RESUMEN
BACKGROUND: The impact of the products of the pol gene, specifically, reverse transcriptase (RT) on HIV-1 replication, evolution, and acquisition of drug resistance has been thoroughly characterized for subtype B. For subtype C, which accounts of almost 60% of HIV cases worldwide, much less is known. It has been reported that subtype C HIV-1 isolates have a lower replication capacity than B; however, the basis of these differences remains unclear. RESULTS: We analyzed the impact of the pol gene products from HIV-1 B and C subtypes on the maturation of HIV virions, accumulation of reverse transcription products, integration of viral DNA, frequency of point mutations in provirus and overall viral replication. Recombinant HIV-1 viruses of B and C subtypes comprising the pol fragments encoding protease, integrase and either the whole RT or a chimeric RT from different isolates of the C and B subtypes, were used for infection of cells expressing CXCR4 or CCR5 co-receptors. The viruses carrying different fragments of pol from the isolates of B and C subtypes did not reveal differences in Gag and GagPol processing and viral RNA incorporation into the virions. However, the presence of the whole RT from subtype C, or the chimeric RT containing either the polymerase or the connection and RNase H domains from C isolates, caused significantly slower viral replication regardless of B or C viral backbone. Subtype C RT carrying viruses displayed lower levels of accumulation of strong-stop cDNA in permeabilized virions during endogenous reverse transcription, and decreased accumulation of both strong-stop and positive strand reverse transcription products in infected cells and in isolated reverse transcription complexes. This decreased accumulation correlated with lower levels of viral DNA integration in cells infected with viruses carrying the whole RT or RT domains from subtype C isolates. The single viral genome assay analysis did not reveal significant differences in the frequency of point mutations between the RT from B or C subtypes. CONCLUSIONS: These data suggest that the whole RT as well as distinct polymerase and connection-RNase H domains from subtype C HIV-1 confer a lower level of accumulation of reverse transcripts in the virions and reverse transcription complexes as compared to subtype B, resulting in a lower overall level of virus replication.
Asunto(s)
Productos del Gen pol/fisiología , Infecciones por VIH/virología , Transcriptasa Inversa del VIH/metabolismo , VIH-1/fisiología , Virus Reordenados/fisiología , Línea Celular , Transcriptasa Inversa del VIH/química , VIH-1/química , VIH-1/clasificación , Humanos , Mutación Puntual , Estructura Terciaria de Proteína/fisiología , Provirus/genética , Virus Reordenados/química , Virus Reordenados/clasificación , Ribonucleasa H del Virus de la Inmunodeficiencia Humana/fisiología , Integración Viral/fisiología , Replicación ViralRESUMEN
Viral infection leads to induction of pattern-recognition receptor signaling, which leads to interferon regulatory factor (IRF) activation and ultimately interferon (IFN) production. To establish infection, many viruses have strategies to evade the innate immunity. For the hepatitis B virus (HBV), which causes chronic infection in the liver, the evasion strategy remains uncertain. We now show that HBV polymerase (Pol) blocks IRF signaling, indicating that HBV Pol is the viral molecule that effectively counteracts host innate immune response. In particular, HBV Pol inhibits TANK-binding kinase 1 (TBK1)/IkappaB kinase-epsilon (IKKepsilon), the effector kinases of IRF signaling. Intriguingly, HBV Pol inhibits TBK1/IKKepsilon activity by disrupting the interaction between IKKepsilon and DDX3 DEAD box RNA helicase, which was recently shown to augment TBK1/IKKepsilon activity. This unexpected role of HBV Pol may explain how HBV evades innate immune response in the early phase of the infection. A therapeutic implication of this work is that a strategy to interfere with the HBV Pol-DDX3 interaction might lead to the resolution of life-long persistent infection.
