Regulation of Sindbis virus RNA replication: uncleaved P123 and nsP4 function in minus-strand RNA synthesis, whereas cleaved products from P123 are required for efficient plus-strand RNA synthesis

Abstract

Nonstructural proteins of Sindbis virus, nsPl, nsP2, nsP3, and nsP4, as well as intermediate polyproteins, are produced from two precursor polyproteins, Pl23 and Pl234, by a proteolytic enzyme encoded in the C-terminal half of nsP2. We studied the requirements for and the functions of the intermediate and mature processing products for Sindbis virus RNA synthesis by using site-directed mutants which have a defect(s) in processing the 1/2, 2/3, or 3/4 cleavage sites either singly or in various combinations. A mutant defective in cleaving both the 1/2 and 2/3 sites, which makes only uncleavable Pl23 and mature nsP4 as final products, produced 10-3 as much virus as did the wild-type virus after 10 hat 30°C and was nonviable at 40°C. A mutant defective in processing the 2/3 site, which makes nsPl, nsP4, and P23 as well as precursor Pl23, grew 10-1 as efficiently as wild-type virus at 30°C and 10-3 as efficiently at 40°C. Early minus-strand RNA synthesis by these mutants was as efficient as that by wild-type virus, whereas plus-strand RNA synthesis was substantially decreased compared with that by wild-type virus. A mutant defective in processing the 3/4 site was nonviable at either 30 or 40°C. The 3/4 site mutant could be complemented by the mutant unable to cleave either the 1/2 or 2/3 site, which can provide mature nsP4. We interpret these results to signify that (i) mature nsP4 is required for RNA replication, (ii) nsP4 and uncleaved Pl23 function in minus-strand RNA synthesis, and (iii) cleavage of Pl23 is required for efficient plus-strand RNA synthesis. We propose that Sindbis virus RNA replication is regulated by differential proteolysis of Pl23. Early in infection, nsP4 and uncleaved Pl23 form transient minus-strand RNA replication complexes which vanish upon cleavage of Pl23. Later in infection, an elevated level of viral proteinase activity eliminates de novo synthesis of Pl23, and no further synthesis of minus-strand RNA is possible. In contrast, nsP4 and cleavage products from Pl23 form plus-strand RNA replication complexes which are stable and remain active throughout the infection cycle