Cys(9), Cys(104) and Cys(207) 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 t he stabilization of the simian virus 40 (SV40) capsid. To identify the cyst eine residues involved in intermolecular disulfide interactions, systematic oligo-directed mutagenesis of cysteine codons to serine codons was initiat ed. 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 nonreducing SDS-PAGE and via sucrose-gradi ent sedimentation, Wild-type Vp1 forms 7S pentamers followed by 12S disulfi de-linked multi-pentameric complexes in cell-free lysates, Mutagenesis of a ll seven cysteine codons abolished Vp1 12S complexes, but did not affect pe ntamer formation. A quadruple Vp1 mutant at Cys(49), Cys(87), Cys(254) and Cys(267) continued to form 12S complexes, whereas the major products of the Cys(9), Cys(104) and Cys(207) 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 Cys(9), Cys(104) and Cys(207) are essential and sufficient for st abilization of Vp1 complexes in cell-free lysates. These results are in agr eement with previous structural studies of SV40 that implicated the same th ree residues in disulfide linkage in the capsid, Possible parameters for th e involvement of the three cysteines are discussed.