RESUMEN
Proper folding of newly synthesized viral proteins in the cytoplasm is a prerequisite for the formation of infectious virions. The major capsid protein Vp1 of simian virus 40 forms a series of disulfide-linked intermediates during folding and capsid formation. In addition, we report here that Vp1 is associated with cellular chaperones (HSP70) and a cochaperone (Hsp40) which can be coimmunoprecipitated with Vp1. Studies in vitro demonstrated the ATP-dependent interaction of Vp1 and cellular chaperones. Interestingly, viral cochaperones LT and ST were essential for stable interaction of HSP70 with the core Vp1 pentamer Vp1 (22-303). LT and ST also coimmunoprecipitated with Vp1 in vivo. In addition to these identified (co)chaperones, stable, covalently modified forms of Vp1 were identified for a folding-defective double mutant, C49A-C87A, and may represent a "trapped" assembly intermediate. By a truncation of the carboxyl arm of Vp1 to prevent the Vp1 folding from proceeding beyond pentamers, we detected several apparently modified Vp1 species, some of which were absent in cells transfected with the folding-defective mutant DNA. These results suggest that transient covalent interactions with known or unknown cellular and viral proteins are important in the assembly process.
Asunto(s)
Antígenos Virales de Tumores/metabolismo , Proteínas HSP70 de Choque Térmico/metabolismo , Virus 40 de los Simios/fisiología , Proteínas Estructurales Virales/metabolismo , Ensamble de Virus , Animales , Línea Celular , Chlorocebus aethiops , Proteínas del Choque Térmico HSP40/metabolismo , Inmunoprecipitación , Mutación Puntual , Unión Proteica , Eliminación de Secuencia , Proteínas no Estructurales Virales/metabolismoRESUMEN
The exposure of molecular signals for simian virus 40 (SV40) cell entry and nuclear entry has been postulated to involve calcium coordination at two sites on the capsid made of Vp1. The role of calcium-binding site 2 in SV40 infection was examined by analyzing four single mutants of site 2, the Glu160Lys, Glu160Arg, Glu157Lys (E157K), and Glu157Arg mutants, and an E157K-E330K combination mutant. The last three mutants were nonviable. All mutants replicated viral DNA normally, and all except the last two produced particles containing all three capsid proteins and viral DNA. The defect of the site 1-site 2 E157K-E330K double mutant implies that at least one of the sites is required for particle assembly in vivo. The nonviable E157K particles, about 10% larger in diameter than the wild type, were able to enter cells but did not lead to T-antigen expression. Cell-internalized E157K DNA effectively coimmunoprecipitated with anti-Vp1 antibody, but little of the DNA did so with anti-Vp3 antibody, and none was detected in anti-importin immunoprecipitate. Yet, a substantial amount of Vp3 was present in anti-Vp1 immune complexes, suggesting that internalized E157K particles are ineffective at exposing Vp3. Our data show that E157K mutant infection is blocked at a stage prior to the interaction of the Vp3 nuclear localization signal with importins, consistent with a role for calcium-binding site 2 in postentry steps leading to the nuclear import of the infecting SV40.
Asunto(s)
Calcio/metabolismo , Infecciones por Polyomavirus/metabolismo , Virus 40 de los Simios/metabolismo , Infecciones Tumorales por Virus/metabolismo , Sustitución de Aminoácidos/genética , Animales , Sitios de Unión/genética , Proteínas de la Cápside/genética , Proteínas de la Cápside/metabolismo , Línea Celular , Chlorocebus aethiops , Mutagénesis Sitio-Dirigida , Infecciones por Polyomavirus/virología , Virus 40 de los Simios/genética , Infecciones Tumorales por Virus/virología , Ensamble de Virus/genéticaRESUMEN
We investigated the roles of simian virus 40 capsid proteins in the viral life cycle by analyzing point mutants in Vp1 and Vp2/3, as well as a deletion mutant lacking the Vp2/3 coding sequence. The Vp1 mutants (V243E and L245E) and the Vp2/3 mutants (F157E-I158E and P164R-G165E-G166R) were previously shown to be defective in Vp1-Vp2/3 interaction and to be noninfectious or poorly infectious, respectively. Here, we show that all these point mutants form stable particles following DNA transfection into cells. The Vp2/3-mutant particles contained very low levels of Vp2/3, whereas the Vp1 mutant particles contained no detectable Vp2/3. As expected, the deletion mutant also formed particles that were noninfectious. We further characterized the two Vp1 point mutants and the deletion mutant. All three mutant particles comprised Vp1 and histone-associated viral DNA, and all were able to enter cells. However, the mutant complexes failed to associate with host importins (owing to the loss of the Vp2/3 nuclear localization signal), and the mutant viral DNAs prematurely dissociated from the Vp1s, suggesting that the nucleocapsids did not enter the nucleus. Consistently, all three mutant particles failed to express large T antigen. Together, our results demonstrate unequivocally that Vp2/3 is dispensable for the formation of nucleocapsids. Further, the nucleocapsids' ability to enter cells implies that Vp1 contains the major determinants for cell attachment and entry. We propose that the major role of Vp2/3 in infectivity is to mediate the nuclear entry of viral DNA.
