RESUMO
The interferon-induced host cell protein Shiftless (SFL) inhibits -1 programmed ribosomal frameshifting (-1PRF) required for the expression of HIV-1 Gal-Pol and the formation of infectious HIV-1 particles. However, the specific regions in SFL required for antiviral activity and the mechanism by which SFL inhibits -1PRF remain unclear. Employing alanine scanning mutagenesis, we found that basic amino acids in the predicted zinc ribbon motif of SFL are essential for the suppression of Gag-Pol expression but dispensable for anti-HIV-1 activity. We have shown that SFL inhibits the expression of the murine leukemia virus (MLV) Gag-Pol polyprotein and the formation of infectious MLV particles, although Gag-Pol expression of MLV is independent of -1PRF but requires readthrough of a stop codon. These findings indicate that SFL might inhibit HIV-1 infection by more than one mechanism and that SFL might target programmed translational readthrough as well as -1PRF signals, both of which are regulated by mRNA secondary structure elements.
Assuntos
Proteínas de Fusão gag-pol , Infecções por HIV , HIV-1 , Proteínas de Ligação a RNA , Humanos , Mudança da Fase de Leitura do Gene Ribossômico , Proteínas de Fusão gag-pol/genética , Proteínas de Fusão gag-pol/metabolismo , Regulação Viral da Expressão Gênica , Células HEK293 , Infecções por HIV/virologia , Infecções por HIV/genética , Infecções por HIV/metabolismo , HIV-1/genética , HIV-1/fisiologia , Vírus da Leucemia Murina/genética , Vírus da Leucemia Murina/fisiologia , Replicação Viral , Proteínas de Ligação a RNA/metabolismoRESUMO
Human immunodeficiency virus 1 (HIV-1) viral protease (PR) is one of the most studied viral enzymes and a crucial antiviral target. Despite its well-characterized role in virion maturation, an increasing body of research is starting to focus on its ability to cleave host cell proteins. Such findings are apparently in contrast with the dogma of HIV-1 PR activity being restricted to the interior of nascent virions and suggest catalytic activity within the host cell environment. Given the limited amount of PR present in the virion at the time of infection, such events mainly occur during late viral gene expression, mediated by newly synthesized Gag-Pol polyprotein precursors, rather than before proviral integration. HIV-1 PR mainly targets proteins involved in three different processes: those involved in translation, those controlling cell survival, and restriction factors responsible for innate/intrinsic antiviral responses. Indeed, by cleaving host cell translation initiation factors, HIV-1 PR can impair cap-dependent translation, thus promoting IRES-mediated translation of late viral transcripts and viral production. By targeting several apoptotic factors, it modulates cell survival, thus promoting immune evasion and viral dissemination. Additionally, HIV-1 PR counteracts restriction factors incorporated in the virion that would otherwise interfere with nascent virus vitality. Thus, HIV-1 PR appears to modulate host cell function at different times and locations during its life cycle, thereby ensuring efficient viral persistency and propagation. However, we are far from having a complete picture of PR-mediated host cell modulation, which is emerging as a field that needs further investigation.
Assuntos
Proteínas de Fusão gag-pol , Protease de HIV , Humanos , Protease de HIV/genética , Protease de HIV/metabolismo , Proteólise , Proteínas de Fusão gag-pol/metabolismo , Endopeptidases/metabolismo , Vírion/metabolismo , AntiviraisRESUMO
HIV-1 protease (PR) is a viral enzyme that cleaves the Gag and Gag-Pol polyprotein precursors to convert them into their functional forms, a process which is essential to generate infectious viral particles. Due to its broad substrate specificity, HIV-1 PR can also cleave certain host cell proteins. Several studies have identified host cell substrates of HIV-1 PR and described the potential impact of their cleavage on HIV-1-infected cells. Of particular interest is the interaction between PR and the caspase recruitment domain-containing protein 8 (CARD8) inflammasome. A recent study demonstrated that CARD8 can sense HIV-1 PR activity and induce cell death. While PR typically has low levels of intracellular activity prior to viral budding, premature PR activation can be achieved using certain non-nucleoside reverse transcriptase inhibitors (NNRTIs), resulting in CARD8 cleavage and downstream pyroptosis. Used together with latency reversal agents, the induction of premature PR activation to trigger CARD8-mediated cell killing may help eliminate latent reservoirs in people living with HIV. This represents a novel strategy of utilizing PR as an antiviral target through premature activation rather than inhibition. In this review, we discuss the viral and host substrates of HIV-1 protease and highlight potential applications and advantages of targeting CARD8 sensing of HIV-1 PR.
