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1.
Nat Commun ; 10(1): 2753, 2019 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-31266936

RESUMO

Elimination of HIV-1 requires clearance and removal of integrated proviral DNA from infected cells and tissues. Here, sequential long-acting slow-effective release antiviral therapy (LASER ART) and CRISPR-Cas9 demonstrate viral clearance in latent infectious reservoirs in HIV-1 infected humanized mice. HIV-1 subgenomic DNA fragments, spanning the long terminal repeats and the Gag gene, are excised in vivo, resulting in elimination of integrated proviral DNA; virus is not detected in blood, lymphoid tissue, bone marrow and brain by nested and digital-droplet PCR as well as RNAscope tests. No CRISPR-Cas9 mediated off-target effects are detected. Adoptive transfer of human immunocytes from dual treated, virus-free animals to uninfected humanized mice fails to produce infectious progeny virus. In contrast, HIV-1 is readily detected following sole LASER ART or CRISPR-Cas9 treatment. These data provide proof-of-concept that permanent viral elimination is possible.


Assuntos
Fármacos Anti-HIV/administração & dosagem , Sistemas CRISPR-Cas , Infecções por HIV/terapia , HIV-1/genética , Transferência Adotiva , Animais , Terapia Combinada , DNA Viral/genética , DNA Viral/imunologia , Edição de Genes , Infecções por HIV/tratamento farmacológico , Infecções por HIV/imunologia , Infecções por HIV/virologia , HIV-1/imunologia , HIV-1/fisiologia , Humanos , Camundongos , Resultado do Tratamento , Latência Viral
2.
Adv Exp Med Biol ; 1140: 69-83, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31317496

RESUMO

Productive HIV infection requires integration of viral genes into the host genome. But how viral DNA gets to the nucleus in the first place remains one of the most controversial yet deceptively simple questions in HIV post-entry biology. This is illustrated in cartoons of viral entry, which often depict the entry process as an 'explosion' of the HIV capsid in the cytosol and independent movement of viral DNA through nuclear pores and into the nucleus. HIV enters the cell cytosol with two encapsidated RNA strands and must undergo reverse transcription (RT) to synthesise DNA. Even here there is no consensus for where, when or how RT happens. HIV must get into the nucleus, which in a non-dividing cell requires transport through the nuclear pore. Finally, the virus must 'uncoat': shed its protein capsid to allow its DNA to be spliced with that of the host. Where the virus uncoats and whether this is a single or multi-step process are similarly hotly debated. Understanding these processes is further complicated by three broad factors. First, that there are inter-relationships between these processes that may ensure HIV undergoes the right step at the right place at the right time. Second, the host has cofactors which the virus is dependent upon and must recruit but also immune factors that can sense and inhibit virus and so must be avoided. Third, HIV post-entry biology is cell-type dependent-meaning that factors which are essential in one cell type can be redundant in another.


Assuntos
Capsídeo , Infecções por HIV , HIV-1 , Capsídeo/metabolismo , Proteínas do Capsídeo/metabolismo , Infecções por HIV/virologia , HIV-1/fisiologia , Humanos , RNA Viral/genética
3.
BMC Bioinformatics ; 20(1): 398, 2019 Jul 17.
Artigo em Inglês | MEDLINE | ID: mdl-31315557

RESUMO

BACKGROUND: Utilization of quantitative proteomics data on the network level is still a challenge in proteomics data analysis. Currently existing models use sophisticated, sometimes hard to implement analysis techniques. Our aim was to generate a relatively simple strategy for quantitative proteomics data analysis in order to utilize as much of the data generated in a proteomics experiment as possible. RESULTS: In this study, we applied label-free proteomics, and generated a network model utilizing both qualitative, and quantitative data, in order to examine the early host response to Human Immunodeficiency Virus type 1 (HIV-1). A weighted network model was generated based on the amount of proteins measured by mass spectrometry, and analysis of weighted networks and functional sub-networks revealed upregulation of proteins involved in translation, transcription, and DNA condensation in the early phase of the viral life-cycle. CONCLUSION: A relatively simple strategy for network analysis was created and applied to examine the effect of HIV-1 on host cellular proteome. We believe that our model may prove beneficial in creating algorithms, allowing for both quantitative and qualitative studies of proteome change in various biological and pathological processes by quantitative mass spectrometry.


