essential virion enzymes protease, reverse transcriptase, and integrase. Virion GP levels are traditionally thought to reflect the relative abundances of GP and Gag in cells (∼1:20), dictated by the frequency of a -1 programmed ribosomal frameshifting (PRF) event occurring in gag-pol mRNAs. Here, we exploited a panel of PRF mutant viruses to show that mechanisms in addition to PRF regulate GP incorporation into virions. First, we show that GP is enriched ∼3-fold in virions relative to cells, with viral infectivity being better maintained at subphysiological levels of GP than when GP levels are too high. Second, we report that GP is more efficiently incorporated into virions when Gag and GP are synthesized in cis (i.e., from the same gag-pol mRNA) than in trans, suggesting that Gag/GP translation and assembly are spatially coupled processes. Third, we show that, surprisingly, virions exhibit a strong upper limit to trans-delivered GP incorporation; an adaptation that appears to allow the virus to temper defects to GP/Gag cleavage that may negatively impact reverse transcription. Taking these results together, we propose a "weighted Goldilocks" scenario for HIV-1 GP incorporation, wherein combined mechanisms of GP enrichment and exclusion buffer virion infectivity over a broad range of local GP concentrations. These results provide new insights into the HIV-1 virion assembly pathway relevant to the anticipated efficacy of PRF-targeted antiviral strategies. IMPORTANCE HIV-1 infectivity requires incorporation of the Gag-Pol (GP) precursor polyprotein into virions during the process of virus particle assembly. Mechanisms dictating GP incorporation into assembling virions are poorly defined, with GP levels in virions traditionally thought to solely reflect relative levels of Gag and GP expressed in cells, dictated by the frequency of a -1 programmed ribosomal frameshifting (PRF) event that occurs in gag-pol mRNAs. Herein, we provide experimental support for a "weighted Goldilocks" scenario for GP incorporation, wherein the virus exploits both random and nonrandom mechanisms to buffer infectivity over a wide range of GP expression levels. These mechanistic data are relevant to ongoing efforts to develop antiviral strategies targeting PRF frequency and/or HIV-1 virion maturation.


Assuntos
Mudança da Fase de Leitura do Gene Ribossômico , Regulação Viral da Expressão Gênica , Infecções por HIV/virologia , HIV-1/fisiologia , Montagem de Vírus , Produtos do Gene gag do Vírus da Imunodeficiência Humana/genética , Produtos do Gene gag do Vírus da Imunodeficiência Humana/metabolismo , Inibidores da Protease de HIV/farmacologia , HIV-1/efeitos dos fármacos , Humanos , Sequências Repetidas Invertidas , Modelos Biológicos , Conformação de Ácido Nucleico , Estabilidade de RNA , RNA Viral/química , RNA Viral/genética , Vírion , Replicação Viral
4.
PLoS Pathog ; 17(2): e1009364, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33635925

RESUMO

Previously, we reported that cellular transcription factor ZASC1 facilitates DNA-dependent/RNA-independent recruitment of HIV-1 TAT and the cellular elongation factor P-TEFb to the HIV-1 promoter and is a critical factor in regulating HIV-1 transcriptional elongation (PLoS Path e1003712). Here we report that cellular transcription factor ZBTB2 is a novel repressor of HIV-1 gene expression. ZBTB2 strongly co-immunoprecipitated with ZASC1 and was dramatically relocalized by ZASC1 from the cytoplasm to the nucleus. Mutations abolishing ZASC1/ZBTB2 interaction prevented ZBTB2 nuclear relocalization. We show that ZBTB2-induced repression depends on interaction of cellular histone deacetylases (HDACs) with the ZBTB2 POZ domain. Further, ZASC1 interaction specifically recruited ZBTB2 to the HIV-1 promoter, resulting in histone deacetylation and transcription repression. Depleting ZBTB2 by siRNA knockdown or CRISPR/CAS9 knockout in T cell lines enhanced transcription from HIV-1 vectors lacking Vpr, but not from these vectors expressing Vpr. Since HIV-1 Vpr activates the viral LTR by inducing the ATR kinase/DNA damage response pathway, we investigated ZBTB2 response to Vpr and DNA damaging agents. Expressing Vpr or stimulating the ATR pathway with DNA damaging agents impaired ZASC1's ability to localize ZBTB2 to the nucleus. Moreover, the effects of DNA damaging agents and Vpr on ZBTB2 localization could be blocked by ATR kinase inhibitors. Critically, Vpr and DNA damaging agents decreased ZBTB2 binding to the HIV-1 promoter and increased promoter histone acetylation. Thus, ZBTB2 is recruited to the HIV-1 promoter by ZASC1 and represses transcription, but ATR pathway activation leads to ZBTB2 removal from the promoter, cytoplasmic sequestration and activation of viral transcription. Together, our data show that ZASC1/ZBTB2 integrate the functions of TAT and Vpr to maximize HIV-1 gene expression.


Assuntos
Dano ao DNA , Proteínas de Ligação a DNA/metabolismo , Infecções por HIV/genética , HIV-1/genética , Proteínas Repressoras/metabolismo , Produtos do Gene tat do Vírus da Imunodeficiência Humana/metabolismo , Produtos do Gene vpr do Vírus da Imunodeficiência Humana/metabolismo , Acetilação , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Sistemas CRISPR-Cas , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/genética , Infecções por HIV/metabolismo , Infecções por HIV/patologia , Infecções por HIV/virologia , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Humanos , Regiões Promotoras Genéticas , Proteínas Repressoras/antagonistas & inibidores , Proteínas Repressoras/genética , Transcrição Gênica , Replicação Viral , Produtos do Gene tat do Vírus da Imunodeficiência Humana/genética , Produtos do Gene vpr do Vírus da Imunodeficiência Humana/genética
5.
PLoS Pathog ; 9(10): e1003712, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-24204263

RESUMO

Transcription from the HIV-1 LTR promoter efficiently initiates but rapidly terminates because of a non-processive form of RNA polymerase II. This premature termination is overcome by assembly of an HIV-1 TAT/P-TEFb complex at the transactivation response region (TAR), a structured RNA element encoded by the first 59 nt of HIV-1 mRNA. Here we have identified a conserved DNA-binding element for the cellular transcription factor, ZASC1, in the HIV-1 core promoter immediately upstream of TAR. We show that ZASC1 interacts with TAT and P-TEFb, co-operating with TAT to regulate HIV-1 gene expression, and promoting HIV-1 transcriptional elongation. The importance of ZASC1 to HIV-1 transcription elongation was confirmed through mutagenesis of the ZASC1 binding sites in the LTR promoter, shRNAs targeting ZASC1 and expression of dominant negative ZASC1. Chromatin immunoprecipitation analysis revealed that ZASC1 recruits Tat and P-TEFb to the HIV-1 core promoter in a TAR-independent manner. Thus, we have identified ZASC1 as novel regulator of HIV-1 gene expression that functions through the DNA-dependent, RNA-independent recruitment of TAT/P-TEFb to the HIV-1 promoter.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Infecções por HIV/metabolismo , Repetição Terminal Longa de HIV , HIV-1/metabolismo , Proteínas Nucleares/metabolismo , Fator B de Elongação Transcricional Positiva/metabolismo , Regiões Promotoras Genéticas , Elongação da Transcrição Genética , Produtos do Gene tat do Vírus da Imunodeficiência Humana/metabolismo , Proteínas de Ligação a DNA/genética , Infecções por HIV/genética , Infecções por HIV/patologia , HIV-1/genética , Células HeLa , Humanos , Células Jurkat , Proteínas Nucleares/genética , Fator B de Elongação Transcricional Positiva/genética , Produtos do Gene tat do Vírus da Imunodeficiência Humana/genética
6.
mBio ; 14(5): e0042023, 2023 Oct 31.
Artigo em Inglês | MEDLINE | ID: mdl-37676006

RESUMO

IMPORTANCE: Unlike humans, mice are unable to support HIV-1 infection. This is due, in part, to a constellation of defined minor, species-specific differences in conserved host proteins needed for viral gene expression. Here, we used precision CRISPR/Cas9 gene editing to engineer a "mousified" version of one such host protein, cyclin T1 (CCNT1), in human T cells. CCNT1 is essential for efficient HIV-1 transcription, making it an intriguing target for gene-based inactivation of virus replication. We show that isogenic cell lines engineered to encode CCNT1 bearing a single mouse-informed amino acid change (tyrosine in place of cysteine at position 261) exhibit potent, durable, and broad-spectrum resistance to HIV-1 and other pathogenic lentiviruses, and with no discernible impact on host cell biology. These results provide proof of concept for targeting CCNT1 in the context of one or more functional HIV-1 cure strategies.