Assuntos
Protease de HIV , HIV-1 , Proteínas Adaptadoras de Sinalização CARD/metabolismo , Proteínas de Fusão gag-pol/metabolismo , Protease de HIV/metabolismo , HIV-1/fisiologia , Humanos , Proteínas de Neoplasias/metabolismo , Inibidores da Transcriptase Reversa/farmacologiaRESUMO
BACKGROUND: HIV-1 pol, which encodes enzymes required for virus replication, is initially translated as a Gag-Pol fusion protein. Gag-Pol is incorporated into virions via interactions with Gag precursor Pr55gag. Protease (PR) embedded in Gag-Pol mediates the proteolytic processing of both Pr55gag and Gag-Pol during or soon after virus particle release from cells. Since efficient Gag-Pol viral incorporation depends on interaction with Pr55gag via its N-terminal Gag domain, the prevention of premature Gag cleavage may alleviate Gag-Pol packaging deficiencies associated with cleavage enhancement from PR. RESULTS: We engineered PR cleavage-blocking Gag mutations with the potential to significantly reduce Gag processing efficiency. Such mutations may mitigate the negative effects of enhanced PR activation on virus assembly and Gag-Pol packaging due to an RT dimerization enhancer or leucine zipper dimerization motif. When co-expressed with Pr55gag, we noted that enhanced PR activation resulted in reduced Gag-Pol cis or trans incorporation into Pr55gag particles, regardless of whether or not Gag cleavage sites within Gag-Pol were blocked. CONCLUSIONS: Our data suggest that the amount of HIV-1 Gag-Pol or Pol viral incorporation is largely dependent on virus particle production, and that cleavage blocking in the Gag-Pol N-terminal Gag domain does not exert significant impacts on Pol packaging.
Assuntos
HIV-1 , Proteínas de Fusão gag-pol/genética , Proteínas de Fusão gag-pol/metabolismo , HIV-1/genética , Zíper de Leucina/genética , Vírion , Montagem de VírusRESUMO
A genetic selection system for activity of HIV protease is described that is based on a synthetic substrate constructed as a modified AraC regulatory protein that when cleaved stimulate l-arabinose metabolism in an Escherichia coli araC strain. Growth stimulation on selective plates was shown to depend on active HIV protease and the scissile bond in the substrate. In addition, the growth of cells correlated well with the established cleavage efficiency of the sites in the viral polyprotein, Gag, when these sites were individually introduced into the synthetic substrate of the selection system. Plasmids encoding protease variants selected based on stimulation of cell growth in the presence of saquinavir or cleavage of a site not cleaved by wild-type protease, were indistinguishable with respect to both phenotypes. Also, both groups of selected plasmids encoded side chain substitutions known from clinical isolates or displayed different side chain substitutions but at identical positions. One highly frequent side chain substitution, E34V, not regarded as a major drug resistance substitution was found in variants obtained under both selective conditions and is suggested to improve protease processing of the synthetic substrate. This substitution is away from the substrate-binding cavity and together with other substitutions in the selected reading frames supports the previous suggestion of a substrate-binding site extended from the active site binding pocket itself.
Assuntos
Fármacos Anti-HIV/farmacocinética , Farmacorresistência Viral/genética , Protease de HIV/genética , Substituição de Aminoácidos , Fator de Transcrição AraC/genética , Arabinose/metabolismo , Quimosina/metabolismo , Escherichia coli , Proteínas de Escherichia coli/genética , Proteínas de Fusão gag-pol/metabolismo , Produtos do Gene gag/metabolismo , Genes araC , Protease de HIV/química , Protease de HIV/isolamento & purificação , Protease de HIV/metabolismo , Modelos Moleculares , Mutação de Sentido Incorreto , Mutação Puntual , Conformação Proteica , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Saquinavir/antagonistas & inibidores , Saquinavir/farmacologia , Seleção Genética , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
The genetic manipulation of cells followed by their selection is indispensable for cell biological research. Although antibiotics-resistant genes are commonly used as selection markers, optimization of the condition for each selective agent is required. Here we utilized split-inteins and the drug-selectable marker puromycin N-acetyltransferase (PAC) to develop a system that enables the selection of cells simultaneously or sequentially transfected with multiple genetic constructs, using only puromycin. The active PAC enzyme was reconstituted by intein-mediated trans-splicing at several inherent or engineered serine/cysteine residues. Multiple splitting and reconstitution of active PAC was readily achieved by selecting optimum division sites based on the cellular tolerance to various puromycin concentrations. To achieve the stepwise selection method, PAC-intein fragments were transduced into cells using a virus-like particle (VLP) composed of HIV-1 gag-pol and VSV-G. The PAC-intein-VLP successfully conferred sufficient PAC activity for puromycin selection, which was quickly diminished in the absence of the VLP. Our findings demonstrate a versatile strategy for establishing markers for all-at-once or stepwise selection of multiple genetic manipulations, which will be useful in many fields of biology.