Assuntos
HIV-1/fisiologia , Proteômica/métodos , HIV-1/genética , Humanos , Espectrometria de Massas , Mapeamento de Interação de Proteínas , Proteoma/metabolismo , Transdução Genética
4.
BMC Infect Dis ; 19(1): 588, 2019 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-31277590

RESUMO

BACKGROUND: HIV controllers (HICs) are a rare group of HIV-1-infected individuals able to naturally control viral replication. Several studies have identified the occurrence of HIV dual infections in seropositive individuals leading to disease progression. In HICs, however, dual infections with divergent outcomes in pathogenesis have been described. CASE PRESENTATION: Here, we present a case report of a HIC diagnosed in late 1999 who displayed stable CD4+ T cell levels and low plasmatic viral load across 12 years of follow-up. In early 2013, the patient started to present an increase in viral load, reaching a peak of 10,000 copies/ml in early 2014, followed by an oscillation of viremia at moderate levels in the following years. The genetic diversity of env proviral quasispecies from peripheral blood mononuclear cells (PBMCs) was studied by single genome amplification (SGA) at six timepoints across 2009-2017. Phylogenetic analyses of env sequences from 2009 and 2010 samples showed the presence of a single subtype B variant (called B1). Analyses of sequences from 2011 and after revealed an additional subtype B variant (called B2) and a subsequent dominance shift in the proviral quasispecies frequencies, with the B2 variant becoming the most frequent from 2014 onwards. Latent syphilis related to unprotected sexual intercourse was diagnosed a year before the first detection of B2, evidencing risk behavior and supporting the superinfection hypothesis. Immunologic analyses revealed an increase in CD8+ and CD4+ T cell immune activation following viremia increase and minor T cell subset alterations during follow-up. HIV-specific T cell responses remained low throughout the follow-up period. CONCLUSIONS: Altogether, these results show that loss of viremia control in the HIC was associated with superinfection. These data alert to the negative consequences of reinfection on HIV pathogenesis, even in patients with a long history of viremia control and an absence of disease progression, reinforcing the need for continued use of adequate prevention strategies.


Assuntos
Infecções por HIV/virologia , HIV-1/fisiologia , Superinfecção/virologia , Replicação Viral/fisiologia , Adulto , Fármacos Anti-HIV/uso terapêutico , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/virologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/virologia , Feminino , Seguimentos , Infecções por HIV/tratamento farmacológico , HIV-1/isolamento & purificação , HIV-1/patogenicidade , Antígenos HLA-B/genética , Humanos , Leucócitos Mononucleares/virologia , Masculino , Filogenia , RNA Viral/sangue , Sífilis/diagnóstico , Carga Viral , Viremia/tratamento farmacológico , Viremia/virologia
5.
Nat Commun ; 10(1): 2144, 2019 05 13.
Artigo em Inglês | MEDLINE | ID: mdl-31086185

RESUMO

Pathogens face varying microenvironments in vivo, but suitable experimental systems and analysis tools to dissect how three-dimensional (3D) tissue environments impact pathogen spread are lacking. Here we develop an Integrative method to Study Pathogen spread by Experiment and Computation within Tissue-like 3D cultures (INSPECT-3D), combining quantification of pathogen replication with imaging to study single-cell and cell population dynamics. We apply INSPECT-3D to analyze HIV-1 spread between primary human CD4 T-lymphocytes using collagen as tissue-like 3D-scaffold. Measurements of virus replication, infectivity, diffusion, cellular motility and interactions are combined by mathematical analyses into an integrated spatial infection model to estimate parameters governing HIV-1 spread. This reveals that environmental restrictions limit infection by cell-free virions but promote cell-associated HIV-1 transmission. Experimental validation identifies cell motility and density as essential determinants of efficacy and mode of HIV-1 spread in 3D. INSPECT-3D represents an adaptable method for quantitative time-resolved analyses of 3D pathogen spread.