Assuntos
Infecções por HIV , Soropositividade para HIV , HIV-1 , Humanos , Camundongos , Animais , HIV-1/fisiologia , Roedores , Linhagem Celular , Ciclina T/genética , Ciclina T/metabolismo , Expressão Gênica , Linfócitos T
7.
J Virol ; 84(15): 7473-83, 2010 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-20484494

RESUMO

To identify cellular processes involved in retroviral infection, we employed a high-volume forward genetic screen of insertionally mutagenized somatic cells using a murine leukemia virus (MLV) vector. This approach identified a clonal cell line that exhibited approximately 10-fold reduced gene expression from MLV vectors following infection despite supporting normal levels of MLV reverse transcription and integration. The defect in this cell line was specific for the MLV long terminal repeat (LTR) promoter, as normal levels of reporter gene expression were obtained from both an internal cytomegalovirus (CMV) promoter contained within an LTR-defective MLV vector and LTR expression from an avian sarcoma and leukosis virus (ASLV) vector. Complementation and shRNA knockdown experiments demonstrated that the defective gene in these cells is ZASC1 (ZNF639), a transcription factor with strong links to cancer and inherited ataxias. We demonstrated that ZASC1 is a sequence-specific DNA binding protein with three closely related binding sites located within the MLV LTR promoter, but it does not bind to the ASLV promoter. Mutating these putative ZASC1 binding sites significantly reduced levels of MLV gene expression. While wild-type ZASC1 activated expression from the MLV promoter, a green fluorescent protein-ZASC1 fusion protein showed dominant-negative inhibition of MLV gene expression. These studies identify the cellular transcription factor ZASC1 as an activator of MLV gene expression and provide tools that should be useful in studying the links between ZASC1 and human diseases.


Assuntos
Interações Hospedeiro-Patógeno , Vírus da Leucemia Murina/fisiologia , Fatores de Transcrição/fisiologia , Transcrição Gênica , Replicação Viral , Animais , Linhagem Celular , Cricetinae , Cricetulus , Deleção de Genes , Inativação Gênica , Teste de Complementação Genética , Mutagênese Insercional , Sequências Repetidas Terminais , Fatores de Transcrição/genética
8.
PLoS Pathog ; 4(11): e1000207, 2008 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-19008949

RESUMO

The early steps of retrovirus replication leading up to provirus establishment are highly dependent on cellular processes and represent a time when the virus is particularly vulnerable to antivirals and host defense mechanisms. However, the roles played by cellular factors are only partially understood. To identify cellular processes that participate in these critical steps, we employed a high volume screening of insertionally mutagenized somatic cells using a murine leukemia virus (MLV) vector. This approach identified a role for 3'-phosphoadenosine 5'-phosphosulfate synthase 1 (PAPSS1), one of two enzymes that synthesize PAPS, the high energy sulfate donor used in all sulfonation reactions catalyzed by cellular sulfotransferases. The role of the cellular sulfonation pathway was confirmed using chemical inhibitors of PAPS synthases and cellular sulfotransferases. The requirement for sulfonation was mapped to a stage during or shortly after MLV provirus establishment and influenced subsequent gene expression from the viral long terminal repeat (LTR) promoter. Infection of cells by an HIV vector was also shown to be highly dependent on the cellular sulfonation pathway. These studies have uncovered a heretofore unknown regulatory step of retroviral replication, have defined a new biological function for sulfonation in nuclear gene expression, and provide a potentially valuable new target for HIV/AIDS therapy.


Assuntos
Complexos Multienzimáticos/metabolismo , Provírus/fisiologia , Infecções por Retroviridae/etiologia , Sulfato Adenililtransferase/metabolismo , Sulfatos/metabolismo , Animais , Linhagem Celular , Regulação Viral da Expressão Gênica , Vetores Genéticos , HIV/genética , Humanos , Vírus da Leucemia Murina/genética , Transfecção , Replicação Viral
9.
Virology ; 471-473: 1-12, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25310595

RESUMO

Long-lived pools of latently infected cells are a significant barrier to the development of a cure for HIV-1 infection. A better understanding of the mechanisms of reactivation from latency is needed to facilitate the development of novel therapies that address this problem. Here we show that chemical inhibitors of the sulfonation pathway prevent virus reactivation, both in latently infected J-Lat and U1 cell lines and in a primary human CD4+ T cell model of latency. In each of these models, sulfonation inhibitors decreased transcription initiation from the HIV-1 promoter. These inhibitors block transcription initiation at a step that lies downstream of nucleosome remodeling and affects RNA polymerase II recruitment to the viral promoter. These results suggest that the sulfonation pathway acts by a novel mechanism to regulate efficient virus transcription initiation during reactivation from latency, and further that augmentation of this pathway could be therapeutically useful.