Asunto(s)
ARN Helicasas DEAD-box/metabolismo , Productos del Gen pol/fisiología , Virus de la Hepatitis B/inmunología , Evasión Inmune , Receptores de Reconocimiento de Patrones/antagonistas & inhibidores , Línea Celular , Productos del Gen pol/inmunología , Virus de la Hepatitis B/enzimología , Humanos , Quinasa I-kappa B , Inmunidad Innata , Factores Reguladores del Interferón/antagonistas & inhibidores , Factores Reguladores del Interferón/genética , Proteínas Serina-Treonina Quinasas , Transducción de Señal/inmunologíaRESUMEN
Hepatitis B virus (HBV) infection remains one of the most serious health problems worldwide. Whilst studies have shown that HBV impairs interferon (IFN) production from dendritic cells in chronic hepatitis B patients, it remains unknown whether HBV inhibits IFN production in human hepatocytes. Using transient transfection assays in a primary human hepatocyte cell line (PH5CH8), this study demonstrated that HBV polymerase inhibits IFN-beta promoter activity induced by Newcastle disease virus, Sendai virus or poly(I : C) in a dose-dependent manner, whilst ectopic expression of the HBV core and X proteins had no effect on IFN-beta promoter activity. In addition, HBV polymerase blocked cellular IFN-beta expression and consequent antiviral immunity revealed by an infection protection assay. Furthermore, overexpression of key molecules on the IFN-beta induction axis, together with HBV polymerase, resulted in a block of IFN-beta promoter activity triggered by RIG-I, IPS-1, TRIF, TBK1 and IKKepsilon, but not by an IFN regulatory factor 3 dominant-positive mutant (IRF3-5D), suggesting that HBV polymerase prevents IFN-beta expression at the TBK1/IKKepsilon level. Further studies showed that HBV polymerase inhibited phosphorylation, dimerization and nuclear translocation of IRF3, in response to Sendai virus infection. Finally, it was shown that HBV polymerase-mediated dampening of the interaction between TBK1/IKKepsilon and DDX3 may be involved in the inhibitory effect on IFN-beta induction. Taken together, these findings reveal a novel role of HBV polymerase in HBV counteraction of IFN-beta production in human hepatocytes.
Asunto(s)
ARN Helicasas DEAD-box/inmunología , Productos del Gen pol/fisiología , Virus de la Hepatitis B/patogenicidad , Factor 3 Regulador del Interferón/antagonistas & inhibidores , Interferón beta/biosíntesis , Receptor Toll-Like 3/inmunología , Factores de Virulencia/fisiología , Línea Celular , Proteína 58 DEAD Box , ARN Helicasas DEAD-box/metabolismo , Virus de la Hepatitis B/inmunología , Hepatocitos/virología , Humanos , Quinasa I-kappa B/metabolismo , Virus de la Enfermedad de Newcastle/inmunología , Poli I-C/inmunología , Proteínas Serina-Treonina Quinasas/metabolismo , Receptores Inmunológicos , Virus Sendai/inmunología , TransfecciónRESUMEN
It has previously been shown that three amino acid changes, one each in the fusion (F; Ala/Thr-91-->Thr), haemagglutinin-neuraminidase (HN; Ser-466-->Asn) and polymerase (L; Ile-736-->Val) proteins, are associated with attenuation of a neurovirulent clinical isolate of mumps virus (88-1961) following serial passage in vitro. Here, using full-length cDNA plasmid clones and site-directed mutagenesis, it was shown that the single amino acid change in the HN protein and to a lesser extent, the change in the L protein, resulted in neuroattenuation, as assessed in rats. The combination of both amino acid changes caused neuroattenuation of the virus to levels previously reported for the clinical isolate following attenuation in vitro. The amino acid change in the F protein, despite having a dramatic effect on protein function in vitro, was previously shown to not be involved in the observed neuroattenuation, highlighting the importance of conducting confirmatory in vivo studies. This report provides additional supporting evidence for the role of the HN protein as a virulence factor and, as far as is known, is the first report to associate an amino acid change in the L protein with mumps virus neuroattenuation.
Asunto(s)
Sustitución de Aminoácidos/genética , Productos del Gen pol/fisiología , Proteína HN/fisiología , Virus de la Parotiditis/genética , Virus de la Parotiditis/patogenicidad , Factores de Virulencia/fisiología , Animales , Encéfalo/patología , Encéfalo/virología , Productos del Gen pol/genética , Proteína HN/genética , Hidrocefalia/patología , Mutagénesis Sitio-Dirigida , Mutación Missense , Ratas , Índice de Severidad de la Enfermedad , Virulencia , Factores de Virulencia/genéticaRESUMEN
Effective, vaccine-induced CD8+ T-cell responses should recognize infected cells early enough to prevent production of progeny virions. We have recently shown that Gag-specific CD8+ T cells recognize simian immunodeficiency virus-infected cells at 2 h postinfection, whereas Env-specific CD8+ T cells do not recognize infected cells until much later in infection. However, it remains unknown when other proteins present in the viral particle are presented to CD8+ T cells after infection. To address this issue, we explored CD8+ T-cell recognition of epitopes derived from two other relatively large virion proteins, Pol and Nef. Surprisingly, infected cells efficiently presented CD8+ T-cell epitopes from virion-derived Pol proteins within 2 h of infection. In contrast, Nef-specific CD8+ T cells did not recognize infected cells until 12 h postinfection. Additionally, we show that SIVmac239 Nef downregulated surface major histocompatibility complex class I (MHC-I) molecules beginning at 12 h postinfection, concomitant with presentation of Nef-derived CD8+ T-cell epitopes. Finally, Pol-specific CD8+ T cells eliminated infected cells as early as 6 h postinfection, well before MHC-I downregulation, suggesting a previously underappreciated antiviral role for Pol-specific CD8+ T cells.
