Subcellular distribution of viral structural proteins during simian virus 40 infection

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Structural proteins of simian virus 40 (SV40), Vp2 and Vp3 (Vp2/3) and Vpl, carry individual nuclear targeting signals, Vp3198_2o6 (Vp2316-324) and Vpll,, respectively, which are encoded in different reading frames of an overlapping region of the genome. How signals coordinate nuclear targeting during virion morphogenesis was examined by using SV40 variants in which there is only one structural gene for Vpl or

Section: Discussionsupporting

confidence: 91%

mentioning

confidence: 99%

Functional complementation of nuclear targeting-defective mutants of simian virus 40 structural proteins

Ishii

Nakanishi

Yamada

et al. 1994

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“…In vivo experiments with SV40 suggest that VP1, VP2, and VP3 interact prior to assembly to ensure the proper stoichiometry of capsid proteins within the nucleus (18,23,24). One hypothesis proposes that VP1 pentamers interact with VP2 and/or VP3 in the cytoplasm for cotransport into the nucleus (7,17).…”

mentioning

confidence: 99%

Expression of the polyomavirus minor capsid proteins VP2 and VP3 in Escherichia coli: in vitro interactions with recombinant VP1 capsomeres

Delos

Cripe

Leavitt

et al. 1995

The polyomavirus VP2 and VP3 capsid proteins were expressed in Escherichia coli. The majority of the expressed proteins were in an insoluble fraction, and they were extracted and initially purified in 8 M urea before renaturation. Soluble VP2 and VP3 were mixed with purified recombinant VP1 capsomeres, and their interactions were assayed by immunoprecipitation and ion-exchange chromatography. Coimmunoprecipitation could be demonstrated with antibodies to either VP1 or VP2/VP3. Mixing recombinant VP1 with VP2 and VP3 modified the recognition of VP1 by domain-specific antipeptide antibodies and altered the chromatographic behavior of the individual proteins. Similar results were observed when a truncated VP1 protein, ⌬NCOVP1, with 62 amino acids deleted from the carboxy terminus was mixed with VP2/VP3. After the mixing, equilibrium dissociation constants for their binding to either VP1 or ⌬NCOVP1 were determined to be 0.37 ؎ 0.23 M for VP2 and 0.18 ؎ 0.21 M for VP3. These studies demonstrate that the recombinant VP2 and VP3 proteins interact with VP1 to affect the biochemical properties of VP1 capsomeres and to change the epitope accessibility of VP1 pentamers. These changes may reflect conformational alterations in VP1 capsomeres which are necessary for viral genome encapsidation.

“…SV40 is composed of a circular 5,243-bp DNA genome, which is enclosed within an icosahedral capsid made up of three virally encoded proteins (32). Seventy-two Vp1 pentamers form the outer shell of the virus, while the minor coat proteins, Vp2 and Vp3, of which there are about 72 copies, lie within this capsid (1,26,27). Vp2 and Vp3 are inferred to form prongs which radiate from a minichromosome core into the central cavities of the Vp1 pentamers and, as such, would place the minor coat proteins in contact with the viral DNA as well as Vp1 (19).…”

mentioning

confidence: 99%

“…How viral assembly proceeds in the nucleus remains unclear. Several studies with both polyomavirus and SV40 have suggested that Vp1 and either Vp2 or Vp3 form ''subvirion'' complexes immediately following the synthesis of the capsid proteins in the cytoplasm and that these complexes are then transported into the nucleus, where virion assembly proceeds (12,14,22,24,27). Once the structural proteins enter the nucleus, the interaction of these proteins with the progeny viral genome has been shown to occur in a sequential manner, with the capsid proteins arranging on the viral minichromosome to form a virion particle (3,4,9,13,15,23).…”

mentioning

confidence: 99%

Essential role of the Vp2 and Vp3 DNA-binding domain in simian virus 40 morphogenesis

Dean

Lee

et al. 1995

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Both a DNA-binding domain and a Vp1 interactive determinant have been mapped to the carboxy-terminal 40 residues of the simian virus 40 (SV40) minor capsid proteins, Vp2 and Vp3 (Vp2/3), with the last 13 residues being necessary for these activities. The role of this DNA-binding domain in SV40 morphogenesis and the ability to separate these two signals were investigated by mutagenesis and assessment of the activity and viability of the mutants. The carboxy-terminal 40 residues of Vp2/3 were expressed as a polyhistidine fusion protein, and five basic residues at the extreme carboxy terminus (Vp3 residues K226, R227, R228, R230, and R233) were mutagenized. The wild-type fusion protein bound DNA with a K d of 3 ؋ 10 ؊8 M, identical to that of the full-length Vp3. Mutant proteins containing either one to three or four amino acid substitutions bound DNA 4-to 7-fold or 20-to 30-fold less well, respectively, than the wild-type protein did. The most severe point mutants showed residual DNA binding similar to that of a truncated protein which lacks the entire 13 carboxy-terminal residues. All of the point mutants were able to interact with Vp1, indicating that the two signals within this region are mediated by different residues. When the mutations were placed into the context of the viral DNA and introduced into cells, all the structural proteins were expressed and localized correctly. Not all, however, were viable: mutant genomes whose Vp2/3 bound DNA with intermediate affinities formed plaques just as well as wild-type SV40 DNA did, but three mutants showing greatly reduced DNA binding failed to form plaques at all. These results are consistent with the hypothesis that Vp2/3 plays an essential role in SV40 virion assembly in the nucleus.