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 or Nicotiana benthamiana protoplasts. 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 that cis-acting RNA sequences within the pX gene, rather than the predicted protein influence genome accumulation. The requirement of the cis-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 or N. benthamiana, failed to interfere substantially with replication in Chenopodium 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 sequences. However, it is possible that very early in the replication cycle of genomic RNA in vivo, the pX gene cis-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.

Tomato Bushy Stunt Virus Genomic RNA Accumulation Is Regulated by Interdependent cis -Acting Elements within the Movement Protein Open Reading Frames

“…The second observation was that deletions affecting pX (nt 4492 to 4699), such as those in pT/NSl (⌬nt 3890 to 4499), pT/HSl (⌬nt 4172 to 4497), and pT/ScSl (⌬nt 4400 to 4498), prohibited efficient viral RNA accumulation ( Fig. 2B and C, lanes 6, 7, and 8), as expected on the basis of previous results (39).…”

“…Subsequently, the HpaI-to-SalI fragment of pKAN157⌬EcoRV-HB19 was used to replace the same restriction fragment of pTBSV-100 to generate pTHB19. To construct pTHS19, a SacI site (underlined in the sequences below) was generated at the 3Ј end of the p19 ORF of pHS49 (39) by oligonucleotide-directed mutagenesis with a primer (5Ј-GA AGGCGAGCTCGACAGACTC-3Ј) to produce pHS49/19S. The NcoI-to-SalI fragment of pTBSV-100 was replaced with the same fragment of pHS49/19S to generate an intermediate construct, pTS19, that contains the SacI site at the 3Ј end of p19.…”

“…MP indicates movement proteins expressed from the overlapping p22 and p19 ORFs. The function of pX is unknown, but its RNA sequences have a host-dependent effect on viral RNA accumulation (39). The solid lines between ORFs indicate presumed untranslated regions.…”

“…This is reminiscent of our observation that an element within the replicase gene differentially affects DI versus genome amplification (29). Another example involves the different effects of a region downstream of region III, termed pX, that showed host-specific activities for RNA accumulation despite its dispensability for DI replication (39). On the basis of these cumulative observations on context-dependent effects, it appears necessary to consider RNA elements adjacent to regions I through IV in order to fully understand their biological activity in the context of the TBSV genome.…”

“…Some of the effects observed in plants could be caused by the influence of mutations on the activity of the P22 or P19 protein or its dosage or by host-specific effects of RNA mutations on infectivity (6,38,39,42). Therefore, to specifically examine the effects on RNA accumulation, each mutant construct was tested in protoplasts.…”