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1.
Biophys J ; 122(11): 2216-2229, 2023 06 06.
Artigo em Inglês | MEDLINE | ID: mdl-36632034

RESUMO

Many transient processes in cells arise from the binding of cytosolic proteins to membranes. Quantifying this membrane binding and its associated diffusion in the living cell is therefore of primary importance. Dynamic photonic microscopies, e.g., single/multiple particle tracking, fluorescence recovery after photobleaching, and fluorescence correlation spectroscopy (FCS), enable non-invasive measurement of molecular mobility in living cells and their plasma membranes. However, FCS with a single beam waist is of limited applicability with complex, non-Brownian, motions. Recently, the development of FCS diffusion laws methods has given access to the characterization of these complex motions, although none of them is applicable to the membrane binding case at the moment. In this study, we combined computer simulations and FCS experiments to propose an FCS diffusion law for membrane binding. First, we generated computer simulations of spot-variation FCS (svFCS) measurements for a membrane binding process combined to 2D and 3D diffusion at the membrane and in the bulk/cytosol, respectively. Then, using these simulations as a learning set, we derived an empirical diffusion law with three free parameters: the apparent binding constant KD, the diffusion coefficient on the membrane D2D, and the diffusion coefficient in the cytosol, D3D. Finally, we monitored, using svFCS, the dynamics of retroviral Gag proteins and associated mutants during their binding to supported lipid bilayers of different lipid composition or at plasma membranes of living cells, and we quantified KD and D2D in these conditions using our empirical diffusion law. Based on these experiments and numerical simulations, we conclude that this new approach enables correct estimation of membrane partitioning and membrane diffusion properties (KD and D2D) for peripheral membrane molecules.


Assuntos
Bicamadas Lipídicas , Membrana Celular/metabolismo , Bicamadas Lipídicas/química , Membranas , Espectrometria de Fluorescência/métodos , Difusão
2.
RNA Biol ; 19(1): 191-205, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35067194

RESUMO

Maturation of the HIV-1 viral particles shortly after budding is required for infectivity. During this process, the Pr55Gag precursor undergoes a cascade of proteolytic cleavages, and whilst the structural rearrangements of the viral proteins are well understood, the concomitant maturation of the genomic RNA (gRNA) structure is unexplored, despite evidence that it is required for infectivity. To get insight into this process, we systematically analysed the interactions between Pr55Gag or its maturation products (NCp15, NCp9 and NCp7) and the 5' gRNA region and their structural consequences, in vitro. We show that Pr55Gag and its maturation products mostly bind at different RNA sites and with different contributions of their two zinc knuckle domains. Importantly, these proteins have different transient and permanent effects on the RNA structure, the late NCp9 and NCp7 inducing dramatic structural rearrangements. Altogether, our results reveal the distinct contributions of the different Pr55Gag maturation products on the gRNA structural maturation.


Assuntos
Regiões 5' não Traduzidas , Regulação Viral da Expressão Gênica , Infecções por HIV/virologia , HIV-1/fisiologia , RNA Viral/genética , Produtos do Gene gag do Vírus da Imunodeficiência Humana/metabolismo , Humanos , Conformação de Ácido Nucleico , Ligação Proteica , Processamento de Proteína Pós-Traducional , RNA Viral/química , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo , Montagem de Vírus , Replicação Viral
4.
J Biol Chem ; 293(9): 3168-3179, 2018 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-29187603