Assuntos
Acetiltransferases/genética , Engenharia Celular/métodos , Proteínas de Fusão gag-pol/genética , Inteínas/genética , Glicoproteínas de Membrana/genética , Seleção Genética , Proteínas do Envelope Viral/genética , Acetiltransferases/metabolismo , Partículas Artificiais Semelhantes a Vírus/química , Partículas Artificiais Semelhantes a Vírus/metabolismo , Linhagem Celular Tumoral , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Proteínas de Fusão gag-pol/metabolismo , Expressão Gênica , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Glicoproteínas de Membrana/metabolismo , Plasmídeos/química , Plasmídeos/metabolismo , Puromicina/farmacologia , Transfecção/métodos , Vírus da Estomatite Vesicular Indiana/genética , Vírus da Estomatite Vesicular Indiana/metabolismo , Proteínas do Envelope Viral/metabolismo , Proteína Vermelha FluorescenteRESUMO
The pol retrovirus gene encodes required enzymes for virus replication and maturation. Unlike HIV-1 Pol (expressed as a Gag-Pol fusion protein), foamy virus (described as an ancient retrovirus) expresses Pol without forming Gag-Pol polyproteins. We placed a "self-cleaving" 2A peptide between HIV-1 Gag and Pol. This construct, designated G2AP, is capable of producing virions with the same density as a wild-type (wt) HIV-1 particle. The 2A peptide allows for Pol to be packaged into virions independently from Gag following co-translationally cleaved from Gag. We found that G2AP exhibited only one-third the virus infectivity of the wt, likely due, at least in part, to defects in Pol packaging. Attenuated protease (PR) activity, or a reduction in Pol expression due to the placement of 2A-mediated Pol in a normal Gag-Pol frameshift context, resulted in significant increases in virus yields and/or titers. This suggests that reduced G2AP virus yields were largely due to increased PR activity associated with overexpressed Pol. Our data suggest that HIV-1 adopts a gag/pol ribosomal frameshifting mechanism to support virus assembly via the efficient modulation of Gag-Pol/Gag expression, as well as to promote viral enzyme packaging. Our results help clarify the molecular basis of HIV-1 gene expression and assembly.
Assuntos
HIV-1/fisiologia , Replicação Viral , Produtos do Gene gag do Vírus da Imunodeficiência Humana/metabolismo , Produtos do Gene pol do Vírus da Imunodeficiência Humana/metabolismo , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/metabolismo , Proteínas de Fusão gag-pol/genética , Proteínas de Fusão gag-pol/metabolismo , Regulação Viral da Expressão Gênica , Células HEK293 , Protease de HIV/genética , Protease de HIV/metabolismo , Transcriptase Reversa do HIV/genética , Transcriptase Reversa do HIV/metabolismo , HIV-1/genética , HIV-1/crescimento & desenvolvimento , HIV-1/metabolismo , Células HeLa , Humanos , Carga Viral , Vírion/metabolismo , Montagem de Vírus , Produtos do Gene gag do Vírus da Imunodeficiência Humana/genética , Produtos do Gene pol do Vírus da Imunodeficiência Humana/genéticaRESUMO
Ty1 is one of the many transposons in the budding yeast Saccharomyces cerevisiae. The life-cycle of Ty1 shows numerous similarities with that of retroviruses, e.g. the initially synthesized polyprotein precursor undergoes proteolytic processing by the protease. The retroviral proteases have become important targets of current antiretroviral therapies due to the critical role of the limited proteolysis of Gag-Pol polyprotein in the replication cycle and they therefore belong to the most well-studied enzymes. Comparative analyses of retroviral and retroviral-like proteases can help to explore the key similarities and differences which may help understanding how resistance is developed against protease inhibitors, but the available information about the structural and biochemical characteristics of retroviral-like, and especially retrotransposon, proteases is limited. To investigate the main characteristics of Ty1 retrotransposon protease of Saccharomyces cerevisiae, untagged and His6-tagged forms of Ty1 protease were expressed in E. coli. After purification of the recombinant proteins, activity measurements were performed using synthetic oligopeptide and fluorescent recombinant protein substrates, which represented the wild-type and the modified forms of naturally occurring cleavage sites of the protease. We investigated the dependence of enzyme activity on different reaction conditions (pH, temperature, ionic strength, and urea concentration), and determined enzyme kinetic parameters for the studied substrates. Inhibitory potentials of 10 different protease inhibitors were also tested. Ty1 protease was not inhibited by the inhibitors which have been designed against human immunodeficiency virus type 1 protease and are approved as antiretroviral therapeutics. A quaternary structure of homodimeric Ty1 protease was proposed based on homology modeling, and this structure was used to support interpretation of experimental results and to correlate some structural and biochemical characteristics with that of other retroviral proteases.