Assuntos
Linfócitos T CD4-Positivos/virologia , HIV-1/patogenicidade , Modelos Biológicos , Cultura Primária de Células/métodos , Fenômenos Fisiológicos Virais , Linfócitos T CD4-Positivos/fisiologia , Movimento Celular , Células Cultivadas , Simulação por Computador , Células HEK293 , HIV-1/fisiologia , Voluntários Saudáveis , Humanos
6.
Nat Immunol ; 20(6): 711-723, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31061530

RESUMO

Resting CD4+ T cells are highly resistant to the production of human immunodeficiency virus type 1 (HIV-1). However, the mechanism by which resting CD4+ T cells restrict such production in the late viral replication phase of infection has remained unclear. In this study, we found that the cell membrane metalloprotease TRAB domain-containing protein 2A (TRABD2A) inhibited this production in resting CD4+ T cells by degrading the virion structural precursor polyprotein Gag at the plasma membrane. Depletion or inhibition of metalloprotease activity by TRABD2A profoundly enhanced HIV-1 production in resting CD4+ T cells. TRABD2A expression was much higher in resting CD4+ T cells than in activated CD4+ T cells and was considerably reduced by T cell activation. Moreover, reexpressing TRABD2A reinforced the resistance of activated CD4+ T cells to the production of HIV-1 progeny. Collectively, these results elucidate the molecular mechanism employed by resting CD4+ T cells to potently restrict the assembly and production of HIV-1 progeny.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Infecções por HIV/genética , Infecções por HIV/imunologia , Infecções por HIV/virologia , HIV-1/fisiologia , Metaloproteases/genética , Replicação Viral , Produtos do Gene gag do Vírus da Imunodeficiência Humana/metabolismo , Animais , Linfócitos T CD4-Positivos/metabolismo , Cátions , Linhagem Celular , Ativação Enzimática , Expressão Gênica , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Humanos , Proteínas de Membrana/metabolismo , Metaloproteases/antagonistas & inibidores , Metaloproteases/metabolismo , Família Multigênica , Proteólise , Proteína 1 com Domínio SAM e Domínio HD/genética , Proteína 1 com Domínio SAM e Domínio HD/metabolismo , Carga Viral
7.
BMC Bioinformatics ; 20(1): 271, 2019 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-31138124

RESUMO

BACKGROUND: Networks have been widely used to model the structures of various biological systems. The ultimate aim of research on biological networks is to steer biological system structures to desired states by manipulating signals. Despite great advances in the linear control of single-layer networks, it has been observed that many complex biological systems have a multilayer networked structure and extremely complicated nonlinear processes. RESULT: In this study, we propose a general framework for controlling nonlinear dynamical systems with multilayer networked structures by formulating the problem as a minimum union optimization problem. In particular, we offer a novel approach for identifying the minimal driver nodes that can steer a multilayered nonlinear dynamical system toward any desired dynamical attractor. Three disease-related biology multilayer networks are used to demonstrate the effectiveness of our approaches. Moreover, in the set of minimum driver nodes identified by the algorithm we proposed, we confirmed that some nodes can act as drug targets in the biological experiments. Other nodes have not been reported as drug targets; however, they are also involved in important biological processes from existing literature. CONCLUSIONS: The proposed method could be a promising tool for determining higher drug target enrichment or more meaningful steering nodes for studying complex diseases.


Assuntos
Doença , Redes Reguladoras de Genes , Algoritmos , Comunicação Celular , Colite/complicações , Colite/genética , Neoplasias do Colo/complicações , Neoplasias do Colo/genética , Bases de Dados como Assunto , HIV-1/fisiologia , Humanos , Dinâmica não Linear
8.
Cell Mol Life Sci ; 76(18): 3583-3600, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31129856

RESUMO

35 years since identification of HIV as the causative agent of AIDS, and 35 million deaths associated with this disease, significant effort is now directed towards the development of potential cures. Current anti-retroviral (ART) therapies for HIV/AIDS can suppress virus replication to undetectable levels, and infected individuals can live symptom free so long as treatment is maintained. However, removal of therapy allows rapid re-emergence of virus from a highly stable reservoir of latently infected cells that exist as a barrier to elimination of the infection with current ART. Prospects of a cure for HIV infection are significantly encouraged by two serendipitous cases where individuals have entered remission following stem cell transplantation from compatible HIV-resistant donors. However, development of a routine cure that could become available to millions of infected individuals will require a means of specifically purging cells harboring latent HIV, preventing replication of latent provirus, or destruction of provirus genomes by gene editing. Elimination of latently infected cells will require a means of exposing this population, which may involve identification of a natural specific biomarker or therapeutic intervention to force their exposure by reactivation of virus expression. Accordingly, the proposed "Shock and Kill" strategy involves treatment with latency-reversing agents (LRA) to induce HIV provirus expression thus exposing these cells to killing by cellular immunity or apoptosis. Current efforts to enable this strategy are directed at developing improved combinations of LRA to produce broad and robust induction of HIV provirus and enhancing the elimination of cells where replication has been reactivated by targeted immune modulation. Alternative strategies may involve preventing re-emergence virus from latently infected cells by "Lock and Block" intervention, where transcription of provirus is inhibited to prevent virus spread or disruption of the HIV provirus genome by genome editing.