Asunto(s)
Linfocitos T CD8-positivos/virología , Regulación Viral de la Expresión Génica , Productos del Gen pol/fisiología , Antígenos de Histocompatibilidad Clase I/metabolismo , Síndrome de Inmunodeficiencia Adquirida del Simio/metabolismo , Virus de la Inmunodeficiencia de los Simios/metabolismo , Proteínas Reguladoras y Accesorias Virales/metabolismo , Animales , Células Cultivadas , Regulación hacia Abajo , Epítopos/química , Epítopos de Linfocito T/química , Productos del Gen pol/metabolismo , Macaca mulatta , Complejo Mayor de Histocompatibilidad , Factores de TiempoRESUMEN
Multiple studies have described a reduction in the replicative fitness of HIV-1 isolates harboring mutations that confer resistance to antiretroviral drugs. Contradictory results, however, have been obtained depending on the methodology used in each study (Quinones-Mateu, M.E., Arts, E.J., 2002. Fitness of drug resistant HIV-I: methodology and clinical implications. Drug Resist. Update 5, 224-233), affecting our understanding of the potential relationship of viral replicative fitness with HIV-1 disease. It has been demonstrated previously that both pol and env genes play a major role in HIV-1 replicative fitness of clinical isolates. Therefore, measuring clinically relevant replicative fitness using recombinant viruses where a single mutation and/or viral gene have been introduced does not seem like a reasonable approach in this era of multi-target antiretroviral therapy. A novel method was developed to measure HIV-1 replicative fitness based on recombinant viruses expressing the enhanced green fluorescent (EGFP) or the Discosoma sp. red fluorescent (DsRed2) proteins in a HIV-1NL4-3 backbone. Contrary to previous designs to analyze HIV-1 fitness, these replication competent viruses were created in an intact viral genetic background (without deleting or affecting the expression of any viral gene). This new system was used to evaluate the contribution of drug-resistance mutations in the pol and env genes to overall viral replicative fitness (in the presence and absence of drug pressure) using direct growth competition experiments. Mutations in pol showed a stronger effect on HIV-1 replicative fitness than mutations in the env gene associated with resistance to enfuvirtide, corroborating the plasticity of the later gene to accept mutations and the sensibility of the protease and reverse transcriptase enzymes to drug-associated primary mutations. In conclusion, a new protocol was used to measure HIV-1 replicative fitness in either the presence or absence of antiretroviral drugs, which may be used as a high-throughput assay to help us understand the clinical significance of viral fitness.
Asunto(s)
Productos del Gen pol/fisiología , VIH-1/fisiología , Proteínas del Envoltorio Viral/fisiología , Replicación Viral/genética , Línea Celular , Farmacorresistencia Viral/genética , Productos del Gen pol/genética , Genes Reporteros , Proteínas Fluorescentes Verdes/análisis , Proteínas Fluorescentes Verdes/genética , VIH-1/genética , Humanos , Proteínas Luminiscentes/análisis , Proteínas Luminiscentes/genética , Coloración y Etiquetado , Proteínas del Envoltorio Viral/genética , Proteína Fluorescente RojaRESUMEN
Our goal is to understand how enzymes adapt to utilize novel substrates. We and others have shown that directed evolution tends to generate enzyme variants with broadened substrate specificity. Broad-specificity enzymes are generally deleterious to living cells, so this observed trend might be an artifact of the most commonly employed high throughput screens. Here, we demonstrate a more natural and effective screening strategy for directed evolution. The gene encoding model enzyme HIV protease was randomly mutated, and the resulting library was expressed in Escherichia coli cells to eliminate cytotoxic broad-specificity variants. The surviving variants were screened for clones with activity against a reporter enzyme. The wild-type human immunodeficiency virus type I protease (HIV PR) is cytotoxic and exhibits no detectable activity in reactions with beta-galactosidase (BGAL). In contrast, the selected variants were nontoxic and exhibited greater activity and specificity against BGAL than did the wild-type HIV PR in reactions with any substrate. A single round of whole gene random mutagenesis and conventional high-throughput screening does not usually effect complete inversions of substrate specificity. This suggests that a combination of positive and purifying selection engenders more rapid adaptation than positive selection alone.