Assuntos
Endopeptidases/química , Retroelementos , Proteínas de Saccharomyces cerevisiae/química , Endopeptidases/metabolismo , Estabilidade Enzimática , Proteínas de Fusão gag-pol/metabolismo , Temperatura Alta , Cinética , Concentração Osmolar , Inibidores de Proteases/farmacologia , Proteólise , Saccharomyces cerevisiae/enzimologia , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Mouse APOBEC3 (mA3) inhibits murine leukemia virus (MuLV) replication by a deamination-independent mechanism in which the reverse transcription is considered the main target process. However, other steps in virus replication that can be targeted by mA3 have not been examined. We have investigated the possible effect of mA3 on MuLV protease-mediated processes and found that mA3 binds both mature viral protease and Pr180gag-pol precursor polyprotein. Using replication-competent MuLVs, we also show that mA3 inhibits the processing of Pr65 Gag precursor. Furthermore, we demonstrate that the autoprocessing of Pr180gag-pol is impeded by mA3, resulting in reduced production of mature viral protease. This reduction appears to link with the above inefficient Pr65gag processing in the presence of mA3. Two major isoforms of mA3, exon 5-containing and -lacking ones, equally exhibit this antiviral activity. Importantly, physiologically expressed levels of mA3 impedes both Pr180gag-pol autocatalysis and Pr65gag processing. This blockade is independent of the deaminase activity and requires the C-terminal region of mA3. These results suggest that the above impairment of Pr180gag-pol autoprocessing may significantly contribute to the deaminase-independent antiretroviral activity exerted by mA3.
Assuntos
Citidina Desaminase/metabolismo , Proteínas de Fusão gag-pol/metabolismo , Vírus da Leucemia Murina/metabolismo , Infecções por Retroviridae/metabolismo , Replicação Viral/fisiologia , Animais , Produtos do Gene gag/metabolismo , Leucemia Experimental/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Infecções Tumorais por Vírus/metabolismoRESUMO
HIV-1 protease (PR) is the viral protein responsible for virion maturation, and its mechanisms of action remain incompletely understood. PR is dimeric and contains two flexible, symmetry-related flaps, which act as a gate to inhibit access to the binding pocket and hold the polypeptide substrate in the binding pocket once bound. Wide flap opening, a conformational change assumed to be necessary for substrate binding, is a rare event in the closed and bound form. In this study, we use molecular dynamics (MD) simulations and advanced MD techniques including temperature acceleration and string method in collective variables to study the conformational changes associated with substrate unbinding of both wild-type and F99Y mutant PR. The F99Y mutation is shown via MD to decouple the closing of previously unrecognized distal pockets from substrate unbinding. To determine whether or not the F99Y mutation affects the energetic cost of wide flap opening, we use string method in collective variables to determine the minimum free-energy mechanism for wide flap opening in concert with distal pocket closing. The results indicate that the major energetic cost in flap opening is disengagement of the two flap-tip Ile50 residues from each other and is not affected by the F99Y mutation.
Assuntos
Protease de HIV/metabolismo , Sítios de Ligação , Proteínas de Fusão gag-pol/química , Proteínas de Fusão gag-pol/metabolismo , Protease de HIV/química , Protease de HIV/genética , HIV-1/enzimologia , Simulação de Dinâmica Molecular , Mutação , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Ligação Proteica , Conformação Proteica , TermodinâmicaRESUMO
Synthesis of HIV GagPol involves a proportion of ribosomes translating a U6A shift site at the distal end of the gag gene performing a programmed -1 ribosomal frameshift event to enter the overlapping pol gene. In vitro studies here show that at the same shift motif HIV reverse transcriptase generates -1 and +1 indels with their ratio being sensitive to the relative concentration ratio of dNTPs specified by the RNA template slippage-prone sequence and its 5' adjacent base. The GGG sequence 3' adjacent to the U6A shift/slippage site, which is important for ribosomal frameshifting, is shown here to limit reverse transcriptase base substitution and indel 'errors' in the run of A's in the product. The indels characterized here have either 1 more or less A, than the corresponding number of template U's. cDNA with 5 A's may yield novel Gag product(s), while cDNA with an extra base, 7 A's, may only be a minor contributor to GagPol polyprotein. Synthesis of a proportion of non-ribosomal frameshift derived GagPol would be relevant in efforts to identify therapeutically useful compounds that perturb the ratio of GagPol to Gag, and pertinent to the extent in which specific polymerase slippage is utilized in gene expression.
