Some double-stranded RNA viruses from fungi utilize a small, pseudoknot-containing RNA cassette to promote stop/restart translation of their RNA polymerase
In 1996, when those of us in the Ghabrial lab reported the sequence of prototype Helminthosporium victoriae virus 190S (HvV190S), of what is now genus Victorivirus in family Totiviridae, we were surprised to find that its two open reading frames (ORF’s) were minimally overlapping, in the motif AUGA, where UGA is the stop codon of ORF1 encoding the capsid protein (CP) and AUG is the start codon of ORF2 encoding the RNA-dependent RNA polymerase (RdRp) (Huang and Ghabrial, 1996). Moreover, we found that the RdRp is expressed as a physically separate protein, not fused to the CP as in many other Totiviridae members, which have more substantially overlapping CP and RdRp ORF’s and take advantage of ribosomal frameshifting to express the CP/RdRp fusion protein. Subsequently, expression of RdRp from an HvV190S-like bicistronic plasmid construct was obtained in the yeast Schizosaccharomyces pombe (Soldevila and Ghabrial, 2000). Those findings suggested a distinct mechanism for RdRp expression by HvV190S, reminiscent of translationally coupled termination–reinitiation (stop/restart translation), as had been described at that time only for the M1 and BM2 proteins of influenzavirus B (Horvath et al., 1990).
Forward to 2008, when graduate student Hua Li committed to dissecting the mechanism of HvV190S RdRp expression. By that time, stop/restart translation had been identified in other viruses, including caliciviruses and pneumoviruses (see review by Powell, 2010); moreover, sequences of other victoriviruses had further suggested the use of this mechanism among them. Hua Li proceeded to use molecular genetic approaches to show that translation of HvV190S RdRp from ORF2 indeed depends on translation of CP from ORF1, with translational termination at the CP stop codon needing to occur within a limited primary sequence distance from the RdRp start codon (Li et al., 2011). In addition, among other findings in that study was evidence for a small RNA pseudoknot located nearby upstream of the stop/restart codons in most victoriviruses.
Many questions remained, however, including several resulting from the experimental system we had used in the preceding study, such as limited quantifiability and a possibility that reduced RdRp expression with certain mutant constructs might have reflected effects on RdRp packaging into virus-like particles instead of on its expression. For the current study, we therefore developed a new system that overcame previous limitations and additionally allowed us to identify the minimal RNA sequences from HvV190S sufficient for stop/restart translation. This new system employed green and red fluorescent protein genes to replace large portions of HvV190S ORF1 and ORF2 (see the figure), allowing fluorometric quantification of ORF2 expression using ORF1 expression as an internal standard. Key new results included that the previously predicted RNA pseudoknot is indeed required and that a small RNA cassette (44 nt in HvV190S), encompassing the pseudoknot and the stop/restart codons, is sufficient for stop/restart translation in this system. In addition to setting the stage for further mechanistic studies, the results identify this small cassette as having potential utility in other bi- or multicistronic eukaryotic vectors.
(A) Expression of hexahistidine-tagged green and red fluorescent proteins (GFP-His and RFP-His) from HvV190S-derived monocistronic plasmid constructs in filamentous fungus Helminthosporium victoriae, the natural host of HvV190S. (B) Top, HvV190S-derived bicistronic plasmid construct for expression of CP-GFP and RFP-His in H. victoriae, the latter dependent on stop/restart translation promoted by HvV190S-derived sequences. Middle, mutant constructs for which data are shown in panel C. Bottom, minimally sized region of HvV190S (44 nt, including 38 nt upstream of the AUGA motif; see construct CP5´d5) that was found to promote stop/restart translation of CP-GFP and RFP-His in this system. (C) Fluorometric quantification of RFP-His expression relative to CP-GFP expression from each bicistronic construct, shown as a percentage of the value obtained for the “wild-type” construct pCP-GFP:RFP-His.
Introducing the Authors
Hua Li is now a postdoctoral researcher at Northwestern University in Evanston, Illinois, USA. Max L. Nibert is a Professor of Microbiology and Immunobiology at Harvard Medical School in Boston, Massachusetts, USA. Said A. Ghabrial is now an Emeritus Professor of Plant Pathology at the University of Kentucky in Lexington, Kentucky, USA. Wendy H. Havens, formerly a Principal Research Analyst in the Ghabrial lab, was also an author but is not pictured.
About the Research
An RNA cassette from Helminthosporium victoriae virus 190S necessary and sufficient for stop/restart translation
Virology, Volume 474, January 2015, Pages 131-143
Hua Li, Wendy H. Havens, Max L. Nibert, Said A. Ghabrial