Cys9, Cys104 and Cys207 of simian virus 40 Vp1 are essential for inter-pentamer disulfide-linkage and stabilization in cell-free lysates

Previous studies have implicated disulfide bonds between Vp1 molecules in the stabilization of the simian virus 40 (SV40) capsid. To identify the cysteine residues involved in intermolecular disulfide interactions, systematic oligo-directed mutagenesis of cysteine codons to serine codons was initiated. Wild-type and mutant Vp1 proteins were produced in rabbit reticulocyte lysates and were allowed to interact post-translationally. Disulfide-linked Vp1 complexes were assessed via non-reducing SDS–PAGE and via sucrose-gradient sedimentation. Wild-type Vp1 forms 7S pentamers followed by 12S disulfide-linked multi-pentameric complexes in cell-free lysates. Mutagenesis of all seven cysteine codons abolished Vp1 12S complexes, but did not affect pentamer formation. A quadruple Vp1 mutant at Cys49, Cys87, Cys254 and Cys267 continued to form 12S complexes, whereas the major products of the Cys9, Cys104 and Cys207 triple mutant Vp1 were 7S pentamers. Single and double mutant Vp1 proteins at the three cysteines affected continued to form 12S complexes, but to a lesser extent. Thus, inter-pentamer disulfide bonds at Cys9, Cys104 and Cys207 are essential and sufficient for stabilization of Vp1 complexes in cell-free lysates. These results are in agreement with previous structural studies of SV40 that implicated the same three residues in disulfide linkage in the capsid. Possible parameters for the involvement of the three cysteines are discussed.

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Role of Simian Virus 40 Vp1 Cysteines in Virion Infectivity

Journal of Virology ◽

10.1128/jvi.74.23.11388-11393.2000 ◽

2000 ◽

Vol 74 (23) ◽

pp. 11388-11393 ◽

Cited By ~ 25

Author(s):

Peggy P. Li ◽

Akira Nakanishi ◽

Mary A. Tran ◽

Adler M. Salazar ◽

Robert C. Liddington ◽

...

Keyword(s):

Life Cycle ◽

Disulfide Bonds ◽

Three Dimensional ◽

Simian Virus 40 ◽

Experimental System ◽

Simian Virus ◽

Dimensional Structure ◽

Wild Type

ABSTRACT We have developed a new nonoverlapping infectious viral genome (NO-SV40) in order to facilitate structure-based analysis of the simian virus 40 (SV40) life cycle. We first tested the role of cysteine residues in the formation of infectious virions by individually mutating the seven cysteines in the major capsid protein, Vp1. All seven cysteine mutants—C9A, C49A, C87A, C104A, C207S, C254A, and C267L—retained viability. In the crystal structure of SV40, disulfide bridges are formed between certain Cys104 residues on neighboring pentamers. However, our results show that none of these disulfide bonds are required for virion infectivity in culture. We also introduced five different mutations into Cys254, the most strictly conserved cysteine across the polyomavirus family. We found that C254L, C254S, C254G, C254Q, and C254R mutants all showed greatly reduced (around 100,000-fold) plaque-forming ability. These mutants had no apparent defect in viral DNA replication. Mutant Vp1's, as well as wild-type Vp2/3, were mostly localized in the nucleus. Further analysis of the C254L mutant revealed that the mutant Vp1 was able to form pentamers in vitro. DNase I-resistant virion-like particles were present in NO-SV40-C254L-transfected cell lysate, but at about 1/18 the amount in wild-type-transfected lysate. An examination of the three-dimensional structure reveals that Cys254 is buried near the surface of Vp1, so that it cannot form disulfide bonds, and is not involved in intrapentamer interactions, consistent with the normal pentamer formation by the C254L mutant. It is, however, located at a critical junction between three pentamers, on a conserved loop (G2H) that packs against the dual interpentamer Ca2+-binding sites and the invading C-terminal helix of an adjacent pentamer. The substitution by the larger side chains is predicted to cause a localized shift in the G2H loop, which may disrupt Ca2+ ion coordination and the packing of the invading helix, consistent with the defect in virion assembly. Our experimental system thus allows dissection of structure-function relationships during the distinct steps of the SV40 life cycle.