Assuntos
Mudança da Fase de Leitura do Gene Ribossômico , Proteínas de Fusão gag-pol/metabolismo , Genes gag , Genes pol , Transcriptase Reversa do HIV/metabolismo , HIV-1/genética , Mutação INDEL , Sequência de Aminoácidos , Sequência de Bases , Sequência Conservada , DNA Complementar/genética , Farmacorresistência Viral , Proteínas de Fusão gag-pol/genética , Inibidores da Protease de HIV/farmacologia , Transcriptase Reversa do HIV/genética , Conformação de Ácido Nucleico , Análise de Sequência de DNA , Especificidade por SubstratoRESUMO
HIV-1 protease (PR) functions as a homodimer mediating virus maturation following virus budding. Gag-Pol dimerization is believed to trigger embedded PR activation by promoting PR dimer formation. Early PR activation can lead to markedly reduced virus yields due to premature Gag cleavage. The p6* peptide, located between Gag and PR, is believed to ensure virus production by preventing early PR maturation. Studies aimed at finding supporting evidence for this proposal are limited due to a reading frame overlap between p6* and the p6gag budding domain. To determine if p6* affects virus production via the modulation of PR activation, we engineered multiple constructs derived from Dp6*PR (an assembly- and processing-competent construct with Pol fused at the inactivated PR C terminus). The data indicated that a p6* deletion adjacent to active PR significantly impaired virus processing. We also observed that the insertion of a leucine zipper (LZ) dimerization motif in the deleted region eliminated virus production in a PR activity-dependent manner, suggesting that the LZ insertion triggered premature PR activation by facilitating PR dimer formation. As few as four C-terminal p6* residues remaining at the p6*/PR junction were sufficient to restore virus yields, with a Gag processing profile similar to that of the wild type. Our study provides supporting evidence in a virus assembly context that the C-terminal p6* tetrapeptide plays a role in preventing premature PR maturation.IMPORTANCE Supporting evidence for the assumption that p6* retards PR maturation in the context of virus assembly is lacking. We found that replacing p6* with a leucine zipper peptide abolished virus assembly due to the significant enhancement of Gag cleavage. However, as few as four C-terminal p6* residues remaining in the deleted region were sufficient for significant PR release, as well as for counteracting leucine zipper-incurred premature Gag cleavage. Our data provide evidence that (i) p6* ensures virus assembly by preventing early PR activation and (ii) four C-terminal p6* residues are critical for modulating PR activation. Current PR inhibitor development efforts are aimed largely at mature PR, but there is a tendency for HIV-1 variants that are resistant to multiple protease inhibitors to emerge. Our data support the idea of modulating PR activation by targeting PR precursors as an alternative approach to controlling HIV-1/AIDS.
Assuntos
Protease de HIV/metabolismo , Zíper de Leucina , Produtos do Gene gag do Vírus da Imunodeficiência Humana/genética , Produtos do Gene gag do Vírus da Imunodeficiência Humana/metabolismo , Sequência de Aminoácidos , Proteínas de Fusão gag-pol/genética , Proteínas de Fusão gag-pol/metabolismo , Protease de HIV/genética , HIV-1/enzimologia , HIV-1/fisiologia , Zíper de Leucina/genética , Deleção de Sequência , Montagem de VírusRESUMO
INI1/hSNF5/SMARCB1/BAF47 is an HIV-specific integrase (IN)-binding protein that influences HIV-1 transcription and particle production. INI1 binds to SAP18 (Sin3a-associated protein, 18 kDa), and both INI1 and SAP18 are incorporated into HIV-1 virions. To determine the significance of INI1 and the INI1-SAP18 interaction during HIV-1 replication, we isolated a panel of SAP18-interaction-defective (SID)-INI1 mutants using a yeast reverse two-hybrid screen. The SID-INI1 mutants, which retained the ability to bind to IN, cMYC, and INI1 but were impaired for binding to SAP18, were tested for their effects on HIV-1 particle production. SID-INI1 dramatically reduced the intracellular Gag/Gag-Pol protein levels and, in addition, decreased viral particle production. The SID-INI1-mediated effects were less dramatic in trans complementation assays using IN deletion mutant viruses with Vpr-reverse transcriptase (RT)-IN. SID-INI1 did not inhibit long-terminal-repeat (LTR)-mediated transcription, but it marginally decreased the steady-state gag RNA levels, suggesting a posttranscriptional effect. Pulse-chase analysis indicated that in SID-INI1-expressing cells, the pr55Gag levels decreased rapidly. RNA interference analysis indicated that small hairpin RNA (shRNA)-mediated knockdown of INI1 reduced the intracellular Gag/Gag-Pol levels and further inhibited HIV-1 particle production. These results suggest that SID-INI1 mutants inhibit multiple stages of posttranscriptional events of HIV-1 replication, including intracellular Gag/Gag-Pol RNA and protein levels, which in turn inhibits assembly and particle production. Interfering INI1 leads to a decrease in particle production and Gag/Gag-Pol protein levels. Understanding the role of INI1 and SAP18 in HIV-1 replication is likely to provide novel insight into the stability of Gag/Gag-Pol, which may lead to the development of novel therapeutic strategies to inhibit HIV-1 late events. IMPORTANCE: Significant gaps exist in our current understanding of the mechanisms and host factors that influence HIV-1 posttranscriptional events, including gag RNA levels, Gag/Gag-Pol protein levels, assembly, and particle production. Our previous studies suggested that the IN-binding host factor INI1 plays a role in HIV-1 assembly. An ectopically expressed minimal IN-binding domain of INI1, S6, potently and selectively inhibited HIV-1 Gag/Gag-Pol trafficking and particle production. However, whether or not endogenous INI1 and its interacting partners, such as SAP18, are required for late events was unknown. Here, we report that endogenous INI1 and its interaction with SAP18 are necessary to maintain intracellular levels of Gag/Gag-Pol and for particle production. Interfering INI1 or the INI1-SAP18 interaction leads to the impairment of these processes, suggesting a novel strategy for inhibiting posttranscriptional events of HIV-1 replication.