Assuntos
Reservatórios de Doenças/virologia , Infecções por HIV/terapia , HIV-1/fisiologia , Antirretrovirais/uso terapêutico , Edição de Genes , Infecções por HIV/tratamento farmacológico , Infecções por HIV/imunologia , Humanos , Imunidade Celular , Imunoterapia , Proteínas Recombinantes/uso terapêutico , Latência Viral
9.
BMC Res Notes ; 12(1): 242, 2019 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-31036079

RESUMO

OBJECTIVE: Resting CD4+ T cells are major reservoirs of latent HIV-1 infection, and may be formed during the early phase of the infection. Although CCR5-tropic (R5) HIV-1 is highly transmissible during the early phase, newly infected individuals have usually been exposed to a mixture of R5 and CXCR4-tropic (X4) viruses, and X4 viral DNA is also detectable in the host. Our aim was to identify which subsets of resting CD4+ T cells contribute to forming the latent reservoir in the presence of both X4 and R5 viruses. RESULTS: Primary resting CD4+ naïve T (TN) cells, CCR5- memory T (TM) cells, and CCR5+ TM cells isolated by flow cytometry were infected simultaneously with X4 and R5 HIV-1, which harbored different reporter genes, and were cultured in the resting condition. Flow cytometry at 3 days post-infection demonstrated that X4 HIV-1+ cells were present in all three subsets of cells, whereas R5 HIV-1+ cells were present preferentially in CCR5+ TM cells, but not in TN cells. Following CD3/CD28-mediated activation at 3 days post-infection, numbers of R5 HIV-1+ cells and X4 HIV-1+ cells increased significantly only in the CCR5+ TM subset, suggesting that it provides a major reservoir of replication-competent, latently infected viruses.


Assuntos
Linfócitos T CD4-Positivos/virologia , DNA Viral/genética , HIV-1/fisiologia , Receptores CCR5/genética , Receptores CXCR4/genética , Latência Viral , Adulto , Linfócitos T CD4-Positivos/imunologia , DNA Viral/metabolismo , Expressão Gênica , HIV-1/patogenicidade , Voluntários Saudáveis , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Humanos , Memória Imunológica , Cultura Primária de Células , Receptores CCR5/imunologia , Receptores CXCR4/imunologia , Tropismo Viral , Replicação Viral
11.
Retrovirology ; 16(1): 12, 2019 04 29.
Artigo em Inglês | MEDLINE | ID: mdl-31036027

RESUMO

BACKGROUND: The different interactions between viral proteins and cellular host proteins are required for efficient replication of HIV-1. Various reports implicated host cellular proteins as a key factor that either interact directly with HIV-1 integrase (IN) or get involved in the integration process of virus resulting in the modulation of integration step. Polypyrimidine tract binding protein and associated splicing factor (PSF) has diverse functions inside the cell such as transcriptional regulation, DNA repair, acts as nucleic acids binding protein and regulate replication and infectivity of different viruses. RESULTS: The protein binding study identified the association of host protein PSF with HIV-1 integrase. The siRNA knockdown (KD) of PSF resulted in increased viral replication in TZM-bl cells, suggesting PSF has negative influence on viral replication. The quantitative PCR of virus infected PSF knockdown TZM-bl cells showed more integrated DNA and viral cDNA as compared to control cells. We did not observe any significant difference between the amount of early reverse transcription products as well as infectivity of virus in the PSF KD and control TZM-bl cells. Molecular docking study supported the argument that PSF hinders the binding of viral DNA with IN. CONCLUSION: In an attempt to study the host interacting protein of IN, we have identified a new interacting host protein PSF which is a splicing factor and elucidated its role in integration and viral replication. Experimental as well as in silico analysis inferred that the host protein causes not only change in the integration events but also targets the incoming viral DNA or the integrase-viral DNA complex. The role of PSF was also investigated at early reverse transcript production as well as late stages. The PSF is causing changes in integration events, but it does not over all make any changes in the virus infectivity. MD trajectory analyses provided a strong clue of destabilization of Integrase-viral DNA complex occurred due to PSF interaction with the conserved bases of viral DNA ends that are extremely crucial contact points with integrase and indispensable for integration. Thus our study emphasizes the negative influence of PSF on HIV-1 replication.