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Cys9, Cys104 and Cys207 of simian virus 40 Vp1 are essential for infectious virion formation in CV-1 cells

Journal of General Virology ◽

10.1099/0022-1317-82-8-1935 ◽

2001 ◽

Vol 82 (8) ◽

pp. 1935-1939 ◽

Cited By ~ 4

Author(s):

Editte Gharakhanian ◽

Clare L. Fasching ◽

Salvatore J. Orlando ◽

Ana R. Perez

Keyword(s):

Protein Interactions ◽

Virus Assembly ◽

Simian Virus 40 ◽

Simian Virus ◽

Protein Protein Interactions ◽

Triple Mutant ◽

Disulfide Linkage ◽

Sv40 Infection ◽

Virion Formation

Structural studies have implicated Cys9, Cys104 and Cys207 of simian virus 40 (SV40) Vp1 in disulfide bond formation. Recently, we have shown the three cysteines to be essential for disulfide linkage of Vp1 complexes in vitro. Here, the role of the three cysteines was explored during the course of SV40 infection. Single-, double- and triple-mutant Vp1 at Cys9, Cys104 and Cys207 continued to localize to the nuclei of transfected CV-1 cells and to bind DNA, but showed a range of abilities to form plaques. Only mutants containing the Cys9→Ser change showed defects in plaque formation. Single mutants at Cys9 formed small plaques; mutants at Cys9 . Cys104, Cys9 . Cys207 and Cys9 . Cys104 . Cys207 formed no plaques. All three isolated revertants contained back-mutations at the Vp1 Cys9 codon. These results further confirm the involvement of the three Vp1 cysteines in protein–protein interactions during virus assembly. Cys9 is critical for production of wild-type infectious virions, whereas Cys104 and Cys207 play secondary roles.

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Analysis of simian virus 40 wild-type and mutant virions by agarose gel electrophoresis.

Journal of Virology ◽

10.1128/jvi.17.3.916-923.1976 ◽

1976 ◽

Vol 17 (3) ◽

pp. 916-923 ◽

Cited By ~ 5

Author(s):

M Zweig ◽

S Barban ◽

N P Salzman

Keyword(s):

Gel Electrophoresis ◽

Simian Virus 40 ◽

Simian Virus ◽

Agarose Gel ◽

Agarose Gel Electrophoresis ◽

Wild Type

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A splice junction deletion deficient in the transport of RNA does not polyadenylate nuclear RNA.

Molecular and Cellular Biology ◽

10.1128/mcb.3.8.1381 ◽

1983 ◽

Vol 3 (8) ◽

pp. 1381-1388 ◽

Cited By ~ 17

Author(s):

L P Villarreal ◽

R T White

Keyword(s):

Simian Virus 40 ◽

Splice Junction ◽

Simian Virus ◽

The Body ◽

Splice Acceptor Site ◽

Acceptor Site ◽

Wild Type ◽

Normal Position ◽

Nuclear Rna ◽

Late Region

A late region deletion mutant of simian virus 40 (dl5) was previously shown to be deficient in the transport of nuclear RNA. This is a splice junction deletion that has lost the 3' end of an RNA leader, an intervening sequence, and the 5' end of the splice acceptor site on the body of the mRNA. In this report, we analyzed the steady-state structure of the untransported nuclear RNA. The 5' ends of this RNA are heterogeneous but contain a prominent 5' end at the normal position (nucleotide 325) in addition to several other prominent 5' ends not seen in wild-type RNA. The 3' end of this RNA does not occur at the usual position (nucleotide 2674) of polyadenylation; instead, this RNA is non-polyadenylated, with the 3' end occurring either downstream or upstream of the normal position.

