RESUMEN
The human immunodeficiency virus type 1 (HIV-1) Gag protein recruits Tsg101 to facilitate HIV-1 particle budding and release. In uninfected cells, the Hrs protein recruits the ESCRT-I complex to the endosome, also through an interaction with Tsg101, to promote the sorting of host proteins into endosomal vesicles and multivesicular bodies. Here, we show that the overexpression of the C-terminal fragment of Hrs (residues 391 to 777) or Hrs mutants lacking either the N-terminal FYVE domain (mutant dFYVE) or the PSAP (residues 348 to 351) motif (mutant ASAA) all efficiently inhibit HIV-1 Gag particle production. Expression of the dFYVE or ASAA mutants of Hrs had no effect on the release of Moloney murine leukemia virus. Coimmunoprecipitation analysis showed that the expression of Hrs mutant dFYVE or ASAA significantly reduced or abolished the HIV-1 Gag-Tsg101 interaction. Yeast-two hybrid assays were used to identify two new and independent Tsg101 binding sites, one in the Hrs coiled-coil domain and one in the proline/glutamic acid-rich domain. Scanning electron microscopy of HeLa cells expressing HIV-1 Gag and the Hrs ASAA mutant showed viral particles arrested in "lump-like" structures that remained attached to the cell surface. Together, these data indicate that fragments of Hrs containing the C-terminal portion of the protein can potently inhibit HIV-1 particle release by efficiently sequestering Tsg101 away from the Gag polyprotein.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Productos del Gen gag/metabolismo , VIH-1/fisiología , Fosfoproteínas/metabolismo , Factores de Transcripción/metabolismo , Virión/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Sitios de Unión , Línea Celular , Proteínas de Unión al ADN/genética , Complejos de Clasificación Endosomal Requeridos para el Transporte , Productos del Gen gag/genética , Humanos , Datos de Secuencia Molecular , Mutación , Fosfoproteínas/química , Fosfoproteínas/genética , Unión Proteica , Estructura Terciaria de Proteína , Factores de Transcripción/genética , Técnicas del Sistema de Dos Híbridos , Virión/ultraestructuraRESUMEN
Capsid assembly among the herpes-group viruses is coordinated by two related scaffolding proteins. In cytomegalovirus (CMV), the main scaffolding constituent is called the assembly protein precursor (pAP). Like its homologs in other herpesviruses, pAP is modified by proteolytic cleavage and phosphorylation. Cleavage is essential for capsid maturation and production of infectious virus, but the role of phosphorylation is undetermined. As a first step in evaluating the significance of this modification, we have identified the specific sites of phosphorylation in the simian CMV pAP. Two were established previously to be adjacent serines (Ser156 and Ser157) in a casein kinase II consensus sequence. The remaining two, identified here as Thr231 and Ser235, are within consensus sequences for glycogen synthase kinase 3 (GSK-3) and mitogen-activated protein kinase, respectively. Consistent with Thr231 being a GSK-3 substrate, its phosphorylation required a downstream "priming" phosphate (i.e., Ser235) and was reduced by a GSK-3-specific inhibitor. Phosphorylation of Ser235 converts pAP to an electrophoretically slower-mobility isoform, pAP*; subsequent phosphorylation of pAP* at Thr231 converts pAP* to a still-slower isoform, pAP**. The mobility shift to pAP* was mimicked by substituting an acidic amino acid for either Thr231 or Ser235, but the shift to pAP** required that both positions be phosphorylated. Glu did not substitute for pSer235 in promoting phosphorylation of Thr231. We suggest that phosphorylation of Thr231 and Ser235 causes charge-driven conformational changes in pAP, and we demonstrate that preventing these modifications alters interactions of pAP with itself and with major capsid protein, suggesting a functional significance.
Asunto(s)
Proteínas de la Cápside/metabolismo , Citomegalovirus/metabolismo , Glucógeno Sintasa Quinasa 3/química , Precursores de Proteínas/metabolismo , Proteínas Virales/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular , Glucógeno Sintasa Quinasa 3/metabolismo , Humanos , Espectrometría de Masas , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mapeo Peptídico , Fosforilación , Precursores de Proteínas/química , Precursores de Proteínas/genética , Proteínas Virales/química , Proteínas Virales/genéticaRESUMEN
As the most numerous cells in the brain, astrocytes play a critical role in maintaining central nervous system homeostasis, and therefore, infection of astrocytes by human immunodeficiency virus (HIV) or simian immunodeficiency virus (SIV) in vivo could have important consequences for the development of HIV encephalitis. In this study, we establish that astrocytes are infected in macaques during acute SIV infection (10 days postinoculation) and during terminal infection when there is evidence of SIV-induced encephalitis. Additionally, with primary adult rhesus macaque astrocytes in vitro, we demonstrate that the macrophage-tropic, neurovirulent viruses SIV/17E-Br and SIV/17E-Fr replicate efficiently in astrocytes, while the lymphocyte-tropic, nonneurovirulent virus SIV(mac)239 open-nef does not establish productive infection. Furthermore, aminoxypentane-RANTES abolishes virus replication, suggesting that these SIV strains utilize the chemokine receptor CCR5 for entry into astrocytes. Importantly, we show that SIV Nef is required for optimal replication in primary rhesus macaque astrocytes and that normalizing input virus by particle number rather than by infectivity reveals a disparity between the ability of a Nef-deficient virus and a virus encoding a nonmyristoylated form of Nef to replicate in these central nervous system cells. Since the myristoylated form of Nef has been implicated in functions such as CD4 and major histocompatibility complex I downregulation, kinase association, and enhancement of virion infectivity, these data suggest that an as yet unidentified function of Nef may exist to facilitate SIV replication in astrocytes that may have important implications for in vivo pathogenesis.