Dataset Information

Identification of amino acid residues within simian virus 40 capsid proteins Vp1, Vp2, and Vp3 that are required for their interaction and for viral infection.

ABSTRACT: Interaction of simian virus 40 (SV40) major capsid protein Vp1 with the minor capsid proteins Vp2 and Vp3 is an integral aspect of the SV40 architecture. Two Vp3 sequence elements mediate Vp1 pentamer binding in vitro, Vp3 residues 155 to 190, or D1, and Vp3 residues 222 to 234, or D2. Of the two, D1 but not D2 was necessary and sufficient to direct the interaction with Vp1 in vivo. Rational mutagenesis of Vp3 residues (Phe157, Ile158, Pro164, Gly165, Gly166, Leu177, and Leu181) or Vp1 residues (Val243 and Leu245), based on a structural model of the SV40 Vp1 pentamer complexed with Vp3 D1, was carried out to disrupt the interaction between Vp1 and Vp3 and to study the consequences of these mutations for viral viability. Altering these residues to bulky, charged residues blocked the interaction in vitro. When these alterations were introduced into the viral genome, they reduced viral viability. Mutants with alterations in Vp1 Val243, Leu245, or both to glutamate were nearly nonviable, whereas those with Vp3 alterations reduced, but did not eliminate, viability. Our results defined the residues of Vp1 and the minor capsid proteins that are essential for both the interaction of the capsid proteins and viral viability in permissive cells.

SUBMITTER: Nakanishi A

PROVIDER: S-EPMC1563927 | biostudies-literature | 2006 Sep

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Identification of amino acid residues within simian virus 40 capsid proteins Vp1, Vp2, and Vp3 that are required for their interaction and for viral infection.

Nakanishi Akira A Nakamura Akiko A Liddington Robert R Kasamatsu Harumi H

Journal of virology 20060901 18

Interaction of simian virus 40 (SV40) major capsid protein Vp1 with the minor capsid proteins Vp2 and Vp3 is an integral aspect of the SV40 architecture. Two Vp3 sequence elements mediate Vp1 pentamer binding in vitro, Vp3 residues 155 to 190, or D1, and Vp3 residues 222 to 234, or D2. Of the two, D1 but not D2 was necessary and sufficient to direct the interaction with Vp1 in vivo. Rational mutagenesis of Vp3 residues (Phe157, Ile158, Pro164, Gly165, Gly166, Leu177, and Leu181) or Vp1 residues ...[more]

PMID: 16940501

Similar Datasets

Project description:UnlabelledSimian virus 40 (SV40), a polyomavirus that has served as an important model to understand many aspects of biology, induces dramatic cytoplasmic vacuolization late during productive infection of monkey host cells. Although this activity led to the discovery of the virus in 1960, the mechanism of vacuolization is still not known. Pentamers of the major SV40 capsid protein VP1 bind to the ganglioside GM1, which serves as the cellular receptor for the virus. In this report, we show that binding of VP1 to cell surface GM1 plays a key role in SV40 infection-induced vacuolization. We previously showed that SV40 VP1 mutants defective for GM1 binding fail to induce vacuolization, even though they replicate efficiently. Here, we show that interfering with GM1-VP1 binding by knockdown of GM1 after infection is established abrogates vacuolization by wild-type SV40. Vacuole formation during permissive infection requires efficient virus release, and conditioned medium harvested late during SV40 infection rapidly induces vacuoles in a VP1- and GM1-dependent fashion. Furthermore, vacuolization can also be induced by a nonreplicating SV40 pseudovirus in a GM1-dependent manner, and a mutation in BK pseudovirus VP1 that generates GM1 binding confers vacuole-inducing activity. Vacuolization can also be triggered by purified pentamers of wild-type SV40 VP1, but not by GM1 binding-defective pentamers or by intracellular expression of VP1. These results demonstrate that SV40 infection-induced vacuolization is caused by the binding of released progeny viruses to GM1, thereby identifying the molecular trigger for the activity that led to the discovery of SV40.ImportanceThe DNA tumor virus SV40 was discovered more than a half century ago as a contaminant of poliovirus vaccine stocks, because it caused dramatic cytoplasmic vacuolization of permissive host cells. Although SV40 played a historically important role in the development of molecular and cellular biology, restriction mapping, molecular cloning, and whole-genome sequencing, the basis of this vacuolization phenotype was unknown. Here, we show that SV40-induced vacuolization is triggered by the binding of the major viral capsid protein, VP1, to a cell surface ganglioside receptor, GM1. No other viral proteins or virus replication is required for vacuole formation. Other polyomaviruses utilize different ganglioside receptors, but they do not induce vacuolization. This work identifies the molecular trigger for the phenotype that led to the discovery of this important virus and provides the first molecular insight into an unusual and enigmatic cytopathic effect due to virus infection.

Project description:Rotaviruses are 11-segmented double-stranded RNA viruses and important causes of acute gastroenteritis in young children. To investigate the functions of specific viral proteins during the rotavirus lifecycle, temperature-sensitive (ts) mutants were previously created using a cultivatable simian strain (SA11) and chemical mutagenesis. These ts SA11 mutants replicate more efficiently at the permissive temperature of 31 °C than at the non-permissive temperature of 39 °C. Prototype strains SA11-tsC, SA11-tsF, and SA11-tsG were mapped to the genes encoding structural proteins VP1, VP2, and VP6, respectively, and putative ts lesions were identified using Sanger sequencing. However, additional background mutations in their genomes had hampered validation of the ts lesions and confounded their use in mechanistic studies. Here, we employed plasmid only-based reverse genetics to engineer recombinant (r) SA11 rotaviruses containing only the putative ts lesions of SA11-tsC (L138P change in VP1), SA11-tsF (A387D change in VP2) or SA11-tsG (S10T, D13H, and A121G changes in VP6). For simplicity, we refer to these newly-engineered, isogenic viruses as rSA11-tsVP1, rSA11-tsVP2, and rSA11-tsVP6. Single-cycle growth assays revealed that these mutants indeed exhibit ts phenotypes with significantly diminished titers (>1.5-logs) at 39 °C versus 31 °C. The rSA11 ts mutants proved genetically stable at the population-level following 3 sequential passages at 39 °C, but individual revertant clones were detected in plaque assays. Heat sensitivity experiments showed that pre-incubation of rSA11-tsVP1 or rSA11-tsVP2, but not rSA11-tsVP6, at 39 °C diminished replication at 31 °C. This result indicates that the ts lesions in VP1 and VP2 affect the incoming virion but those in VP6 affect a later stage of the viral lifecycle. In silico molecular dynamics simulations predicted temperature-dependent, long-range effects of the S10T, D13H, and/or A121G changes on the VP6 structure. Altogether, our results confirm the ts lesions of the original SA11-tsC, SA11-tsF, and SA11-tsG mutants, provide a new set of isogenic strains for investigating aspects of rotavirus replication, and shed light on how the ts lesions might impact VP1, VP2, or VP6 functions.

Dataset Information

Identification of amino acid residues within simian virus 40 capsid proteins Vp1, Vp2, and Vp3 that are required for their interaction and for viral infection.

Publications

Identification of amino acid residues within simian virus 40 capsid proteins Vp1, Vp2, and Vp3 that are required for their interaction and for viral infection.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets