RNA Sequence Determinants of a Coupled Termination-Reinitiation Strategy for Downstream Open Reading Frame Translation in Helminthosporium victoriae Virus 190S and Other Victoriviruses (Family Totiviridae)

Abstract

The genome-length, dicistronic mRNA of the double-stranded RNA fungal virus Helminthosporium victoriae virus 190S (genus Victorivirus, family Totiviridae) contains two long open reading frames (ORFs) that overlap in the tetranucleotide AUGA. Translation of the downstream ORF, which encodes the RNA-dependent RNA polymerase (RdRp), has been proposed to depend on ribosomal reinitiation following termination of the upstream ORF, which encodes the capsid protein. In the current study, we examined the RNA sequence determinants for RdRp translation in this virus and demonstrated that a coupled termination-reinitiation (stop-restart) strategy is indeed used. Signals for termination-reinitiation are found within a 32-nucleotide stretch of RNA immediately upstream of the AUGA motif, including a predicted pseudoknot structure. The close proximity in which this predicted structure is followed by the upstream ORF's stop codon appears to be especially important for promoting translation of the downstream ORF. The normal strong preferences for an AUG start codon and the canonical sequence context to favor translation initiation appear somewhat relaxed for the downstream ORF. Similar sequence motifs and predicted RNA structures in other victoriviruses suggest that they all share a related stop-restart strategy for RdRp translation. Members of the genus Victorivirus thus provide new and unique opportunities for exploring the molecular mechanisms of translational coupling, which remain only partly understood in this and other systems.The vast majority of eukaryotic mRNAs are monocistronic and, hence, contain only one translated open reading frame (ORF). Some, however, contain two or more ORFs and have an associated mechanism, such as ribosomal frameshifting or coupled termination-reinitiation (stop-restart), to promote regulated translation of the downstream ORF(s). Such multicistronic mRNAs are especially common among eukaryotic RNA viruses.In coupled termination-reinitiation, ribosomes translate the upstream ORF of an mRNA, but upon termination, a proportion of the 40S subunits remain bound to the mRNA and go on to reinitiate at the start codon of the downstream ORF (see review in reference 39). This strategy has been shown to promote the translation of downstream ORFs in the mRNAs of several different plus-or minus-stranded RNA mammalian viruses, including those that encode protein VP2 or VP10 of certain caliciviruses (30,33,34), the BM2 protein of influenza B virus (21, 40), and the M2-2 proteins of respiratory syncytial virus (RSV) and other pneumoviruses (1,15,16). One example of a plus-stranded RNA fungal virus that employs such translational coupling has also been described (17). A stop-restart strategy has been previously proposed for downstream ORF translation from the dicistronic mRNA of the double-stranded RNA (dsRNA) fungal virus Helminthosporium victoriae virus 190S (HvV190S) (45), and identifying RNA sequence determinants for that strategy in HvV190S and related viruses is the focus of this report.HvV190S is the pr...

A Novel Victorivirus from a Phytopathogenic Fungus, Rosellinia necatrix, Is Infectious as Particles and Targeted by RNA Silencing

“…Although the positive strand of the RnVV1 genome possesses the pentamer (UAAUG) separating the two ORFs, in place of the tetramer, it has the stem I and stem II (Fig. 1E) that are well conserved in victoriviruses (54). Furthermore, three stemloop structures can be formed immediately downstream of the pentamer, which may also be important for the coupled translation.…”

“…Like other members of the genus Victorivirus, RnVV1 likely expresses RdRp as an independent protein product probably via the termination-reinitiation mechanism. As shown by Li et al (54), many victoriviruses have a tetranucleotide (AUGA) in which the first triplet is considered to serve as the start codon of the downstream RdRp ORF and the last triplet serves as the stop codon of the upstream CP ORF. The same authors demonstrated that the 32-nt sequence immediately upstream of the tetramer that potentially forms a pseudoknot structure is important for the reinitiation translation of HvV190S RdRp (54).…”

“…As shown by Li et al (54), many victoriviruses have a tetranucleotide (AUGA) in which the first triplet is considered to serve as the start codon of the downstream RdRp ORF and the last triplet serves as the stop codon of the upstream CP ORF. The same authors demonstrated that the 32-nt sequence immediately upstream of the tetramer that potentially forms a pseudoknot structure is important for the reinitiation translation of HvV190S RdRp (54). Although the positive strand of the RnVV1 genome possesses the pentamer (UAAUG) separating the two ORFs, in place of the tetramer, it has the stem I and stem II (Fig.…”

“…1E). This pentamer is considered to be a key motif for translation of the downstream ORF by coupled translation termination and reinitiation (53,54). Furthermore, this motif is followed by three of hairpin structures (Fig.…”

Characterization of a Novel Megabirnavirus from Sclerotinia sclerotiorum Reveals Horizontal Gene Transfer from Single-Stranded RNA Virus to Double-Stranded RNA Virus

“…The pseudoknot structure usually facilitates pausing of the translating ribosome and increasing the frequency of frameshifting (35). Moreover, the pseudoknot structure commonly exists in dsRNA mycoviruses belonging to the family Totiviridae (36). Thus, LdsRNA probably represents a novel dsRNA mycovirus and was tentatively named SsMBV1 (Sclerotinia sclerotiorum megabirnavirus 1).…”