RESUMO

The type I interferons (IFNs) are a family of cytokines with diverse biological activities, including antiviral, antiproliferative, and immunoregulatory functions. The discovery of the hormonally regulated, constitutively expressed IFNϵ has suggested a function for IFNs in reproductive tract homeostasis and protection from infections, but its intrinsic activities are untested. We report here the expression, purification, and functional characterization of murine IFNϵ (mIFNϵ). Recombinant mIFNϵ (rmIFNϵ) exhibited an α-helical fold characteristic of type I IFNs and bound to IFNα/ß receptor 1 (IFNAR1) and IFNAR2, but, unusually, it had a preference for IFNAR1. Nevertheless, rmIFNϵ induced typical type I IFN signaling activity, including STAT1 phosphorylation and activation of canonical type I IFN signaling reporters, demonstrating that it uses the JAK-STAT signaling pathway. We also found that rmIFNϵ induces the activation of T, B, and NK cells and exhibits antiviral, antiproliferative, and antibacterial activities typical of type I IFNs, albeit with 100-1000-fold reduced potency compared with rmIFNα1 and rmIFNß. Surprisingly, although the type I IFNs generally do not display cross-species activities, rmIFNϵ exhibited high antiviral activity on human cells, suppressing HIV replication and inducing the expression of known HIV restriction factors in human lymphocytes. Our findings define the intrinsic properties of murine IFNϵ, indicating that it distinctly interacts with IFNAR and elicits pathogen-suppressing activity with a potency enabling host defense but with limited toxicity, appropriate for a protein expressed constitutively in a sensitive mucosal site, such as the reproductive tract.


Assuntos
Interferon Tipo I/química , Interferon Tipo I/metabolismo , Animais , Antibacterianos/química , Antibacterianos/metabolismo , Antibacterianos/farmacologia , Antivirais/química , Antivirais/metabolismo , Antivirais/farmacologia , Proliferação de Células/efeitos dos fármacos , Chlamydia/efeitos dos fármacos , Feminino , Humanos , Imunidade nas Mucosas , Interferon Tipo I/farmacologia , Camundongos , Fosforilação , Conformação Proteica em alfa-Hélice , Células RAW 264.7 , Receptores de Interferon/metabolismo , Reprodução , Fator de Transcrição STAT1/metabolismo , Transdução de Sinais
5.
J Virol ; 92(14)2018 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-29743369

RESUMO

Induction of persistent antibody responses by vaccination is generally thought to depend on efficient help by T follicular helper cells. Since the T helper cell response to HIV Env may not be optimal, we explored the possibility of improving the HIV Env antibody response to virus-like particle (VLP) vaccines by recruiting T helper cells induced by commonly used licensed vaccines to provide help for Env-specific B cells. B cells specific for the surface protein of a VLP can internalize the entire VLP and thus present peptides derived from the surface and core proteins on their major histocompatibility complex class II (MHC-II) molecules. This allows T helper cells specific for the core protein to provide intrastructural help for B cells recognizing the surface protein. Consistently, priming mice with an adjuvanted Gag protein vaccine enhanced the HIV Env antibody response to subsequent booster immunizations with HIV VLPs. To harness T helper cells induced by the licensed Tetanolpur vaccines, HIV VLPs that contained T helper cell epitopes of tetanus toxoid were generated. Tetanol-immunized mice raised stronger antibody responses to immunizations with VLPs containing tetanus toxoid T helper cell epitopes but not to VLPs lacking these epitopes. Depending on the priming immunization, the IgG subtype response to HIV Env after the VLP immunization could also be modified. Thus, harnessing T helper cells induced by other vaccines appears to be a promising approach to improve the HIV Env antibody response to VLP vaccines.IMPORTANCE Induction of HIV Env antibodies at sufficient levels with optimal Fc effector functions for durable protection remains a challenge. Efficient T cell help may be essential to induce such a desirable antibody response. Here, we provide proof of concept that T helper cells induced by a licensed vaccine can be harnessed to provide help for HIV Env-specific B cells and to modulate the Env-specific IgG subtype response.


