THE TOMATO BUSHY STUNT VIRUS REPLICASE PROTEINS ARE COORDINATELY EXPRESSED AND MEMBRANE-ASSOCIATED

Two open reading frames at the 5'-end of the tomato bushy stunt virus genomic RNA are predicted to encode a 33-kDa (p33) protein and its 92- kDa (p92) readthrough product. From amino acid sequence comparisons wi th other small single-stranded RNA viruses, these proteins resemble vi ral components of the replicase-transcriptase complex. To investigate the accumulation of these proteins in the infected cell, two chimeric proteins were produced that expressed either a portion of p33 or the c arboxy-terminal ''half'' of p92 fused with glutathione S-transferase, and polyclonal ascites fluids specific to p33 or p92 were elicited in mice. As expected, the anti-p33 antibody recognized p33 and the p92 re adthrough protein, but the anti-p92 antibody was specific for p92. Imm unoblot analyses revealed that at an early stage of infection both pro teins were associated with the membrane fractions isolated from virus- infected plants, but later in the infection, prior to collapse of the tissues, these proteins were also associated with the cytoplasmic frac tion. At all time points in plants and protoplasts p33 was about 20-fo ld more abundant than p92. A series of mutations derived from an infec tious cDNA clone demonstrated that both the p33 and the p92 proteins w ere required for replication in protoplasts and the ratio of the two p roteins was maintained in the replication-competent mutants. The wild- type amber (UAG) and in vitro-generated ochre (UAA) readthrough codon derivatives replicated in protoplasts. However, the tyrosine mutants ( UAC or UAU) that were predicted to express only p92 were not viable in protoplasts. (C) 1995 Academic Press, Inc.