Asunto(s)
Evolución Molecular Dirigida , Productos del Gen pol/genética , Proteasa del VIH/fisiología , VIH/enzimología , VIH/genética , Adaptación Biológica/genética , Clonación Molecular , Activación Enzimática/genética , Escherichia coli/enzimología , Escherichia coli/genética , Productos del Gen pol/fisiología , VIH/fisiología , Proteasa del VIH/genética , Humanos , Mutagénesis Sitio-Dirigida , Estructura Terciaria de Proteína/genética , Especificidad por Sustrato/genética , beta-Galactosidasa/metabolismoRESUMEN
Reports of multiple sclerosis developing after hepatitis B vaccination have led to the concern that this vaccine might be a cause of multiple sclerosis in previously healthy subjects. Some articles evidenced that minor Hepatitis B virus (HBV) polymerase proteins could be produced by alternative transcriptional or translational strategies. Their detection is very difficult because they are in minute concentration and probably enzymatically inactive, however, it was shown that they could be exposed on the outside of the virus particles and also be immunogenic. In addition, HBV polymerase shares significant amino acid similarities with the human myelin basic protein. We hypothesise that some of the apparent adverse reactions to the vaccine could be due to a process called of molecular mimicry, the HBV polymerase, which could be a contaminant in the recombinant or plasma-derived vaccines, could act as autoantigens and induce autoimmune demyelinating diseases such as multiple sclerosis.
Asunto(s)
Productos del Gen pol/química , Productos del Gen pol/fisiología , Vacunas contra Hepatitis B/efectos adversos , Virus de la Hepatitis B/enzimología , Imitación Molecular/inmunología , Esclerosis Múltiple/inmunología , Esclerosis Múltiple/virología , Secuencia de Aminoácidos , Secuencia de Bases , Productos del Gen pol/genética , Virus de la Hepatitis B/inmunología , Virus de la Hepatitis B/patogenicidad , Humanos , Datos de Secuencia MolecularRESUMEN
Foamy viruses (FVs) generate their Pol protein precursor molecule independently of the Gag protein from a spliced mRNA. This mode of expression raises the question of the mechanism of Pol protein incorporation into the viral particle (capsid). We previously showed that the packaging of (pre)genomic RNA is essential for Pol encapsidation (M. Heinkelein, C. Leurs, M. Rammling, K. Peters, H. Hanenberg, and A. Rethwilm, J. Virol. 76:10069-10073, 2002). Here, we demonstrate that distinct sequences in the RNA, which we termed Pol encapsidation sequences (PES), are required to incorporate Pol protein into the FV capsid. Two PES were found, which are contained in the previously identified cis-acting sequences necessary to transfer an FV vector. One PES is located in the U5 region of the 5' long terminal repeat and one at the 3' end of the pol gene region. Neither element has any significant effect on RNA packaging. However, deletion of either PES resulted in a significant reduction in Pol encapsidation. On the protein level, we show that only the Pol precursor, but not the individual reverse transcriptase (RT) and integrase (IN) subunits, is incorporated into FV particles. However, enzymatic activities of the protease (PR), RT, or IN are not required. Our results strengthen the view that in FVs, (pre)genomic RNA functions as a bridging molecule between Gag and Pol precursor proteins.
Asunto(s)
Productos del Gen pol/genética , Productos del Gen pol/fisiología , ARN Viral/genética , ARN Viral/metabolismo , Spumavirus/genética , Spumavirus/fisiología , Línea Celular , Mapeo Cromosómico , Genes Virales , Humanos , Spumavirus/crecimiento & desarrollo , Proteínas Virales/genética , Proteínas Virales/fisiología , Virión/genética , Virión/crecimiento & desarrollo , Virión/fisiología , Ensamble de Virus/genética , Ensamble de Virus/fisiologíaRESUMEN
All retroviruses contain two copies of genomic RNA that are linked noncovalently. The dimeric RNA of human immunodeficiency virus type 1 (HIV-1) undergoes rearrangement during virion maturation, whereby the dimeric RNA genome assumes a more stable conformation. Previously, we have shown that the packaging of the HIV-1 polymerase (Pol) proteins reverse transcriptase (RT) and integrase (IN) is essential for the generation of the mature RNA dimer conformation. Analysis of HIV-1 mutants that are defective in processing of Pol showed that these mutant virions contained altered dimeric RNA conformation, indicating that the mature RNA dimer conformation in HIV-1 requires the correct proteolytic processing of Pol. The HIV-1 Pol proteins are multimeric in their mature enzymatically active forms; RT forms a heterodimer, and IN appears to form a homotetramer. Using RT and IN multimerization defective mutants, we have found that dimeric RNA from these mutant virions has the same stability and conformation as wild-type RNA dimers, showing that the mature enzymatically active RT and IN proteins are dispensable for the generation of mature RNA dimer conformation. This also indicated that formation of the mature RNA dimer structure occurs prior to RT or IN maturation. We have also investigated the requirement of Pol for RNA dimerization in both Mason-Pfizer monkey virus (M-PMV) and Moloney murine leukemia virus (MoMuLV) and found that in contrast to HIV-1, Pol is dispensable for RNA dimer maturation in M-PMV and MoMuLV, demonstrating that the requirement of Pol in retroviral RNA dimer maturation is not conserved among all retroviruses.
Asunto(s)
Integrasas/fisiología , ARN Viral/metabolismo , ADN Polimerasa Dirigida por ARN/fisiología , Retroviridae/metabolismo , Dimerización , Productos del Gen pol/fisiologíaAsunto(s)
Productos del Gen pol/fisiología , Transactivadores/fisiología , Proteínas Virales/fisiología , Replicación Viral/genética , Animales , ADN Viral , Productos del Gen pol/genética , Genoma Viral , Proteínas de Choque Térmico/fisiología , Humanos , Activación Transcripcional/genética , Proteínas Reguladoras y Accesorias ViralesRESUMEN
Four heavily antiretroviral-experienced HIV-infected patients had significant plasma HIV-RNA reductions (>1 log) after beginning an Enfuvirtide (ENF)-based rescue regimen. However, all had viral rebound shortly thereafter, sustaining high levels of plasma viremia over 80 weeks. These patients developed rapidly genotypic and phenotypic resistance to ENF. Mutations within the HR1 env region were selected (N43D in three and G36V/D in one), resulting in high-level phenotypic resistance to ENF. Interestingly, two patients had a sustained CD4+ T-cell increase and two maintained stable CD4+ T-cell counts despite virologic failure under ENF. The possible mechanisms involved in this response were examined. Changes in virus tropism from R5 to R5/X4 were observed in two patients, in parallel with increases in ENF phenotypic resistance. Low levels of T-cell activation, T-cell turnover, and cytotoxic T lymphocyte (CTL) activity were found in all four patients. An overall increase in the proportion of viruses released from cells of the macrophage lineage was observed. In summary, single mutations at the HR1 env region result in significant loss of susceptibility to ENF. Despite virologic failure, these patients may maintain elevated CD4+ counts through a reduction in their overall immune activation.
Asunto(s)
Proteína gp41 de Envoltorio del VIH/farmacología , Proteína gp41 de Envoltorio del VIH/uso terapéutico , Inhibidores de Fusión de VIH/uso terapéutico , Infecciones por VIH/tratamiento farmacológico , VIH/efectos de los fármacos , Fragmentos de Péptidos/farmacología , Fragmentos de Péptidos/uso terapéutico , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Terapia Antirretroviral Altamente Activa , Recuento de Linfocito CD4 , Farmacorresistencia Viral/genética , Enfuvirtida , Productos del Gen pol/fisiología , Genes Virales , Genes env , Genes pol , VIH/genética , VIH/crecimiento & desarrollo , VIH/inmunología , Proteína gp41 de Envoltorio del VIH/administración & dosificación , Proteína gp41 de Envoltorio del VIH/fisiología , Inhibidores de Fusión de VIH/administración & dosificación , Inhibidores de Fusión de VIH/farmacología , Infecciones por VIH/inmunología , Infecciones por VIH/virología , Humanos , Activación de Linfocitos , Macrófagos/virología , Datos de Secuencia Molecular , Mutación Missense , Fragmentos de Péptidos/administración & dosificación , ARN Viral/sangre , Receptores CCR5/fisiología , Receptores CXCR4/fisiología , Terapia Recuperativa , Linfocitos T Citotóxicos/inmunología , ViremiaRESUMEN
The human immunodeficiency virus type 1 p6 region encodes p6(Gag) and the transframe p6(Pol) protein. The Gag frame encodes an N-terminal late assembly L domain and a C-terminal Vpr binding domain. In the Pol frame, substitution at a C-terminal motif decreases protease autocleavage. The role of the highly polymorphic central region of p6, comprising amino acids S14-I31 (p6(Gag)) and R20-D39 (p6(Pol)), is unclear. Analysis of this central region demonstrated that 35 % of p6(Gag) appears to be dispensable for virus propagation in vitro and smaller deletion and insertion polymorphisms can be tolerated in vivo. Extensive Pol deletion (deltaR20-D39, 42 % of p6(Pol)) did not alter protease autocleavage.