Assuntos
DNA Viral/metabolismo , Integrase de HIV/metabolismo , Interações entre Hospedeiro e Microrganismos , Proteína de Ligação a Regiões Ricas em Polipirimidinas/metabolismo , Fatores de Processamento de RNA/metabolismo , Replicação Viral , DNA Viral/genética , Técnicas de Silenciamento de Genes , Células HEK293 , Integrase de HIV/genética , HIV-1/fisiologia , Humanos , Simulação de Acoplamento Molecular , Proteína de Ligação a Regiões Ricas em Polipirimidinas/genética , Ligação Proteica , Processamento de RNA , Fatores de Processamento de RNA/genética , RNA Interferente Pequeno , Transcrição Reversa , Integração Viral
12.
BMC Infect Dis ; 19(1): 467, 2019 May 24.
Artigo em Inglês | MEDLINE | ID: mdl-31126239

RESUMO

BACKGROUND: The circumstances of prescription of tropism tests clinically relevant in treatment-experienced patients are unclear. METHODS: We performed a monocentric retrospective analysis of all tropism tests performed between 2006 and 2015 in HIV-infected patients on antiretroviral therapy (ART) without MVC. The motivation of tropism determination was collected. Factors associated with MVC prescription were determined using logistic regression analysis. RESULTS: Five hundred sixty-three tests were performed in experienced patients not receiving MVC. Reasons for tropism performance were: virological failure (44%), side effects or drug-interactions (37%), simplification or sparing strategies (11%), immunological failure (5%), and improvement of neurological diffusion (3%). MVC was prescribed in 110 cases (20%), though 366 tests (65%) revealed a tropism CCR5. MVC was more often prescribed before 2011 (OR 3.65, 95% CI 2.17-6.13) and in patients with multiple previous ART regimens (less than 4 ART regimens compare to more than 10 ART regimens (OR 0.34, 95% CI 0.15-0.74)). CONCLUSIONS: In experienced patients not receiving MVC, tropism test prescription should be restricted to patients with virological failure and limited therapeutic options such as patients already treated with a wide range of ART regimens.


Assuntos
Fármacos Anti-HIV/uso terapêutico , Infecções por HIV/tratamento farmacológico , Infecções por HIV/virologia , HIV-1/fisiologia , Tropismo Viral , Adulto , Antagonistas dos Receptores CCR5/uso terapêutico , Feminino , HIV-1/genética , Humanos , Masculino , Maraviroc/uso terapêutico , Pessoa de Meia-Idade , Estudos Retrospectivos , Falha de Tratamento , Resultado do Tratamento
13.
Comput Biol Chem ; 80: 419-432, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31146118

RESUMO

Determination of HIV-1 coreceptor usage is strongly recommended before starting the coreceptor-specific inhibitors for HIV treatment. Currently, the genotypic assays are the most interesting tools due to they are more feasible than phenotypic assays. However, most of prediction models were developed and validated by data set of HIV-1 subtype B and C. The present study aims to develop a powerful and reliable model to accurately predict HIV-1 coreceptor usage for CRF01_AE subtype called HIVCoR. HIVCoR utilized random forest and support vector machine as the prediction model, together with amino acid compositions, pseudo amino acid compositions and relative synonymous codon usage frequencies as the input feature. The overall success rate of 93.79% was achieved from the external validation test on the objective benchmark dataset. Comparison results indicated that HIVCoR was superior to other bioinformatics tools and genotypic predictors. For the convenience of experimental scientists, a user-friendly webserver has been established at http://codes.bio/hivcor/.


Assuntos
Biologia Computacional/métodos , Proteína gp120 do Envelope de HIV/metabolismo , HIV-1/classificação , Interações Hospedeiro-Patógeno , Receptores CCR5/metabolismo , Receptores CXCR4/metabolismo , Algoritmos , Sequência de Aminoácidos , Bases de Dados de Proteínas , Genótipo , Proteína gp120 do Envelope de HIV/química , Proteína gp120 do Envelope de HIV/genética , HIV-1/genética , HIV-1/fisiologia , Interações Hospedeiro-Patógeno/fisiologia , Humanos , Modelos Biológicos , Ligação Proteica , Máquina de Vetores de Suporte
14.
Nanomedicine (Lond) ; 14(9): 1095-1107, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31066644