Assuntos
Formação de Anticorpos/imunologia , Anticorpos Anti-HIV/imunologia , Infecções por HIV/imunologia , HIV-1/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Vacinas de Partículas Semelhantes a Vírus/administração & dosagem , Produtos do Gene env do Vírus da Imunodeficiência Humana/imunologia , Vacinas contra a AIDS/administração & dosagem , Animais , Células HEK293 , Infecções por HIV/prevenção & controle , Infecções por HIV/virologia , Humanos , Imunização Secundária , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Vacinação
6.
PLoS Pathog ; 13(2): e1006221, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-28222188

RESUMO

The interactions that occur during HIV Pr55Gag oligomerization and genomic RNA packaging are essential elements that facilitate HIV assembly. However, mechanistic details of these interactions are not clearly defined. Here, we overcome previous limitations in producing large quantities of full-length recombinant Pr55Gag that is required for isothermal titration calorimetry (ITC) studies, and we have revealed the thermodynamic properties of HIV assembly for the first time. Thermodynamic analysis showed that the binding between RNA and HIV Pr55Gag is an energetically favourable reaction (ΔG<0) that is further enhanced by the oligomerization of Pr55Gag. The change in enthalpy (ΔH) widens sequentially from: (1) Pr55Gag-Psi RNA binding during HIV genome selection; to (2) Pr55Gag-Guanosine Uridine (GU)-containing RNA binding in cytoplasm/plasma membrane; and then to (3) Pr55Gag-Adenosine(A)-containing RNA binding in immature HIV. These data imply the stepwise increments of heat being released during HIV biogenesis may help to facilitate the process of viral assembly. By mimicking the interactions between A-containing RNA and oligomeric Pr55Gag in immature HIV, it was noted that a p6 domain truncated Pr50Gag Δp6 is less efficient than full-length Pr55Gag in this thermodynamic process. These data suggest a potential unknown role of p6 in Pr55Gag-Pr55Gag oligomerization and/or Pr55Gag-RNA interaction during HIV assembly. Our data provide direct evidence on how nucleic acid sequences and the oligomeric state of Pr55Gag regulate HIV assembly.


Assuntos
HIV-1/fisiologia , Precursores de Proteínas/química , RNA Viral/química , Montagem de Vírus/fisiologia , Sequência de Aminoácidos , Sequência de Bases , Western Blotting , Calorimetria , Cromatografia , Imunoprecipitação , Microscopia Eletrônica , Termodinâmica
7.
J Struct Biol ; 203(2): 94-101, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29630922

RESUMO

Cryo-Electron Microscopy (cryo-EM) has become an invaluable tool for structural biology. Over the past decade, the advent of direct electron detectors and automated data acquisition has established cryo-EM as a central method in structural biology. However, challenges remain in the reliable and efficient preparation of samples in a manner which is compatible with high time resolution. The delivery of sample onto the grid is recognized as a critical step in the workflow as it is a source of variability and loss of material due to the blotting which is usually required. Here, we present a method for sample delivery and plunge freezing based on the use of Surface Acoustic Waves to deploy 6-8 µm droplets to the EM grid. This method minimises the sample dead volume and ensures vitrification within 52.6 ms from the moment the sample leaves the microfluidics chip. We demonstrate a working protocol to minimize the atomised volume and apply it to plunge freeze three different samples and provide proof that no damage occurs due to the interaction between the sample and the acoustic waves.


Assuntos
Acústica , Microscopia Crioeletrônica/métodos , Manejo de Espécimes/métodos , Congelamento , Microfluídica , Vitrificação
8.
Retrovirology ; 15(1): 23, 2018 Feb 23.
Artigo em Inglês | MEDLINE | ID: mdl-29471854

RESUMO

Cryo-electron microscopy has undergone a revolution in recent years and it has contributed significantly to a number of different areas in biological research. In this manuscript, we will describe some of the recent advancements in cryo-electron microscopy focussing on the advantages that this technique can bring rather than on the technology. We will then conclude discussing how the field of retrovirology has benefited from cryo-electron microscopy.