Assuntos
Montagem e Desmontagem da Cromatina/genética , Cromatina/genética , Proteínas de Fusão gag-pol/genética , HIV-1/genética , Processamento Pós-Transcricional do RNA/genética , Proteína SMARCB1/genética , Produtos do Gene gag do Vírus da Imunodeficiência Humana/genética , Proteínas de Transporte/metabolismo , Linhagem Celular , Proteínas Correpressoras , Replicação do DNA/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Fusão gag-pol/metabolismo , Células HEK293 , Integrase de HIV/genética , Integrase de HIV/metabolismo , HIV-1/metabolismo , Humanos , Proteínas de Ligação a RNA , Proteína SMARCB1/metabolismo , Replicação Viral/genética , Produtos do Gene gag do Vírus da Imunodeficiência Humana/metabolismoRESUMO
Retroviral reverse transcriptase (RT) of Moloney murine leukemia virus (MoMLV) is expressed in the form of a large Gag-Pol precursor protein by suppression of translational termination in which the maximal efficiency of stop codon read-through depends on the interaction between MoMLV RT and peptidyl release factor 1 (eRF1). Here, we report the crystal structure of MoMLV RT in complex with eRF1. The MoMLV RT interacts with the C-terminal domain of eRF1 via its RNase H domain to sterically occlude the binding of peptidyl release factor 3 (eRF3) to eRF1. Promotion of read-through by MoMLV RNase H prevents nonsense-mediated mRNA decay (NMD) of mRNAs. Comparison of our structure with that of HIV RT explains why HIV RT cannot interact with eRF1. Our results provide a mechanistic view of how MoMLV manipulates the host translation termination machinery for the synthesis of its own proteins.
Assuntos
Vírus da Leucemia Murina de Moloney/metabolismo , Terminação Traducional da Cadeia Peptídica , Fatores de Terminação de Peptídeos/metabolismo , DNA Polimerase Dirigida por RNA/metabolismo , Animais , Calorimetria , Códon de Terminação , Proteínas de Fusão gag-pol/metabolismo , Células HEK293 , Transcriptase Reversa do HIV/metabolismo , Células HeLa , Humanos , Camundongos , Mutação , Degradação do RNAm Mediada por Códon sem Sentido , Ligação Proteica , Domínios Proteicos , RNA Mensageiro/metabolismo , Ribonuclease H/química , Ribonuclease H/metabolismoRESUMO
UNLABELLED: Human immunodeficiency virus (HIV) replication is strongly dependent upon a programmed ribosomal frameshift. Here we investigate the relationships between the thermodynamic stability of the HIV type 1 (HIV-1) RNA frameshift site stem-loop, frameshift efficiency, and infectivity, using pseudotyped HIV-1 and HEK293T cells. The data reveal a strong correlation between frameshift efficiency and local, but not overall, RNA thermodynamic stability. Mutations that modestly increase the local stability of the frameshift site RNA stem-loop structure increase frameshift efficiency 2-fold to 3-fold in cells. Thus, frameshift efficiency is determined by the strength of the thermodynamic barrier encountered by the ribosome. These data agree with previous in vitro measurements, suggesting that there are no virus- or host-specific factors that modulate frameshifting. The data also indicate that there are no sequence-specific requirements for the frameshift site stem-loop. A linear correlation between Gag-polymerase (Gag-Pol) levels in cells and levels in virions supports the idea of a stochastic virion assembly mechanism. We further demonstrate that the surrounding genomic RNA secondary structure influences frameshift efficiency and that a mutation that commonly arises in response to protease inhibitor therapy creates a functional but inefficient secondary slippery site. Finally, HIV-1 mutants with enhanced frameshift efficiencies are significantly less infectious, suggesting that compounds that increase frameshift efficiency by as little as 2-fold may be effective at suppressing HIV-1 replication. IMPORTANCE: HIV, like many retroviruses, utilizes a -1 programmed ribosomal frameshift to generate viral enzymes in the form of a Gag-Pol polyprotein precursor. Thus, frameshifting is essential for viral replication. Here, we utilized a panel of mutant HIV strains to demonstrate that in cells, frameshifting efficiency is correlated with the stability of the local thermodynamic barrier to ribosomal translocation. Increasing the stability of the frameshift site RNA increases the frameshift efficiency 2-fold to 3-fold. Mutant viruses with increased frameshift efficiencies have significantly reduced infectivity. These data suggest that this effect might be exploited in the development of novel antiviral strategies.