RESUMO

Aim: Polyanionic carbosilane dendrimers have been shown to be safe and block human immunodeficiency virus type 1 (HIV-1) infection in a multifunctional manner. The aim of this study is to evaluate the appearance of HIV-1 resistance mutations after treatment with polyanionic carbosilane dendrimers. Materials & methods: A resistance mutation assay was performed on MT2 cells, viral quantity was measured by ELISA HIVp24gag and titration was carried out on TZM.bl. Next generation sequencing for HIV-1 Env was performed on G1-S4 or G2-S16 dendrimers supernatants. Results: Data showed the appearance of mutation resistance to G1-S4 treatment, inducing three significant mutations. G2-S16 did not generate any mutations and, furthermore, inhibited G1-S4-resistant viruses. Conclusion: G1-S4 treatment generates significant mutations in HIV-1NL4.3. G2-S16 does not generate resistance-associated mutation, suggesting that G2-S16 is safe as a HIV-entry inhibitor.


Assuntos
Fármacos Anti-HIV/farmacologia , Dendrímeros/farmacologia , HIV-1/efeitos dos fármacos , Silanos/farmacologia , Linhagem Celular , Dendrímeros/química , Farmacorresistência Viral , Proteína gp120 do Envelope de HIV/genética , HIV-1/fisiologia , Humanos , Mutação , Silanos/química , Falha de Tratamento , Internalização do Vírus/efeitos dos fármacos
15.
Virol J ; 16(1): 42, 2019 04 02.
Artigo em Inglês | MEDLINE | ID: mdl-30940169

RESUMO

BACKGROUND: HIV integrase (IN) and its cellular cofactors, including lens-epithelium-derived growth factor (LEDGF/p75), Ku70, p300, and Rad52, are subject to small ubiquitin-like modifier (SUMO) modification. In addition to covalent SUMOylation, SUMO paralogs can also noncovalently bind proteins through SUMO-interacting motifs (SIMs). However, little is known about whether HIV IN contains SIMs and the roles of these motifs. RESULTS: We searched for the amino acid sequence of HIV IN and investigated three putative SIMs of IN: SIM1 72VILV75, SIM2 200IVDI203 and SIM3 257IKVV260. Our mutational analysis showed that 200IVDI203 and 257IKVV260 are two bona fide SIMs that mediate IN-SUMO noncovalent interactions. Additionally, a cell-based SUMOylation assay revealed that IN SIMs negatively regulate the SUMOylation of IN, as well as the interaction between IN and SUMO E2 conjugation enzyme Ubc9. Conversely, IN SIMs are required for its interactions with LEDGF/p75 but not with Ku70. Furthermore, our study reveals that SIM2 and SIM3 are required for the nuclear localization of IN. Finally, we investigated the impact of IN SIM2 and SIM3 on HIV single cycle replication in CD4+ C8166 T cells, and the results showed that viruses carrying IN SIM mutants are replication defective at the steps of the early viral life cycle, including reverse transcription, nuclear import and integration. CONCLUSION: Our data suggested that the INSIM-SUMO interaction constitutes a new regulatory mechanism of IN functions and might be important for HIV-1 replication.


Assuntos
Integrase de HIV/metabolismo , HIV-1/fisiologia , Proteína SUMO-1/metabolismo , Sumoilação , Replicação Viral , Motivos de Aminoácidos , Células HEK293 , Integrase de HIV/genética , HIV-1/enzimologia , Humanos , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Reação em Cadeia da Polimerase em Tempo Real
16.
Virol Sin ; 34(2): 119-134, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31028522

RESUMO

Human immunodeficiency virus-1 capsid (HIV-1 CA) is involved in different stages of the viral replication cycle. During virion assembly, CA drives the formation of the hexameric lattice in immature viral particles, while in mature virions CA monomers assemble in cone-shaped cores surrounding the viral RNA genome and associated proteins. In addition to its functions in late stages of the viral replication cycle, CA plays key roles in a number of processes during early phases of HIV-1 infection including trafficking, uncoating, recognition by host cellular proteins and nuclear import of the viral pre-integration complex. As a result of efficient cooperation of CA with other viral and cellular proteins, integration of the viral genetic material into the host genome, which is an essential step for productive viral infection, successfully occurs. In this review, we will summarize available data on CA functions in HIV-1 replication, describing in detail its roles in late and early phases of the viral replication cycle.