Assuntos
Fármacos Anti-HIV/farmacologia , Cloratos/farmacologia , Guaiacol/farmacologia , HIV-1/efeitos dos fármacos , Ativação Viral/efeitos dos fármacos , Fármacos Anti-HIV/administração & dosagem , Linfócitos T CD4-Positivos/virologia , Linhagem Celular , Cloratos/administração & dosagem , Quimioterapia Combinada , Regulação Viral da Expressão Gênica/fisiologia , Guaiacol/administração & dosagem , Repetição Terminal Longa de HIV , HIV-1/metabolismo , Humanos , NF-kappa B/genética , NF-kappa B/metabolismo , RNA Polimerase II/metabolismo , Ácidos Sulfônicos/antagonistas & inibidores , Ácidos Sulfônicos/metabolismo , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo , Latência Viral/fisiologia
10.
J Virol ; 79(20): 12969-78, 2005 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-16188999

RESUMO

In order to identify cellular proteins required for early stages of retroviral replication, a high volume screening with mammalian somatic cells was performed. Ten pools of chemically mutagenized Chinese hamster ovary (CHO-K1) cells were challenged with a murine leukemia virus (MLV) vector pseudotyped with the vesicular stomatitis virus glycoprotein (VSV-G), and cells that failed to be transduced were enriched by cell sorting. Each pool yielded a clonally derived cell line with a 5-fold or greater resistance to virus infection, and five cell lines exhibited a >50-fold resistance. These five cell lines were efficiently infected by a human immunodeficiency virus vector pseudotyped with VSV-G. When engineered to express the TVA receptor for subgroup A avian sarcoma and leukosis virus (ASLV-A), the five cell lines were resistant to infection with a MLV vector pseudotyped with the ASLV-A envelope protein but were fully susceptible to infection with an ASLV-A vector. Thus, the defect in these cells resides after virus-cell membrane fusion and, unlike those in other mutant cell lines that have been described, is specific for the MLV core. To identify the specific stages of MLV infection that are impaired in the resistant cell lines, real-time quantitative PCR analyses were employed and two phenotypic groups were identified. Viral infection of three cell lines was restricted before reverse transcription; in the other two cell lines, it was blocked after reverse transcription, nuclear localization, and two-long terminal repeat circle formation but before integration. These data provide genetic evidence that at least two distinct intracellular gene products are required specifically for MLV infection. These cell lines are important tools for the biochemical and genetic analysis of early stages in retrovirus infection.


Assuntos
Regulação Viral da Expressão Gênica , Vírus da Leucemia Murina/fisiologia , Animais , Células CHO , Células Clonais , Cricetinae , Vírus da Leucemia Murina/genética , Transcrição Reversa , Integração Viral , Replicação Viral
11.
J Virol ; 76(1): 195-207, 2002 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-11739685

RESUMO

Expression of most viral genes during productive infection by herpes simplex virus is regulated by the viral protein ICP4 (also called IE175 or Vmw175). The N-terminal portion of ICP4 contains well-defined transactivation, DNA binding, and dimerization domains that contribute to promoter regulation. The C-terminal half of ICP4 contributes to the activity of ICP4, but the functional motifs have not been well mapped. To localize functional motifs in the C-terminal half of ICP4, we have compared the relative specific activities of ICP4 variants in transient-transfection assays. Deletion of the C-terminal 56 residues reduces the specific activity more than 10-fold. Mutational analysis identified three consecutive residues (1252 to 1254) that are conserved in ICP4 orthologs and are essential for full activity, especially in the context of ICP4 variants with a deletion in the N-terminal transactivation domain. Recombinant viruses that encode variants of ICP4 with mutations in the N-terminal transactivation domain and/or the extreme C terminus were constructed. The phenotypes of these recombinant viruses support the hypothesis that efficient promoter activation by ICP4 requires motifs at both the N and C termini. The data suggest that the C terminus of ICP4 functions not as an independent transactivation domain but as an enhancer of the ICP4 N-terminal transactivation domain. The data provide further support for the hypothesis that some ICP4 motifs required for promoter activation are not required for promoter repression and suggest that ICP4 utilizes different cellular factors for activation or repression of viral promoters.


Assuntos
Proteínas Imediatamente Precoces/fisiologia , Simplexvirus/fisiologia , Sequência de Aminoácidos , Animais , Chlorocebus aethiops , Deleção de Genes , Proteínas Imediatamente Precoces/química , Proteínas Imediatamente Precoces/genética , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Alinhamento de Sequência , Simplexvirus/genética , Transcrição Gênica , Ativação Transcricional , Transfecção , Células Vero , Replicação Viral
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