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The number of ribosomes on simian virus 40 late 16S mRNA is determined in part by the nucleotide sequence of its leader

Molecular and Cellular Biology ◽

10.1128/mcb.4.4.813-816.1984 ◽

1984 ◽

Vol 4 (4) ◽

pp. 813-816

Author(s):

A Barkan ◽

J E Mertz

Keyword(s):

Nucleotide Sequence ◽

Direct Evidence ◽

Simian Virus 40 ◽

Simian Virus ◽

The Body ◽

Size Distributions ◽

Wild Type ◽

Leader Protein ◽

16S Rna

The size distributions of polyribosomes containing each of three simian virus 40 late 16S mRNA species that differ in nucleotide sequence only within their leaders were determined. The two 16S RNA species with shorter leaders were incorporated into polysomes that were both larger (on average) and narrower in size distribution than was the predominant wild-type 16S RNA. Therefore, the nucleotide sequence of the leader can influence the number of ribosomes present on the body of an mRNA molecule. We propose a model in which the excision from leaders of sizeable translatable regions permits more frequent utilization of internally located translation initiation signals, thereby enabling genes encoded within the bodies of polygenic mRNAs to be translated at higher rates. In addition, the data provide the first direct evidence that VP1 can, indeed, be synthesized in vivo from the species of 16S mRNA that also encodes the 61-amino acid leader protein.

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E1A induces phosphorylation of the retinoblastoma protein independently of direct physical association between the E1A and retinoblastoma products

Molecular and Cellular Biology ◽

10.1128/mcb.11.8.4253-4265.1991 ◽

1991 ◽

Vol 11 (8) ◽

pp. 4253-4265

Author(s):

H G Wang ◽

G Draetta ◽

E Moran

Keyword(s):

Cell Cycle ◽

Simian Virus 40 ◽

Simian Virus ◽

Binding Activity ◽

T Antigen ◽

Physical Interaction ◽

Resting Cells ◽

Wild Type ◽

Quiescent Cells ◽

Kinase Expression

We have studied the initial effects of adenovirus E1A expression on the retinoblastoma (RB) gene product in normal quiescent cells. Although binding of the E1A products to pRB could, in theory, make pRB phosphorylation unnecessary for cell cycle progression, we have found that the 12S wild-type E1A product is capable of inducing phosphorylation of pRB in normal quiescent cells. The induction of pRB phosphorylation correlates with E1A-mediated induction of p34cdc2 expression and kinase activity, consistent with the possibility that p34cdc2 is a pRB kinase. Expression of simian virus 40 T antigen induces similar effects. Induction of pRB phosphorylation is independent of the pRB binding activity of the E1A products; E1A domain 2 mutants do not bind detectable levels of pRB but remain competent to induce pRB phosphorylation and to activate cdc2 protein kinase expression and activity. Although the kinetics of induction are slower, domain 2 mutants induce wild-type levels of pRB phosphorylation and host cell DNA synthesis and yet fail to induce cell proliferation. These results imply that direct physical interaction between the RB and E1A products does not play a required role in the early stages of E1A-mediated cell cycle induction and that pRB phosphorylation is not, of itself, sufficient to allow quiescent cells to divide. These results suggest that the E1A products do not need to bind pRB in order to stimulate resting cells to enter the cell cycle. Indeed, a more important role of the RB binding activity of the E1A products may be to prevent dividing cells from returning to G0.

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Assembly of T-Antigen Double Hexamers on the Simian Virus 40 Core Origin Requires Only a Subset of the Available Binding Sites

Molecular and Cellular Biology ◽

10.1128/mcb.18.5.2677 ◽

1998 ◽

Vol 18 (5) ◽

pp. 2677-2687 ◽

Cited By ~ 37

Author(s):

Woo S. Joo ◽

Henry Y. Kim ◽

John D. Purviance ◽

K. R. Sreekumar ◽

Peter A. Bullock

Keyword(s):

Structural Changes ◽

Simian Virus 40 ◽

Simian Virus ◽

T Antigen ◽

Wild Type ◽

The Core ◽

Structural Distortions ◽

In The Wild ◽

Sv40 Dna ◽

Initial Assembly

ABSTRACT Initiation of simian virus 40 (SV40) DNA replication is dependent upon the assembly of two T-antigen (T-ag) hexamers on the SV40 core origin. To further define the oligomerization mechanism, the pentanucleotide requirements for T-ag assembly were investigated. Here, we demonstrate that individual pentanucleotides support hexamer formation, while particular pairs of pentanucleotides suffice for the assembly of T-ag double hexamers. Related studies demonstrate that T-ag double hexamers formed on “active pairs” of pentanucleotides catalyze a set of previously described structural distortions within the core origin. For the four-pentanucleotide-containing wild-type SV40 core origin, footprinting experiments indicate that T-ag double hexamers prefer to bind to pentanucleotides 1 and 3. Collectively, these experiments demonstrate that only two of the four pentanucleotides in the core origin are necessary for T-ag assembly and the induction of structural changes in the core origin. Since all four pentanucleotides in the wild-type origin are necessary for extensive DNA unwinding, we concluded that the second pair of pentanucleotides is required at a step subsequent to the initial assembly process.