Assuntos
Proteínas do Capsídeo/genética , Capsídeo/metabolismo , HIV-1/fisiologia , Replicação Viral , Infecções por HIV , Humanos , Montagem de Vírus
17.
PLoS Comput Biol ; 15(4): e1006849, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30978183

RESUMO

Quantitative viral outgrowth assays (QVOA) use limiting dilutions of CD4+ T cells to measure the size of the latent HIV-1 reservoir, a major obstacle to curing HIV-1. Efforts to reduce the reservoir require assays that can reliably quantify its size in blood and tissues. Although QVOA is regarded as a "gold standard" for reservoir measurement, little is known about its accuracy and precision or about how cell storage conditions or laboratory-specific practices affect results. Owing to this lack of knowledge, confidence intervals around reservoir size estimates-as well as judgments of the ability of therapeutic interventions to alter the size of the replication-competent but transcriptionally inactive latent reservoir-rely on theoretical statistical assumptions about dilution assays. To address this gap, we have carried out a Bayesian statistical analysis of QVOA reliability on 75 split samples of peripheral blood mononuclear cells (PBMC) from 5 antiretroviral therapy (ART)-suppressed participants, measured using four different QVOAs at separate labs, estimating assay precision and the effect of frozen cell storage on estimated reservoir size. We found that typical assay results are expected to differ from the true value by a factor of 1.6 to 1.9 up or down. Systematic assay differences comprised a 24-fold range between the assays with highest and lowest scales, likely reflecting differences in viral outgrowth readout and input cell stimulation protocols. We also found that controlled-rate freezing and storage of samples did not cause substantial differences in QVOA compared to use of fresh cells (95% probability of < 2-fold change), supporting continued use of frozen storage to allow transport and batched analysis of samples. Finally, we simulated an early-phase clinical trial to demonstrate that batched analysis of pre- and post-therapy samples may increase power to detect a three-fold reservoir reduction by 15 to 24 percentage points.


Assuntos
Infecções por HIV/virologia , HIV-1 , Carga Viral/métodos , Latência Viral , Fármacos Anti-HIV/uso terapêutico , Teorema de Bayes , Linfócitos T CD4-Positivos/virologia , Biologia Computacional , Simulação por Computador , Infecções por HIV/tratamento farmacológico , HIV-1/fisiologia , Humanos , Leucócitos Mononucleares/virologia , Funções Verossimilhança , Cadeias de Markov , Método de Monte Carlo , Reprodutibilidade dos Testes , Carga Viral/estatística & dados numéricos , Replicação Viral
18.
Retrovirology ; 16(1): 9, 2019 04 02.
Artigo em Inglês | MEDLINE | ID: mdl-30940160

RESUMO

BACKGROUND: We previously showed that the gM of HSV-1 could restrict the release of infectious HIV-1 from cells. In this study, we analyzed if the four HSV-1 glycoproteins (gD, gB, and gH/gL), which are the minimum glycoproteins required for HSV-1 entry, restricted the release of infectious HIV-1. RESULTS: Of these four glycoproteins, gD and gH/gL restricted the production of infectious HIV-1 from cells transfected with an infectious molecular clone of HIV-1 (strain NL4-3) while gB had no significant effect. Pulse-chase analyses indicated that gD did not affect the biosynthesis and processing of gp160 into gp120/gp41, the transport of the gp120/gp41 to the cell surface, or the release of HIV-1 particles from the cell surface. Our analyses revealed that gD was incorporated into HIV-1 virus particles while gp120/gp41 was excluded from released virus particles. Truncated mutants of gD revealed that the cytoplasmic domain was dispensable but that a membrane bound gD was required for the restriction of release of infectious HIV-1. Finally, cell lines expressing gD also potently restricted the release of infectious virus. CONCLUSIONS: Due to its ability to exclude HIV-1 gp120/gp41 from maturing virus, gD may provide a useful tool in deciphering mechanisms of Env incorporation into maturing virus particles.


