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.
Jao
Christine C
CC
California State University, Long Beach, Department of Biological Sciences, 1250 Bellflower Blvd, Long Beach, CA 90840, USA1.
Weidman
Mary K
MK
California State University, Long Beach, Department of Biological Sciences, 1250 Bellflower Blvd, Long Beach, CA 90840, USA1.
Perez
Ana R
AR
California State University, Long Beach, Department of Biological Sciences, 1250 Bellflower Blvd, Long Beach, CA 90840, USA1.
Gharakhanian
Editte
E
California State University, Long Beach, Department of Biological Sciences, 1250 Bellflower Blvd, Long Beach, CA 90840, USA1.
D000818:Animals; D002213:Capsid / Q000737:chemistry*; D036022:Capsid Proteins; D002474:Cell-Free System; D003545:Cysteine / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry; D011817:Rabbits; D013539:Simian virus 40 / Q000737:chemistry*
D036022:Capsid Proteins; D004220:Disulfides; D003545:Cysteine