Asunto(s)
Productos del Gen gag/genética , Productos del Gen gag/fisiología , VIH-1/genética , VIH-1/fisiología , Secuencia de Aminoácidos , Productos del Gen gag/química , Productos del Gen pol/química , Productos del Gen pol/genética , Productos del Gen pol/fisiología , Infecciones por VIH/virología , VIH-1/aislamiento & purificación , VIH-1/patogenicidad , Humanos , Técnicas In Vitro , Datos de Secuencia Molecular , Fenotipo , Polimorfismo Genético , Recombinación Genética , Eliminación de Secuencia , Virulencia/genética , Virulencia/fisiología , Replicación Viral/genética , Replicación Viral/fisiologíaRESUMEN
Based on the recent studies of HBV strains with different replication efficiency, several new potential targets for anti-HBV replication have been presented. These include the viral and cellular regulatory factors associated with HBV replication and the process for encapsidation of viral genome and budding into endoplasmic reticulum (ER). A putative regulatory domain has been reported at the carboxyl-end of reverse transcriptase (RT) and when serine is substituted for proline at residue 652 of RT, replication efficiency of HBV is decreased. Substitution of proline for threonine at the 2798 nucleotide of the terminal protein (TP) gene, renders the mutant completely replication deficient. Expression of TP blocks the interferon (IFN) pathway and inhibits the responsive state of cells to interferons ( IFN) alpha and gamma. Interference of HBV capsid assembly drastically affects the encapsidation of viral genome, a crucial process for reverse transcription and viral DNA synthesis. Small molecules (bis-ANS) have been reported to act as a "wedge" to misdirect the polymerization of capsid, resulting in inhibition of virus replication. Another new group of compounds (HAP) has been shown to inhibit virus replication and also inhibit the assembly of viral capsid (core particle). Finally the capsids containing HBV genome are enveloped by budding into endoplasmic reticulum and release from virus infected cells, and this morphogenesis and secretion of HBV is dependent on glucosidases in the ER of host cells. Competitive inhibition of these glucosidases has been suggested as strategy against HBV replication.
Asunto(s)
Antivirales/farmacología , Virus de la Hepatitis B/efectos de los fármacos , Cápside/efectos de los fármacos , ADN Polimerasa Dirigida por ADN/química , ADN Polimerasa Dirigida por ADN/fisiología , Diseño de Fármacos , Productos del Gen pol/química , Productos del Gen pol/fisiología , Virus de la Hepatitis B/química , Humanos , ADN Polimerasa Dirigida por ARN/química , ADN Polimerasa Dirigida por ARN/fisiología , Proteínas del Envoltorio Viral/antagonistas & inhibidores , Proteínas del Envoltorio Viral/fisiología , Replicación Viral/efectos de los fármacos , Replicación Viral/genéticaRESUMEN
The major role of hepatitis B virus polymerase (HBV pol) is polymerization of nucleotides, but it also participates in protein priming and the packaging of its own genome into capsids. Therefore, HBV pol may require many assistance factors for its roles. Previous reports have shown that Hsp60, a molecular chaperone, activates HBV pol both in vitro and ex vivo, such as inside insect cells. Moreover, HBV pol binds to Hsp60 in the HepG2 host cell line. In this report, we show that Hsp60 plays a role in the in vivo replication of HBV. Antisense oligodeoxynucleotides (A-ODNs) specifically directed against Hsp60 induced its down-regulation, severely reducing the level of replication-competent HBV without influencing cell proliferation and capsid assembly under these conditions. Furthermore, we found that Hsp60 did not encapsidate into nucleocapsids. Our results indicate that Hsp60 is important for HBV replication in vivo, presumably through activation of HBV pol before encapsidation of HBV pol into HBV core particle. In addition, A-ODNs specific for Hsp60 also inhibit replication of a mutant HBV strain that is resistant to the nucleoside analogue 3TC, which is the main drug used for HBV treatment, and we suggest that A-ODNs directed against Hsp60 are possible reagents as anti-HBV drugs. Conclusively, this report shows that the host factor, Hsp60, is essential for in vivo HBV replication and that mechanism of Hsp60 is probably through an activation of HBV pol by Hsp60.