Assuntos
Mutação da Fase de Leitura/genética , Mudança da Fase de Leitura do Gene Ribossômico/genética , Proteínas de Fusão gag-pol/metabolismo , Infecções por HIV/virologia , HIV-1/genética , RNA Viral/genética , Vírion/fisiologia , Pareamento de Bases , Sequência de Bases , Regulação Viral da Expressão Gênica , Células HEK293 , Infecções por HIV/genética , HIV-1/química , HIV-1/metabolismo , Humanos , Conformação de Ácido Nucleico , Estabilidade de RNA , RNA Viral/química , RNA Viral/metabolismo , Montagem de Vírus , Replicação ViralRESUMO
Translational readthrough-promoting drugs enhance the incorporation of amino acids at stop codons and can thus bypass premature termination during protein synthesis. The polymerase (Pol) proteins of Moloney murine leukemia virus (MoMLV) are synthesized as a large GagPol fusion protein, formed by the readthrough of a stop codon at the end of the gag ORF. The downstream pol ORF lacks its own start codon, and Pol protein synthesis is wholly dependent on translation of the upstream gag gene and the readthrough event for expression. Here, we explored the effects of readthrough-promoting drugs aminoglycoside antibiotics and the small molecule ataluren on the efficiency of readthrough of the stop codon in the context of the MoMLV genome. We showed that these compounds increased readthrough of the stop codon at the MoMLV gagpol junction in vivo above the already high basal level and that the resulting elevated gagpol readthrough had deleterious effects on virus replication. We also showed that readthrough efficiency could be driven to even higher levels in vitro, and that the combination of the small molecules and the RNA structure at the MoMLV stop codon could achieve extremely high readthrough efficiencies.
Assuntos
Aminoglicosídeos/farmacologia , Proteínas de Fusão gag-pol/genética , Vírus da Leucemia Murina de Moloney/genética , Oxidiazóis/farmacologia , Biossíntese de Proteínas/efeitos dos fármacos , Aminoglicosídeos/efeitos adversos , Animais , Linhagem Celular , Códon de Terminação , Proteínas de Fusão gag-pol/metabolismo , Regulação Viral da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , Vírus da Leucemia Murina de Moloney/efeitos dos fármacos , Vírus da Leucemia Murina de Moloney/metabolismo , Oxidiazóis/efeitos adversosRESUMO
HIV-1 protease (PR) is encoded by pol, which is initially translated as a Pr160gag-pol polyprotein by a ribosomal frameshift event. Within Gag-Pol, truncated p6gag is replaced by a transframe domain (referred to as p6* or p6pol) located directly upstream of PR. p6* has been proposed as playing a role in modulating PR activation. Overlapping reading frames between p6* and p6gag present a challenge to researchers using genetic approaches to studying p6* biological functions. To determine the role of p6* in PR activation without affecting the gag reading frame, we constructed a series of Gag/Gag-Pol expression vectors by duplicating PR with or without p6* between PR pairs, and observed that PR duplication eliminated virus production due to significant Gag cleavage enhancement. This effect was mitigated when p6* was placed between the two PRs. Further, Gag cleavage enhancement was markedly reduced when either one of the two PRs was mutationally inactivated. Additional reduction in Gag cleavage efficiency was noted following the removal of p6* from between the two PRs. The insertion of a NC domain (wild-type or mutant) directly upstream of PR or p6*PR did not significantly improve Gag processing efficiency. With the exception of those containing p6* directly upstream of an active PR, all constructs were either noninfectious or weakly infectious. Our results suggest that (a) p6* is essential for triggering PR activation, (b) p6* has a role in preventing premature virus processing, and
Assuntos
Proteínas de Fusão gag-pol/metabolismo , Protease de HIV/metabolismo , Protease de HIV/fisiologia , HIV-1/metabolismo , Proteínas de Fusão gag-pol/química , Proteínas de Fusão gag-pol/genética , Protease de HIV/química , Produtos do Gene gag do Vírus da Imunodeficiência Humana/química , Produtos do Gene gag do Vírus da Imunodeficiência Humana/genética , Produtos do Gene gag do Vírus da Imunodeficiência Humana/metabolismoRESUMO
The HIV Rev protein forms a complex with a 351 nucleotide sequence present in unspliced and incompletely spliced human immunodeficiency virus (HIV) mRNAs, the Rev response element (RRE), to recruit the cellular nuclear export receptor Crm1 and Ran-GTP. This complex facilitates nucleo-cytoplasmic export of these mRNAs. The precise secondary structure of the HIV-1 RRE has been controversial, since studies have reported alternative structures comprising either four or five stem-loops. The published structures differ only in regions that lie outside of the primary Rev binding site. Using in-gel SHAPE, we have now determined that the wt NL4-3 RRE exists as a mixture of both structures. To assess functional differences between these RRE 'conformers', we created conformationally locked mutants by site-directed mutagenesis. Using subgenomic reporters, as well as HIV replication assays, we demonstrate that the five stem-loop form of the RRE promotes greater functional Rev/RRE activity compared to the four stem-loop counterpart.
Assuntos
HIV-1/genética , RNA Viral/química , Sequências Reguladoras de Ácido Ribonucleico , Replicação Viral/genética , Produtos do Gene rev do Vírus da Imunodeficiência Humana/metabolismo , Proteínas de Fusão gag-pol/metabolismo , Genes env , HIV-1/fisiologia , Mutação , Conformação de Ácido Nucleico , RNA Viral/metabolismoRESUMO
Human T cell leukemia virus, type 1 (HTLV-1) replication and spread are controlled by different viral and cellular factors. Although several anti-HIV cellular microRNAs have been described, such a regulation for HTLV-1 has not been reported. In this study, we found that miR-28-3p inhibits HTLV-1 virus expression and its replication by targeting a specific site within the genomic gag/pol viral mRNA. Because miR-28-3p is highly expressed in resting T cells, which are resistant to HTLV-1 infection, we investigated a potential protective role of miR-28-3p against de novo HTLV-1 infection. To this end, we developed a new sensitive and quantitative assay on the basis of the detection of products of reverse transcription. We demonstrate that miR-28-3p does not prevent virus receptor interaction or virus entry but, instead, induces a post-entry block at the reverse transcription level. In addition, we found that HTLV-1, subtype 1A isolates corresponding to the Japanese strain ATK-1 present a natural, single-nucleotide polymorphism within the miR-28-3p target site. As a result of this polymorphism, the ATK-1 virus sequence was not inhibited by miR-28. Interestingly, genetic studies on the transmission of the virus has shown that the ATK-1 strain, which carries a Thr-to-Cys transition mutation, is transmitted efficiently between spouses, suggesting that miR-28 may play an important role in HTLV-1 transmission.
Assuntos
Infecções por HTLV-I/genética , Vírus Linfotrópico T Tipo 1 Humano/genética , MicroRNAs/genética , Replicação Viral/genética , Animais , Sítios de Ligação/genética , Western Blotting , Células COS , Linhagem Celular , Linhagem Celular Transformada , Chlorocebus aethiops , Proteínas de Fusão gag-pol/genética , Proteínas de Fusão gag-pol/metabolismo , Regulação Viral da Expressão Gênica , Células HEK293 , Infecções por HTLV-I/virologia , Interações Hospedeiro-Patógeno/genética , Vírus Linfotrópico T Tipo 1 Humano/fisiologia , Humanos , Células Jurkat , MicroRNAs/metabolismo , Mutação de Sentido Incorreto , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Viral/genética , RNA Viral/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
UNLABELLED: Translational readthrough--suppression of termination at a stop codon--is exploited in the replication cycles of several viruses and represents a potential target for antiviral intervention. In the gammaretroviruses, typified by Moloney murine leukemia virus (MuLV), gag and pol are in the same reading frame, separated by a UAG stop codon, and termination codon readthrough is required for expression of the viral Gag-Pol fusion protein. Here, we investigated the effect on MuLV replication of modulating readthrough efficiency. We began by manipulating the readthrough signal in the context of an infectious viral clone to generate a series of MuLV variants in which readthrough was stimulated or reduced. In carefully controlled infectivity assays, it was found that reducing the MuLV readthrough efficiency only 4-fold led to a marked defect and that a 10-fold reduction essentially abolished replication. However, up to an â¼ 8.5-fold stimulation of readthrough (up to 60% readthrough) was well tolerated by the virus. These high levels of readthrough were achieved using a two-plasmid system, with Gag and Gag-Pol expressed from separate infectious clones. We also modulated readthrough by silencing expression of eukaryotic release factors 1 and 3 (eRF1 and eRF3) or by introducing aminoglycosides into the cells. The data obtained indicate that gammaretroviruses tolerate a substantial excess of viral Gag-Pol synthesis but are very sensitive to a reduction in levels of this polyprotein. Thus, as is also the case for ribosomal frameshifting, antiviral therapies targeting readthrough with inhibitory agents are likely to be the most beneficial. IMPORTANCE: Many pathogenic RNA viruses and retroviruses use ribosomal frameshifting or stop codon readthrough to regulate expression of their replicase enzymes. These translational "recoding" processes are potential targets for antiviral intervention, but we have only a limited understanding of the consequences to virus replication of modulating the efficiency of recoding, particularly for those viruses employing readthrough. In this paper, we describe the first systematic analysis of the effect of increasing or decreasing readthrough efficiency on virus replication using the gammaretrovirus MuLV as a model system. We find unexpectedly that MuLV replication is only slightly inhibited by substantial increases in readthrough frequency, but as with other viruses that use recoding strategies, replication is quite sensitive to even modest reductions. These studies provide insights into both the readthrough process and MuLV replication and have implications for the selection of antivirals against gammaretroviruses.