Assuntos
Microscopia Crioeletrônica , Retroviridae/fisiologia , Retroviridae/ultraestrutura , Animais , Microscopia Crioeletrônica/métodos , Humanos , Imageamento Tridimensional , Vírion/ultraestrutura
9.
Nat Methods ; 12(9): 866-72, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26237229

RESUMO

RNA regulates many biological processes; however, identifying functional RNA sequences and structures is complex and time-consuming. We introduce a method, mutational interference mapping experiment (MIME), to identify, at single-nucleotide resolution, the primary sequence and secondary structures of an RNA molecule that are crucial for its function. MIME is based on random mutagenesis of the RNA target followed by functional selection and next-generation sequencing. Our analytical approach allows the recovery of quantitative binding parameters and permits the identification of base-pairing partners directly from the sequencing data. We used this method to map the binding site of the human immunodeficiency virus-1 (HIV-1) Pr55(Gag) protein on the viral genomic RNA in vitro, and showed that, by analyzing permitted base-pairing patterns, we could model RNA structure motifs that are crucial for protein binding.


Assuntos
Mutagênese Sítio-Dirigida/métodos , Precursores de Proteínas/química , Precursores de Proteínas/genética , RNA Viral/química , RNA Viral/genética , Análise de Sequência de RNA/métodos , Sequência de Bases , Dados de Sequência Molecular , Mutação/genética , Relação Estrutura-Atividade
11.
RNA Biol ; 15(7): 923-936, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29954247

RESUMO

The Pr55Gag precursor specifically selects the HIV-1 genomic RNA (gRNA) from a large excess of cellular and partially or fully spliced viral RNAs and drives the virus assembly at the plasma membrane. During these processes, the NC domain of Pr55Gag interacts with the gRNA, while its C-terminal p6 domain binds cellular and viral factors and orchestrates viral particle release. Gag∆p6 is a truncated form of Pr55Gag lacking the p6 domain usually used as a default surrogate for wild type Pr55Gag for in vitro analysis. With recent advance in production of full-length recombinant Pr55Gag, here, we tested whether the p6 domain also contributes to the RNA binding specificity of Pr55Gag by systematically comparing binding of Pr55Gag and Gag∆p6 to a panel of viral and cellular RNAs. Unexpectedly, our fluorescence data reveal that the p6 domain is absolutely required for specific binding of Pr55Gag to the HIV-1 gRNA. Its deletion resulted not only in a decreased affinity for gRNA, but also in an increased affinity for spliced viral and cellular RNAs. In contrast Gag∆p6 displayed a similar affinity for all tested RNAs. Removal of the C-terminal His-tag from Pr55Gag and Gag∆p6 uniformly increased the Kd values of the RNA-protein complexes by ~ 2.5 fold but did not affect the binding specificities of these proteins. Altogether, our results demonstrate a novel role of the p6 domain in the specificity of Pr55Gag-RNA interactions, and strongly suggest that the p6 domain contributes to the discrimination of HIV-1 gRNA from cellular and spliced viral mRNAs, which is necessary for its selective encapsidation.


Assuntos
Genoma Viral/genética , HIV-1/fisiologia , Precursores de Proteínas/metabolismo , RNA Viral/metabolismo , Produtos do Gene gag do Vírus da Imunodeficiência Humana/metabolismo , Sítios de Ligação , Corantes Fluorescentes/química , Corantes Fluorescentes/metabolismo , HIV-1/genética , Humanos , Mutação , Plasmídeos , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Precursores de Proteínas/genética , RNA Viral/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Produtos do Gene gag do Vírus da Imunodeficiência Humana/química , Produtos do Gene gag do Vírus da Imunodeficiência Humana/genética
12.
Immunol Cell Biol ; 95(5): 478-483, 2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-28045025