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Differential ability of a T-antigen transport-defective mutant of simian virus 40 to transform primary and established rodent cells.

Molecular and Cellular Biology ◽

10.1128/mcb.5.5.1043 ◽

1985 ◽

Vol 5 (5) ◽

pp. 1043-1050 ◽

Cited By ~ 33

Author(s):

R E Lanford ◽

C Wong ◽

J S Butel

Keyword(s):

Cell Lines ◽

Mouse Embryo ◽

Simian Virus 40 ◽

Simian Virus ◽

T Antigen ◽

Wild Type ◽

Mouse Embryo Fibroblasts ◽

Established Cell Lines ◽

Established Cell ◽

Embryo Fibroblasts

The transforming potential and oncogenicity of a simian virus 40 (SV40) mutant affecting T-antigen (T-ag), SV40(cT)-3, was examined in an effort to dissect T-ag functions in transformation. SV40(cT)-3 has a point mutation at nucleotide 4434 that abolishes the transport of T-ag to the nucleus but does not affect its association with the cell surface. Transfection-transformation assays were performed with primary cells and established cell lines of mouse and rat origin. The efficiency of transformation for established cell lines by SV40(cT)-3 was comparable to that of wild-type SV40, indicating that transformation of established cell lines can occur in the absence of detectable amounts of nuclear T-ag. Transformation of primary mouse embryo fibroblasts by SV40(cT)-3 was markedly influenced by culture conditions; the relative transforming frequency was dramatically reduced in assays involving focus formation in low serum concentrations or anchorage-independent growth. Immunofluorescence tests revealed that the transformed mouse embryo fibroblasts partially transport the mutant cT-ag to the cell nucleus. Transformed cell lines induced by SV40(cT)-3 did not differ in growth properties from wild-type transformants. SV40(cT)-3 was completely defective for the transformation of primary baby rat kidney cells, a primary cell type unable to transport the mutant T-ag to the nucleus. The intracellular localization of cellular protein p53 was found to mimic T-ag distribution in all the transformants analyzed. The mutant virus was weakly oncogenic in vivo: the induction of tumors in newborn hamsters by SV40(cT)-3 was reduced in incidence and delayed in appearance in comparison to wild-type SV40. These observations suggest that cellular transformation is regulated by both nuclear and surface-associated forms of SV40 T-ag.

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Separation of simian virus 40 large-T-antigen-transforming and origin-binding functions from the ability to block differentiation

Molecular and Cellular Biology ◽

10.1128/mcb.8.3.1380-1384.1988 ◽

1988 ◽

Vol 8 (3) ◽

pp. 1380-1384 ◽

Cited By ~ 1

Author(s):

V Cherington ◽

M Brown ◽

E Paucha ◽

J St Louis ◽

B M Spiegelman ◽

...

Keyword(s):

Dehydrogenase Activity ◽

Adipocyte Differentiation ◽

Simian Virus 40 ◽

Simian Virus ◽

Lipid Binding ◽

T Antigen ◽

Large T Antigen ◽

Wild Type ◽

Glycerophosphate Dehydrogenase ◽

Triglyceride Accumulation

Wild-type simian virus 40 large T antigen is very effective at blocking adipocyte differentiation in 3T3-F442A cells as assayed by triglyceride accumulation, induction of glycerophosphate dehydrogenase activity, and expression of mRNAs for glycerophosphate dehydrogenase, the adipocyte serine protease adipsin, and the putative lipid-binding protein adipocyte P2. Point mutants defective for either origin-specific DNA binding or transformation blocked differentiation as completely as wild type.

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