Asunto(s)
Chaperonina 60/antagonistas & inhibidores , Chaperonina 60/genética , Virus de la Hepatitis B/efectos de los fármacos , Virus de la Hepatitis B/fisiología , Oligodesoxirribonucleótidos Antisentido/genética , Oligodesoxirribonucleótidos Antisentido/farmacología , Replicación Viral/efectos de los fármacos , Antivirales/farmacología , Secuencia de Bases , Cápside/efectos de los fármacos , Línea Celular , Chaperonina 60/fisiología , Regulación hacia Abajo , Farmacorresistencia Viral/genética , Activación Enzimática , Productos del Gen pol/fisiología , Humanos , Técnicas In Vitro , Lamivudine/farmacología , Mutación , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ensamble de Virus/efectos de los fármacosRESUMEN
The clinical management of chronic hepatitis B infection has entered a new era with the introduction and widespread use of oral nucleoside analogues such as lamivudine and nucleotides such as adefovir dipivoxil. From this, new challenges have now emerged in terms of preventing antiviral drug resistance, promoting viral clearance and improving long-term survival. For example, the natural history of nucleoside or nucleotide analogue-associated hepatitis B virus resistant mutants has yet to be determined. Furthermore, the increasing prevalence of HBeAg negative disease with its reduced response to current therapies represents an ongoing challenge to attempts to improve standard of care. There is increasing recognition of the pivotal role that viral load and genotype, and their complex interactions with the host immune response, play in determining the outcome of these treatment interventions. The purpose of this paper is to highlight several key factors that should be considered in the context of future clinical research and management of chronic hepatitis B.
Asunto(s)
Hepatitis B Crónica/tratamiento farmacológico , Antivirales/uso terapéutico , ADN Viral/genética , Farmacorresistencia Viral/genética , Productos del Gen pol/química , Productos del Gen pol/genética , Productos del Gen pol/fisiología , Genes Virales , Virus de la Hepatitis B/efectos de los fármacos , Virus de la Hepatitis B/genética , Virus de la Hepatitis B/fisiología , Hepatitis B Crónica/inmunología , Hepatitis B Crónica/virología , Humanos , Mutación , Replicación ViralRESUMEN
The ordered assembly of the herpes simplex virus (HSV) type 1 replication apparatus leading to replication compartments likely involves the initial assembly of five viral replication proteins, ICP8, UL9, and the heterotrimeric helicase-primase complex (UL5-UL8-UL52), into replication foci. The polymerase and polymerase accessory protein are subsequently recruited to these foci. Four stages of viral infection (stages I to IV) have been described previously (J. Burkham, D. M. Coen, and S. K. Weller, J. Virol. 72:10100-10107, 1998). Of these, stage III foci are equivalent to the previously described promyelocytic leukemia protein (PML)-associated prereplicative sites and contain all seven replication proteins. We constructed a series of mutations in the putative primase subunit, UL52, of the helicase-primase and have analyzed the mutant proteins for their abilities to form intermediates leading to the formation of replication compartments. The results shown in this paper are consistent with the model that the five proteins, ICP8, UL5, UL8, UL9, and UL52, form a scaffold and that formation of this scaffold does not rely on enzymatic functions of the helicase and primase. Furthermore, we demonstrate that recruitment of polymerase to this scaffold requires the presence of an active primase subunit. These results suggest that polymerase recruitment to replication foci requires primer synthesis. Furthermore, they support the existence of two types of stage III intermediates in the formation of replication compartments: stage IIIa foci, which form the scaffold, and stage IIIb foci, which contain, in addition, HSV polymerase, the polymerase accessory subunit, and cellular factors such as PML.