RESUMO

Interferon epsilon (IFNɛ) is a type I IFN that is expressed constitutively in the female reproductive tract (FRT), and contributes to protection in models of sexually transmitted infections. Using multiple cell systems, including reporter cell lines and activated peripheral blood lymphocytes (PBLs), we show that recombinant IFNɛ impairs HIV infection at stage(s) post HIV entry and up to the translation of viral proteins. Consistent with this, IFNɛ upregulated a number of host cell restriction factors that block HIV at these stages of the replication cycle. The potency of IFNɛ induction of these HIV restriction factors was comparable to conventional type I IFNs, namely IFNα and IFNß. IFNɛ also significantly reduced the infectivity of progeny virion particles likely by inducing expression of HIV restriction factors, such as IFITM3, which act at that stage of infection. Thus, our data demonstrate that human IFNɛ suppresses HIV replication at multiple stages of infection.


Assuntos
Infecções por HIV/virologia , HIV-1/fisiologia , Interferons/metabolismo , Replicação Viral , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/patologia , Células Epiteliais/virologia , Infecções por HIV/patologia , Células HeLa , Humanos , Interferon-alfa/metabolismo , Fito-Hemaglutininas/farmacologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/patologia , Linfócitos T/virologia , Vírion/efeitos dos fármacos , Vírion/metabolismo , Replicação Viral/efeitos dos fármacos
13.
RNA Biol ; 14(1): 90-103, 2017 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-27841704

RESUMO

The HIV-1 Pr55Gag precursor specifically selects genomic RNA (gRNA) from a large variety of cellular and spliced viral RNAs (svRNAs), however the molecular mechanisms of this selective recognition remains poorly understood. To gain better understanding of this process, we analyzed the interactions between Pr55Gag and a large panel of viral RNA (vRNA) fragments encompassing the main packaging signal (Psi) and its flanking regions by fluorescence spectroscopy. We showed that the gRNA harbors a high affinity binding site which is absent from svRNA species, suggesting that this site might be crucial for selecting the HIV-1 genome. Our stoichiometry analysis of protein/RNA complexes revealed that few copies of Pr55Gag specifically associate with the 5' region of the gRNA. Besides, we found that gRNA dimerization significantly impacts Pr55Gag binding, and we confirmed that the internal loop of stem-loop 1 (SL1) in Psi is crucial for specific interaction with Pr55Gag. Our analysis of gRNA fragments of different length supports the existence of a long-range tertiary interaction involving sequences upstream and downstream of the Psi region. This long-range interaction might promote optimal exposure of SL1 for efficient Pr55Gag recognition. Altogether, our results shed light on the molecular mechanisms allowing the specific selection of gRNA by Pr55Gag among a variety of svRNAs, all harboring SL1 in their first common exon.


Assuntos
Genoma Viral , HIV-1/genética , HIV-1/metabolismo , Precursores de Proteínas/metabolismo , Splicing de RNA , RNA Viral/genética , RNA Viral/metabolismo , Sítios de Ligação , Humanos , Sequências Repetidas Invertidas , Cinética , Mutação , Conformação de Ácido Nucleico , RNA Viral/química , Proteínas de Ligação a RNA/metabolismo
14.
Retrovirology ; 13(1): 49, 2016 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-27459960

