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.
BakerT. S.,
DrakJ.,
BinaM.1988; Reconstruction of the three-dimensional structure of simian virus 40 and visualization of the chromatic core. Proceedings of the National Academy of Sciences USA 85:422–426
BenerzaR.1994; An intermolecular disulfide bond stabilizes E2A homodimers and is required for DNA binding at physiological temperatures. Cell 79:1057–1067
BradyJ. N.,
WinstonV. D.,
ConsigliR. A.1977; Dissociation of polyoma virus by the chelation of calcium ions found associated with purified virions. Journal of Virology 23:717–724
BradyJ. N.,
WinstonV. D.,
ConsigliR. A.1978; Characterization of a DNA–protein complex and capsomere subunits derived from polyoma virus by treatment with ethyleneglycol-bis- N , N ′-tetraacetic acid and dithiothreitol. Journal of Virology 27:193–204
ChangD.,
FungC.,
OuW.,
ChaoP.,
LiS.,
WangM.,
HuangY.,
TzengT.,
TsaiR.1997; Self-assembly of the JC virus major capsid protein, Vp1, expressed in insect cells. Journal of General Virology 78:1435–1439
ChinenovY.,
SchmidtT.,
YangX.,
MartinM. E.1998; Identification of redox-sensitive cysteines in GA-binding protein-α that regulate DNA binding and heterodimerization. Journal of Biological Chemistry 273:6203–6209
FaschingC.1998The specific role of cysteine residues in the nuclear localization of the SV40 Vp1 protein MS thesis California State University; Long Beach, CA, USA:
GharakhanianE.1988Studies on the nuclear transport and the protein–protein interactions of SV40 structural proteins PhD thesis University of California; Los Angeles, CA, USA:
GharakhanianE.,
SajoA. K.,
WeidmanM. K.1995; SV40 Vp1 assembles into disulfide-linked postpentameric complexes in cell-free lysates. Virology 207:251–254
GharakhanianE.,
TakahashiJ.,
CleverJ.,
KasamatsuH.1988; In vitro assay for protein-protein interaction: carboxyl-terminal 40 residues of simian virus 40 structural protein Vp3 contain a determinant for interaction with Vp1. Proceedings of the National Academy of Sciences USA856607–6611
KosukegawaA.,
ArisakaF.,
TakayamaM.,
YajimaH.,
KaidowA.,
HandaH.1996; Purification and characterization of virus-like particles and pentamers produced by the expression of SV40 capsid proteins in insect cells. Biochimica et Biophysica Acta 1290:37–45
LinW. L.,
HataT.,
KasamatsuH.1984; Subcellular distribution of viral structural proteins during simian virus 40 infection. Journal of Virology 50:363–371
MeyerM.,
SchreckR.,
BaeuerleP. A.1993; H2O2 and antioxidants have opposite effects on activation of NF-κB and AP-1 in intact cells: AP-1 as secondary antioxidant-responsive factor. EMBO Journal 12:2005–2015
MonastryskayaK.,
BoothT.,
NelL.,
RoyP.1994; Mutation of either of two cysteine residues or deletion of the amino or carboxy terminus of the non-structural protein NS1 of bluetongue virus abrogates virus-specified tubule formation in insect cells. Journal of Virology 68:2169–2178
PognonecP.,
KatoH.,
RoederR. G.1992; The helix-loop-helix/leucine repeat transcription factor USF can be functionally regulated in a redox-dependent manner. Journal of Biological Chemistry 267:24563–24567
SchreckR.,
RieberP.,
BaeuerleP. A.1991; Reactive oxygen intermediates as apparently widely used messengers in the activation of the NF-κB transcription factor and HIV-1. EMBO Journal 10:2247–2258
VolpersC.,
SchirmacherP.,
StreeckR. E.,
SappM.1994; Assembly of the major and minor capsid protein of human papillomavirus type 33 into virus-like particles and tubular structures in insect cells. Virology 200:504–512
WalterG.,
DeppertW.1974; Intermolecular disulfide bonds: an important structural feature of the polyoma virus capsid. Cold Spring Harbor Symposia on Quantitative Biology 39:255–257
WeidmanM. K.1994Studies on the role of cysteine residues in simian virus 40 Vp1 oligomerization in vitro MS thesis California State University; Long Beach, CA, USA:
XanthoudakisS.,
MiaoG.,
WangF.,
PanY. C.,
CurranT.1992; Redox activation of Fos–Jun DNA binding activity is mediated by a DNA repair enzyme. EMBO Journal 11:3323–3335
YanY.,
StehleT.,
LiddingtonR. C.,
ZhaoH.,
HarrisonS. C.1996; Structure determination of simian virus 40 and murine polyomavirus by a combination of 30-fold and 5-fold electron-density averaging. Structure 4:157–164