Assuntos
HIV-1/fisiologia , Herpesvirus Humano 1/fisiologia , Glicoproteínas de Membrana/metabolismo , Proteínas do Envelope Viral/metabolismo , Internalização do Vírus , Linhagem Celular , Proteína gp120 do Envelope de HIV/genética , Proteína gp120 do Envelope de HIV/metabolismo , Proteína gp160 do Envelope de HIV/genética , Proteína gp160 do Envelope de HIV/metabolismo , Herpesvirus Humano 1/genética , Humanos , Glicoproteínas de Membrana/genética , Proteínas do Envelope Viral/genética
19.
Retrovirology ; 16(1): 8, 2019 04 02.
Artigo em Inglês | MEDLINE | ID: mdl-30940165

RESUMO

BACKGROUND: Persistence of latent, replication-competent provirus is the main impediment towards the cure of HIV infection. One of the critical questions concerning HIV latency is the role of integration site selection in HIV expression. Inhibition of the interaction between HIV integrase and its chromatin tethering cofactor LEDGF/p75 is known to reduce integration and to retarget residual provirus to regions resistant to reactivation. LEDGINs, small molecule inhibitors of the interaction between HIV integrase and LEDGF/p75, provide an interesting tool to study the underlying mechanisms. During early infection, LEDGINs block the interaction with LEDGF/p75 and allosterically inhibit the catalytic activity of IN (i.e. the early effect). When present during virus production, LEDGINs interfere with proper maturation due to enhanced IN oligomerization in the progeny virions (i.e. the late effect). RESULTS: We studied the effect of LEDGINs present during virus production on the transcriptional state of the residual virus. Infection of cells with viruses produced in the presence of LEDGINs resulted in a residual reservoir that was refractory to activation. Integration of residual provirus was less favored near epigenetic markers associated with active transcription. However, integration near H3K36me3 and active genes, both targeted by LEDGF/p75, was not affected. Also in primary cells, LEDGIN treatment induced a reservoir resistant to activation due to a combined early and late effect. CONCLUSION: LEDGINs present a research tool to study the link between integration and transcription, an essential question in retrovirology. LEDGIN treatment during virus production altered integration of residual provirus in a LEDGF/p75-independent manner, resulting in a reservoir that is refractory to activation.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , HIV-1/fisiologia , Fatores de Transcrição/genética , Integração Viral , Latência Viral , Replicação Viral , Linhagem Celular , Células Cultivadas , Integrase de HIV/genética , HIV-1/genética , Humanos , Leucócitos Mononucleares/virologia , Ligação Proteica , Provírus/fisiologia , Ativação Viral
20.
Retrovirology ; 16(1): 10, 2019 04 04.
Artigo em Inglês | MEDLINE | ID: mdl-30947724

RESUMO

BACKGROUND: Efficient HIV-1 replication depends on interaction of the viral capsid with the host protein cyclophilin A (CypA). CypA, a peptidylprolyl isomerase, binds to an exposed loop in the viral CA protein via the enzyme's active site. Recent structural analysis of CypA in complex with CA tubes in conjunction with molecular dynamics simulations identified a secondary CA binding site on CypA that allows a bridging interaction with two hexameric subunits of the assembled CA lattice, leading to capsid stabilization (Liu et al. in Nat Commun 7:10714, 2016). RESULTS: We performed mutational analysis of residues that have been proposed to mediate CA binding at the secondary binding site on CypA (A25, K27, P29 and K30) and tested the effects of the amino acid substitutions using interaction assays and HIV-1 infection assays in cells. The binding of recombinant CypA to self-assembled CA tubes or native HIV-1 capsids was measured in vitro using a quantitative fluorescence microscopy binding assay revealing that affinity and stoichiometry of CypA to the CA lattice was not affected by the substitutions. To test for functionality of the CypA secondary CA-binding site in HIV-1 infection, mutant CypA proteins were expressed in cells in which endogenous CypA was deleted, and the effects on HIV-1 infection were assayed. In normal HeLa-P4 cells, infection with HIV-1 bearing the A92E substitution in CA is inhibited by endogenous CypA and was inhibited to the same extent by expression of CypA mutants in CypA-null HeLa-P4 cells. Expression of the mutant CypA proteins in CypA-null Jurkat cells restored their permissiveness to infection by wild type HIV-1. CONCLUSIONS: The amino acid changes at A25, K27, P29 and K30 did not affect the affinity of CypA for the CA lattice and did not impair CypA function in infection assays suggesting that these residues are not part of a secondary CA binding site on CypA.


Assuntos
Capsídeo/metabolismo , Ciclofilina A/química , HIV-1/fisiologia , Interações entre Hospedeiro e Microrganismos , Replicação Viral , Aminoácidos , Sítios de Ligação , Proteínas do Capsídeo/metabolismo , Ciclofilina A/genética , Células HeLa , Humanos , Células Jurkat , Ligação Proteica , Vírion/fisiologia
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