Asunto(s)
Proteínas de la Cápside/fisiología , ADN Viral/metabolismo , Nucleocápside/metabolismo , Virus 40 de los Simios/fisiología , Ensamble de Virus , Internalización del Virus , Sustitución de Aminoácidos , Animales , Transporte Biológico , Proteínas de la Cápside/genética , Línea Celular , Núcleo Celular/virología , Genoma Viral , Haplorrinos , Carioferinas/metabolismo , Mutación Puntual , Unión Proteica , Eliminación de Secuencia , Virus 40 de los Simios/genéticaRESUMEN
To establish viral infection, SV40 must expose nuclear localization signals (NLSs) that are internal in the virion architecture in order to enter the nucleus via interaction with the host's nuclear import machinery, which includes importin alpha and importin beta. The time course for SV40 association with the importins in infected cells was examined. The viral DNA associated with importin alpha by 1.5h post infection, before associating with the importin beta nuclear import receptor, by 3h post infection. Only a small fraction of cell-internalized SV40 that contained viral DNA was bound by the two importins. This fraction, termed "nuclear entry-competent SV40," was slightly smaller than the virion but, importantly, was larger than the viral chromatin and contained both Vp1 and Vp3. Furthermore, the internalized viral DNA in either anti-importin or anti-Vp3 immune complexes was sensitive to DNase I, whereas the viral DNA in mature virions was resistant. All these results suggest that once SV40 enters the cytoplasm, it undergoes an architectural modification that exposes the virion's NLSs for nuclear entry.
Asunto(s)
ADN Viral/metabolismo , Virus 40 de los Simios/genética , Virus 40 de los Simios/fisiología , Proteínas Virales/metabolismo , alfa Carioferinas/metabolismo , beta Carioferinas/metabolismo , Animales , Proteínas de la Cápside/análisis , Línea Celular , Desoxirribonucleasa I/metabolismo , Haplorrinos , Cinética , Sustancias Macromoleculares/metabolismo , Unión ProteicaRESUMEN
Transient disulfide bonding occurs during the intracellular folding and pentamerization of simian virus 40 (SV40) major capsid protein Vp1 (P. P. Li, A. Nakanishi, S. W. Clark, and H. Kasamatsu, Proc. Natl. Acad. Sci. USA 99:1353-1358, 2002). We investigated the requirement for Vp1 cysteine pairs during SV40 infection. Our analysis identified three Vp1 double-cysteine mutant combinations that abolished viability as assayed by plaque formation. Mutating the Cys49-Cys87 pair or the Cys87-Cys254 pair led to ineffective nuclear localization and diminished accumulation of the mutant Vp1s, and the defect extended in a dominant-negative manner to the wild-type minor capsid proteins Vp2/3 and an affinity-tagged recombinant Vp1 expressed in the same cells. Mutating the Cys87-Cys207 pair preserved the nuclear localization and normal accumulation of the capsid proteins but diminished the production of virus-like particles. Our results are consistent with a role for Cys49, Cys87, and Cys254 in the folding and cytoplasmic-nuclear trafficking of Vp1 and with a role for Cys87 and Cys207 in the assembly of infectious particles. These findings suggest that transient disulfide bond formation between certain Vp1 cysteine residues functions at two stages of SV40 infection: during Vp1 folding and oligomerization in the cytoplasm and during virion assembly in the nucleus.
Asunto(s)
Proteínas de la Cápside/genética , Proteínas de la Cápside/fisiología , Cisteína/genética , Cisteína/fisiología , Virus 40 de los Simios/fisiología , Sustitución de Aminoácidos , Proteínas de la Cápside/análisis , Proteínas de la Cápside/metabolismo , Núcleo Celular/química , Mutación , Transporte de Proteínas , Virus 40 de los Simios/crecimiento & desarrollo , Ensayo de Placa Viral , Ensamble de Virus , Replicación ViralRESUMEN
For polyomaviruses, calcium ions are known to be essential for virion integrity and for the assembly of capsid structures. To define the role of calcium ions in the life cycle of the virus, we analyzed simian virus 40 (SV40) mutants in which structurally deduced calcium-binding amino acids of Vp1 were mutated singly and in combination. Our study provides evidence that calcium ions mediate not only virion assembly but also the initial infection processes of cell entry and nuclear entry. Mutations at Glu48, Glu157, Glu160, Glu216, and/or Glu330 are correlated with different extents of packaging defects. The low packaging ability of mutant E216R suggests the need to position the Glu216 side chain for proper virion formation. All other mutants selected for further analysis produced virus-like particles (VLPs) but were poorly infectious. The VLPs of mutant E330K could not attach to or enter the cell, and mutant E157A-E160A and E216K VLPs entered the cell but failed to enter the nucleus, apparently as a result of premature VLP dissociation. Our results show that five of the seven acidic side chains at the two calcium-binding sites-Glu48 and Glu330 (site 1), Glu157 and Glu160 (site 2), and Glu216 (both sites)-are important for SV40 infection. We propose that calcium coordination imparts not only stability but also structural flexibility to the virion, allowing the acquisition or loss of the ion at the two sites to control virion formation in the nucleus, as well as virion structural alterations at the cell surface and in the cytoplasm early during infection.
Asunto(s)
Calcio/metabolismo , Proteínas de la Cápside/metabolismo , Núcleo Celular/virología , Fusión de Membrana , Virus 40 de los Simios/fisiología , Aminoácidos/metabolismo , Secuencia de Bases , Proteínas de la Cápside/química , Proteínas de la Cápside/genética , Cartilla de ADN , Microscopía Electrónica , Mutagénesis , Plásmidos , Unión Proteica , Replicación ViralRESUMEN
Pentamer formation by Vp1, the major capsid protein of simian virus 40, requires an interdigitation of structural elements from the Vp1 monomers [Liddington, R. C., Yan, Y., Moulai, J., Sahli, R., Benjamin, T. L. & Harrison, S. C. (1991) Nature (London) 354, 278-284]. Our analyses reveal that disulfide-linked Vp1 homooligomers are present in the simian virus 40-infected cytoplasm and that they are derived from a 41-kDa monomeric intermediate containing an intrachain disulfide bond(s). The 41-kDa species, emerging within 5 min of pulse labeling with [(35)S]methionine, is converted into a 45-kDa, disulfide-free Vp1 monomer and disulfide-bonded dimers through pentamers. The covalent oligomer formation is blocked in the presence of a sulfhydryl-modifying reagent. We propose that there are two stages in this Vp1 disulfide bonding. First, the newly synthesized Vp1 monomers acquire intrachain bonds as they fold and begin to interact. Next, these bonds are replaced with intermolecular bonds as the monomers assemble into pentamers. This sequential appearance of transitory disulfide bonds is consistent with a role for sulfhydryl-disulfide redox reactions in the coordinate folding of Vp1 chains into pentamers. The cytoplasmic Vp1 does not colocalize with marker proteins of the endoplasmic reticulum. This paper demonstrates in vivo disulfide formations and exchanges coupled to the folding and oligomerization of a mammalian protein in the cytoplasm, outside the secretory pathway. Such disulfide dynamics may be a general phenomenon for other cysteine-bearing mammalian proteins that fold in the cytoplasm.