THE ENIGMA OF PX - A HOST-DEPENDENT CIS-ACTING ELEMENT WITH VARIABLE EFFECTS ON TOMBUSVIRUS RNA ACCUMULATION

Tomato bushy stunt virus (TBSV) is a small isometric virus that contai ns a single-stranded RNA genome with five major genes. In this study, we have analyzed the importance of an additional small sixth open read ing frame (ORF) of 207 nucleotides, designated pX, which resides at th e 3' end of the genome. Bioassays showed hat deletions or additions of nucleotides at the 5' end of the pX gene that were designed to disrup t the ORF, or site-specific inactivation of its start codon, ail gave rise to TBSV mutants which were unable to accumulate to detectable lev els in cucumber or Nicotiana benthamiana protoplasts. Although these r esults suggested a role for the putative pX protein, introduction of a premature stop codon in the pX gene had no strong negative effect How ever, a comparable mutation that affected the same nucleotides without changing the predicted amino acid sequence greatly reduced RNA accumu lation. Therefore, we hypothesize that cis-acting RNA sequences within the pX gene, rather than the predicted protein influence genome accum ulation. The requirement of the cis-acting pX ORF sequences appears to be host-dependent because comparisons revealed that subtle pX gene mu tations that prohibited accumulation of TBSV RNA in cucumber or N. ben thamiana, failed to interfere substantially with replication in Chenop odium quinoa protoplasts or plants. irrespective of the host; the cis- acting pX gene sequences were dispensable on replicase-deficient RNAs that require helper TBSV for replication in trans. In addition, the pX gene was not essential for in vitro translation of replicase proteins from genomic RNA. These results suggest that neither translation nor polymerase activity of the replicase proteins require pX gene sequence s. However, it is possible that very early in the replication cycle of genomic RNA in vivo, the pX gene cia-acting element is essential for some other unidentified function which involves interaction with one o r more host components whose composition Varies slightly between diffe rent plants. (C) 1997 Academic Press.