RESUMO

BACKGROUND: Eradication of HIV cannot be achieved with combination antiretroviral therapy (cART) because of the persistence of long-lived latently infected resting memory CD4(+) T cells. We previously reported that HIV latency could be established in resting CD4(+) T cells in the presence of the chemokine CCL19. To define how CCL19 facilitated the establishment of latent HIV infection, the role of chemokine receptor signalling was explored. RESULTS: In resting CD4(+) T cells, CCL19 induced phosphorylation of RAC-alpha serine/threonine-protein kinase (Akt), nuclear factor kappa B (NF-κB), extracellular-signal-regulated kinase (ERK) and p38. Inhibition of the phosphoinositol-3-kinase (PI3K) and Ras/Raf/Mitogen-activated protein kinase/ERK kinase (MEK)/ERK signalling pathways inhibited HIV integration, without significant reduction in HIV nuclear entry (measured by Alu-LTR and 2-LTR circle qPCR respectively). Inhibiting activation of MEK1/ERK1/2, c-Jun N-terminal kinase (JNK), activating protein-1 (AP-1) and NF-κB, but not p38, also inhibited HIV integration. We also show that HIV integrases interact with Pin1 in CCL19-treated CD4(+) T cells and inhibition of JNK markedly reduced this interaction, suggesting that CCL19 treatment provided sufficient signals to protect HIV integrase from degradation via the proteasome pathway. Infection of CCL19-treated resting CD4(+) T cells with mutant strains of HIV, lacking NF-κB binding sites in the HIV long terminal repeat (LTR) compared to infection with wild type virus, led to a significant reduction in integration by up to 40-fold (range 1-115.4, p = 0.03). This was in contrast to only a modest reduction of 5-fold (range 1.7-11, p > 0.05) in fully activated CD4(+) T cells infected with the same mutants. Finally, we demonstrated significant differences in integration sites following HIV infection of unactivated, CCL19-treated, and fully activated CD4(+) T cells. CONCLUSIONS: HIV integration in CCL19-treated resting CD4(+) T cells depends on NF-κB signalling and increases the stability of HIV integrase, which allow subsequent integration and establishment of latency. These findings have implications for strategies needed to prevent the establishment, and potentially reverse, latent infection.


Assuntos
Linfócitos T CD4-Positivos/virologia , Quimiocina CCL19/farmacologia , NF-kappa B/metabolismo , Receptores CCR/genética , Integração Viral , Latência Viral , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/fisiologia , Regulação Viral da Expressão Gênica/efeitos dos fármacos , Integrase de HIV/genética , HIV-1/enzimologia , HIV-1/fisiologia , Humanos , NF-kappa B/genética , Receptores CCR/metabolismo , Transdução de Sinais/efeitos dos fármacos , Integração Viral/efeitos dos fármacos , Latência Viral/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos
15.
16.
J Virol ; 88(2): 1113-24, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24198427

RESUMO

Early studies of HIV infection dynamics suggested that virus-producing HIV-infected cells had an average half-life of approximately 1 day. However, whether this average behavior is reflective of the dynamics of individual infected cells is unclear. Here, we use HIV-enhanced green fluorescent protein (EGFP) constructs and flow cytometry sorting to explore the dynamics of cell infection, viral protein production, and cell death in vitro. By following the numbers of productively infected cells expressing EGFP over time, we show that infected cell death slows down over time. Although infected cell death in vivo could be very different, our results suggest that the constant decay of cell numbers observed in vivo during antiretroviral treatment could reflect a balance of cell death and delayed viral protein production. We observe no correlation between viral protein production and death rate of productively infected cells, showing that viral protein production is not likely to be the sole determinant of the death of HIV-infected cells. Finally, we show that all observed features can be reproduced by a simple model in which infected cells have broad distributions of productive life spans, times to start viral protein production, and viral protein production rates. This broad spectrum of the level and timing of viral protein production provides new insights into the behavior and characteristics of HIV-infected cells.


Assuntos
Infecções por HIV/virologia , HIV-1/fisiologia , Apoptose , Células Cultivadas , Infecções por HIV/fisiopatologia , HIV-1/genética , Meia-Vida , Humanos , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/virologia , Proteínas Virais/genética , Proteínas Virais/metabolismo
17.
J Virol ; 88(5): 2891-902, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24371048