Asunto(s)
ADN Helicasas/genética , ADN Helicasas/fisiología , ADN Polimerasa Dirigida por ADN/genética , ADN Polimerasa Dirigida por ADN/fisiología , Exodesoxirribonucleasas/genética , Exodesoxirribonucleasas/fisiología , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/fisiología , Proteínas Nucleares , Proteínas Virales/genética , Proteínas Virales/fisiología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Línea Celular , Chlorocebus aethiops , ADN Helicasas/química , ADN Primasa , ADN Viral/genética , Proteínas de Unión al ADN , Productos del Gen pol/fisiología , Prueba de Complementación Genética , Herpesvirus Humano 1/enzimología , Humanos , Sustancias Macromoleculares , Datos de Secuencia Molecular , Mutación , Proteínas de Neoplasias/fisiología , Proteína de la Leucemia Promielocítica , Homología de Secuencia de Aminoácido , Factores de Transcripción/fisiología , Transfección , Proteínas Supresoras de Tumor , Células Vero , Replicación Viral/genética , Replicación Viral/fisiología , Dedos de Zinc/genéticaRESUMEN
We examine the potential for a broad range of small animal cells, including rodent, mink, and avian cells, from multiple tissues to support postintegration steps of HIV-1 replication. These cells were engineered so as to support a stable expression of human cyclin T1 and were further transduced with HIV-1 gag and pol genes. Viral gene expression was activated by the presence of human cyclin T1, but, with the exception of mink cells, was not at the level seen in human cells. Furthermore, there were considerable defects in p24 CA release, in particular in the case of rodent cells. Fractionation of Gag proteins by sucrose floatation revealed that the Gag in human cells trafficked to membrane fractions and was processed to p24 CA and p17 MA efficiently. Confocal imaging demonstrated that Gag was localized in a punctate pattern at the plasma membrane as well as intracellular membrane trans-Golgi cisternae in these cells. In contrast, the majority of Gag in rodent cells was largely present in cytosolic complexes and remained unprocessed. Labeling with [9,10(n)-(3)H]myristic acid showed a similar degree of N-myristoylated Pr55(gag) in rodent and human cells, indicating that while N-myristoylation of Gag was essential for membrane binding, it was not sufficient to confer membrane targeting specificity. Remarkably, despite the reduced level of intracellular Gag processing, mink Mv.1.Lu cells did not appear to differ significantly from human cells in support of virion assembly and release. Analysis of reciprocal heterokaryons suggested that the cellular factor(s) required for efficient assembly and release of infectious virions is lacking in murine cells but appears to be functionally present in mink as well as human cells. Our findings confirm and extend previous reports of multiple blocks to HIV replication in nonhuman cells that are most profound in murine cells. They also raise the possibility that other small animals, such as mink, could serve as novel model systems for studying HIV-1 infection and disease.
Asunto(s)
Productos del Gen gag/fisiología , VIH-1/fisiología , Integración Viral , Replicación Viral , Células 3T3 , Animales , Línea Celular , Ciclina T , Ciclinas/fisiología , Productos del Gen gag/análisis , Productos del Gen gag/metabolismo , Productos del Gen pol/fisiología , Humanos , Ratones , Visón , Ácido Mirístico/metabolismo , Precursores de Proteínas/metabolismo , Virión/fisiología , Ensamble de VirusRESUMEN
The core particle of HIV-1 assembles at the membrane of the host cell as the virus buds from the surface. The structural proteins and enzymes that comprise the core are translated as part of two polyprotein precursors, Gag and GagPol. The Gag precursor contains the structural proteins of the core and is both necessary and sufficient for directing particle assembly and budding. Over the past few years, significant progress has been made in our understanding of the interactions that drive particle assembly. Specifically, determinants within the Gag precursor that direct membrane association, Gag-Gag interactions and particle budding have been identified and partially characterized. Subdomains of the host cell membrane that favor particle assembly and budding have also been described. Finally, a potential role for cellular processes in mediating the final stages in particle release has recently been proposed and a cellular protein that appears to bind directly to the Gag precursor has been identified. Each of these observations helps to clarify previously obscure aspects of viral replication and points towards potential targets for the design of novel therapies.
Asunto(s)
VIH-1/fisiología , Ensamble de Virus , Productos del Gen gag/fisiología , Productos del Gen pol/fisiología , Transporte de ProteínasRESUMEN
Hepadnaviruses and retroviruses are evolutionarily related families because they both require a process of reverse transcription for genome replication. However, hepadnaviruses produce polymerase (pol) and core proteins separately, while retroviruses synthesize a gag-pol fusion protein that is subsequently cleaved by a virally encoded protease to release a functional polymerase. To test whether an additional sequence at the N-terminus of pol in hepatitis B virus (HBV) interferes with its function, we created two plasmids expressing core-pol fusion proteins, core144-pol and core31-pol. Secreted particles obtained from HuH-7 cells, which were cotransfected with a core-pol fusion protein-expressing plasmid and a core-expressing plasmid, showed a positive signal of HBV DNA by the endogenous polymerase assay, indicating that the core-pol fusion proteins retain DNA priming, polymerization and RNase H activities. The fusion protein was detected in the cytoplasm of transfected cells and in secreted virions by immunoprecipitation. Furthermore, we found by immunofluorescence staining that the HBV core-pol fusion protein colocalized with the hepatitis C virus (HCV) core protein in cytoplasm and in lipid droplets. Immunoprecipitation studies showed that the anti-HCV core complex contained the HBV core-pol fusion protein while the anti-HBV pol complex contained the HCV core protein, which supports the hypothesis that the HCV core protein can form a complex with the HBV core-pol fusion protein.