The Enigma of pX: A Host-Dependentcis-Acting Element with Variable Effects on Tombusvirus RNA Accumulation

Abstract

Tomato bushy stunt virus (TBSV) is a small isometric virus that contains a single-stranded RNA genome with five major genes. In this study, we have analyzed the importance of an additional small sixth open reading frame (ORF) of 207 nucleotides, designated pX, which resides at the 3′ end of the genome. Bioassays showed that deletions or additions of nucleotides at the 5′ end of the pX gene that were designed to disrupt the ORF, or site-specific inactivation of its start codon, all gave rise to TBSV mutants which were unable to accumulate to detectable levels in cucumber orNicotiana benthamianaprotoplasts. Although these results suggested a role for the putative pX protein, introduction of a premature stop codon in the pX gene had no strong negative effect. However, a comparable mutation that affected the same nucleotides without changing the predicted amino acid sequence greatly reduced RNA accumulation. Therefore, we hypothesize thatcis-acting RNA sequences within the pX gene, rather than the predicted protein influence genome accumulation. The requirement of thecis-acting pX ORF sequences appears to be host-dependent because comparisons revealed that subtle pX gene mutations that prohibited accumulation of TBSV RNA in cucumber orN. benthamiana,failed to interfere substantially with replication inChenopodium quinoaprotoplasts or plants. Irrespective of the host, thecis-acting pX gene sequences were dispensable on replicase-deficient RNAs that require helper TBSV for replicationin trans.In addition, the pX gene was not essential forin vitrotranslation of replicase proteins from genomic RNA. These results suggest that neither translation nor polymerase activity of the replicase proteins require pX gene sequences. However, it is possible that very early in the replication cycle of genomic RNAin vivo,the pX genecis-acting element is essential for some other unidentified function which involves interaction with one or more host components whose composition varies slightly between different plants.