RESUMO

UNLABELLED: HIV-1 infection is characterized by the rapid generation of genetic diversity that facilitates viral escape from immune selection and antiretroviral therapy. Despite recombination's crucial role in viral diversity and evolution, little is known about the genomic factors that influence recombination between highly similar genomes. In this study, we use a minimally modified full-length HIV-1 genome and high-throughput sequence analysis to study recombination in gag and pol in T cells. We find that recombination is favored at a number of recombination hot spots, where recombination occurs six times more frequently than at corresponding cold spots. Interestingly, these hot spots occur near important features of the HIV-1 genome but do not occur at sites immediately around protease inhibitor or reverse transcriptase inhibitor drug resistance mutations. We show that the recombination hot and cold spots are consistent across five blood donors and are independent of coreceptor-mediated entry. Finally, we check common experimental confounders and find that these are not driving the location of recombination hot spots. This is the first study to identify the location of recombination hot spots between two similar viral genomes with great statistical power and under conditions that closely reflect natural recombination events among HIV-1 quasispecies. IMPORTANCE: The ability of HIV-1 to evade the immune system and antiretroviral therapy depends on genetic diversity within the viral quasispecies. Retroviral recombination is an important mechanism that helps to generate and maintain this genetic diversity, but little is known about how recombination rates vary within the HIV-1 genome. We measured recombination rates in gag and pol and identified recombination hot and cold spots, demonstrating that recombination is not random but depends on the underlying gene sequence. The strength and location of these recombination hot and cold spots can be used to improve models of viral dynamics and evolution, which will be useful for the design of robust antiretroviral therapies.


Assuntos
Genoma Viral , HIV-1/genética , Recombinação Genética , Variação Genética , Humanos , Modelos Lineares , Dados de Sequência Molecular , Vírus Reordenados/genética , Produtos do Gene gag do Vírus da Imunodeficiência Humana/genética , Produtos do Gene pol do Vírus da Imunodeficiência Humana/genética
18.
J Virol ; 88(7): 3837-49, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24453357

RESUMO

HIV undergoes high rates of mutation and recombination during reverse transcription, but it is not known whether these events occur independently or are linked mechanistically. Here we used a system of silent marker mutations in HIV and a single round of infection in primary T lymphocytes combined with a high-throughput sequencing and mathematical modeling approach to directly estimate the viral recombination and mutation rates. From >7 million nucleotides (nt) of sequences from HIV infection, we observed 4,801 recombination events and 859 substitution mutations (≈1.51 and 0.12 events per 1,000 nt, respectively). We used experimental controls to account for PCR-induced and transfection-induced recombination and sequencing error. We found that the single-cycle virus-induced mutation rate is 4.6 × 10(-5) mutations per nt after correction. By sorting of our data into recombined and nonrecombined sequences, we found a significantly higher mutation rate in recombined regions (P = 0.003 by Fisher's exact test). We used a permutation approach to eliminate a number of potential confounding factors and confirm that mutation occurs around the site of recombination and is not simply colocated in the genome. By comparing mutation rates in recombined and nonrecombined regions, we found that recombination-associated mutations account for 15 to 20% of all mutations occurring during reverse transcription.


Assuntos
Substituição de Aminoácidos , HIV/crescimento & desenvolvimento , HIV/genética , Mutação de Sentido Incorreto , Recombinação Genética , Linfócitos T/virologia , Células Cultivadas , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Modelos Teóricos
19.
PLoS Pathog ; 9(4): e1003218, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23592978

RESUMO

The HIV-1 gp120-gp41 complex, which mediates viral fusion and cellular entry, undergoes rapid evolution within its external glycan shield to enable escape from neutralizing antibody (NAb). Understanding how conserved protein determinants retain functionality in the context of such evolution is important for their evaluation and exploitation as potential drug and/or vaccine targets. In this study, we examined how the conserved gp120-gp41 association site, formed by the N- and C-terminal segments of gp120 and the disulfide-bonded region (DSR) of gp41, adapts to glycan changes that are linked to neutralization sensitivity. To this end, a DSR mutant virus (K601D) with defective gp120-association was sequentially passaged in peripheral blood mononuclear cells to select suppressor mutations. We reasoned that the locations of suppressors point to structural elements that are functionally linked to the gp120-gp41 association site. In culture 1, gp120 association and viral replication was restored by loss of the conserved glycan at Asn¹³6 in V1 (T138N mutation) in conjunction with the L494I substitution in C5 within the association site. In culture 2, replication was restored with deletion of the N¹³9INN sequence, which ablates the overlapping Asn¹4¹-Asn¹4²-Ser-Ser potential N-linked glycosylation sequons in V1, in conjunction with D601N in the DSR. The 136 and 142 glycan mutations appeared to exert their suppressive effects by altering the dependence of gp120-gp41 interactions on the DSR residues, Leu59³, Trp596 and Lys6°¹. The 136 and/or 142 glycan mutations increased the sensitivity of HIV-1 pseudovirions to the glycan-dependent NAbs 2G12 and PG16, and also pooled IgG obtained from HIV-1-infected individuals. Thus adjacent V1 glycans allosterically modulate the distal gp120-gp41 association site. We propose that this represents a mechanism for functional adaptation of the gp120-gp41 association site to an evolving glycan shield in a setting of NAb selection.


Assuntos
Anticorpos Anti-HIV/imunologia , Proteína gp120 do Envelope de HIV/imunologia , Proteína gp41 do Envelope de HIV/imunologia , HIV-1/imunologia , HIV-1/fisiologia , Anticorpos Neutralizantes/imunologia , Sítios de Ligação , Células Cultivadas , Genótipo , Proteína gp120 do Envelope de HIV/química , Proteína gp120 do Envelope de HIV/genética , Proteína gp120 do Envelope de HIV/metabolismo , Proteína gp41 do Envelope de HIV/química , Proteína gp41 do Envelope de HIV/genética , Proteína gp41 do Envelope de HIV/metabolismo , HIV-1/genética , Humanos , Evasão da Resposta Imune , Leucócitos Mononucleares/virologia , Dados de Sequência Molecular , Mutação , Testes de Neutralização , Polissacarídeos/química , Polissacarídeos/imunologia , Ligação Proteica , Conformação Proteica , Ligação Viral , Internalização do Vírus , Replicação Viral
20.
Virol J ; 12: 55, 2015 Apr 09.
Artigo em Inglês | MEDLINE | ID: mdl-25879746

RESUMO

BACKGROUND: Massive, parallel sequencing is a potent tool for dissecting the regulation of biological processes by revealing the dynamics of the cellular RNA profile under different conditions. Similarly, massive, parallel sequencing can be used to reveal the complexity of viral quasispecies that are often found in the RNA virus infected host. However, the production of cDNA libraries for next-generation sequencing (NGS) necessitates the reverse transcription of RNA into cDNA and the amplification of the cDNA template using PCR, which may introduce artefact in the form of phantom nucleic acids species that can bias the composition and interpretation of original RNA profiles. METHOD: Using HIV as a model we have characterised the major sources of error during the conversion of viral RNA to cDNA, namely excess RNA template and the RNaseH activity of the polymerase enzyme, reverse transcriptase. In addition we have analysed the effect of PCR cycle on detection of recombinants and assessed the contribution of transfection of highly similar plasmid DNA to the formation of recombinant species during the production of our control viruses. RESULTS: We have identified RNA template concentrations, RNaseH activity of reverse transcriptase, and PCR conditions as key parameters that must be carefully optimised to minimise chimeric artefacts. CONCLUSIONS: Using our optimised RT-PCR conditions, in combination with our modified PCR amplification procedure, we have developed a reliable technique for accurate determination of RNA species using NGS technology.


Assuntos
Artefatos , Biblioteca Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Linhagem Celular , Infecções por HIV/diagnóstico , Infecções por HIV/virologia , HIV-1/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Sequenciamento de Nucleotídeos em Larga Escala/normas , Humanos , Reação em Cadeia da Polimerase/métodos , Reação em Cadeia da Polimerase/normas , Vírus Reordenados/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/normas , Transfecção/métodos , Transfecção/normas
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