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)

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.     

Characterization of the termination-reinitiation strategy employed in the expression of influenza B virus BM2 protein

Coupled expression of the M1 and BM2 open-reading frames (ORFs) of influenza B from the dicistronic segment 7 mRNA occurs by a process of termination-dependent reinitiation. The AUG start codon of the BM2 ORF overlaps the stop codon of the upstream M1 ORF in the pentanucleotide UAAUG, and BM2 synthesis is dependent upon translation of the M1 ORF and termination at the stop codon. Here, we have investigated the mRNA sequence requirements for BM2 expression. Termination-reinitiation is dependent upon 45 nucleotide (nt) of RNA immediately upstream of the UAAUG pentanucleotide, which includes an essential stretch complementary to 18S rRNA helix 26. Thus, similar to the caliciviruses, base-pairing between mRNA and rRNA is likely to play a role in tethering the 40S subunit to the mRNA following termination at the M1 stop codon. Consistent with this, repositioning of the M1 stop codon more than 24 nt downstream from the BM2 start codon inhibited BM2 expression. RNA structure probing revealed that the RNA upstream of the UAAUG overlap is not highly structured, but upon encountering the M1 stop codon by the ribosome, a stem-loop may form immediately 5' of the ribosome, with the 18S rRNA complementary region in the apical loop and in close proximity to helix 26. Mutational analysis reveals that the normal requirements for start site selection in BM2 expression are suspended, with little effect of initiation codon context and efficient use of noncanonical initiation codons. This suggests that the full complement of initiation factors is not required for the reinitiation process.     

Translation of phage f1 gene VII occurs from an inherently defective initiation site made functional by coupling

Expression of the filamentous phage f1 gene VII is shown to be translationally coupled to that of the upstream gene V. Fusions of the gene VII initiation site to the lacZ coding region were used to determine that initiation at the VII site is completely dependent on the process of translation having proceeded up to a stop codon immediately upstream from the VII site. Coupled expression from the VII site was found to be inefficient, proportional to the level of upstream translation, and very sensitive to the distance from the functional upstream stop codon. Independent expression from the VII site was not observed, even in a deletion series designed to remove potentially masking RNA structure. On the basis of the VII site's dissimilarity to ribosome binding site sequences and its properties overall, we suggest that it inherently lacks the features required for independent recognition by ribosomes, and acquires the ability to initiate synthesis of gene VII protein by virtue of the coupling process.     

Ribosomal frameshifting in plants: a novel signal directs the -1 frameshift in the synthesis of the putative viral replicase of potato leafroll luteovirus.

The 5.8 kb RNA genome of potato leafroll luteovirus (PLRV) contains two overlapping open reading frames, ORF2a and ORF2b, which are characterized by helicase and RNA polymerase motifs, respectively, and possibly represent the viral replicase. Within the overlap, ORF2b lacks an AUG translational start codon and is therefore presumably translated by -1 ribosomal frameshifting as a transframe protein with ORF2a. This hypothesis was studied by introducing the putative frameshift region into an internal position of the beta-glucuronidase (GUS) gene and testing for the occurrence of frameshifting in vivo by transient expression of GUS activity in potato protoplasts as well as in vitro by translation in the reticulocyte system. Both experimental approaches demonstrate that a -1 frameshift occurs at a frequency of approximately 1%. Site-directed mutagenesis identified the frameshift region and the involvement of the novel heptanucleotide motif UUUAAAU in conjunction with an adjacent stem-loop structure. Part of this stem-loop encodes a basic region in the ORF2b moiety of the transframe protein which was shown by binding experiments with PLRV RNA to represent a nucleic acid-binding domain. These data support a possible biological significance of the frameshift to occur at this position of the large overlap by including the putative RNA template-binding site of the PLRV replicase in the ORF2a/ORF2b transframe protein.     

Conditional rather than absolute requirements of the capsid coding sequence for initiation of methionine-independent translation in Plautia stali intestine virus

The positive-stranded RNA genome of Plautia stali intestine virus (PSIV) has an internal ribosome entry site (IRES) in an intergenic region (IGR). The IGR-IRES of PSIV initiates translation of the capsid protein by using CAA, the codon for glutamine. It was previously reported (J. Sasaki and N. Nakashima, J. Virol. 73:1219-1226, 1999) that IGR-IRES extended by several nucleotides into the capsid open reading frame (ORF). Despite the fact that the secondary structure model of the IGR-IRES is highly conserved, we were unable to find structural similarities in the 5' region of the capsid ORFs in related viruses. Therefore, we reevaluated the role of the capsid ORF in IGR-IRES-mediated translation in PSIV. Mutation of the CAA codon with various triplets did not inhibit IGR-IRES-mediated translation. N-terminal amino acid analyses of mutated products showed that the IGR-IRES could initiate translation by using various elongator tRNAs. By replacement of the capsid ORF with exogenous coding sequences having AUG deleted, translation products were produced in most cases, but capsid-exogenous fusion proteins were produced more efficiently than were the translation products. These data indicate that the 5' part of the capsid ORF is not an absolute requirement for the IGR-IRES-mediated translation. RNA structure probing analyses showed that the 5' part of the capsid ORF was a single strand, while that of exogenous reading frames was structured. Exogenous sequences also caused structural distortion in the 3' part of the IGR-IRES. We hypothesize that the single-stranded capsid ORF helps to form the tertiary structure of the IGR-IRES and facilitates precise positioning of ribosomes.     

Expression of the ORF-2 protein of the human respiratory syncytial virus M2 gene is initiated by a ribosomal termination-dependent reinitiation mechanism

Translation of the open reading frame 2 (ORF-2) of the human respiratory syncytial virus M2 gene initiates at one of the three initiation codons located upstream of the termination codon for the first ORF. Replacement of ORF-2 with the major ORF of the chloramphenicol acetyltransferase reporter gene followed by systematic mutagenesis of the putative initiation codons demonstrated the usage of these codons as the translational initiators for ORF-2 expression both in vitro and in vivo. While the efficiency of translation was maintained when only the first and second AUG codons were preserved in vivo, there was no apparent preference in vitro for any of the three codons when only one was present. Mutagenesis studies showed that the location of the termination codon of ORF-1 protein plays a crucial role in directing translation of ORF-2 from the upstream initiation codons in vivo. This indicates that the second ORF is accessed by the ribosomes that are departing from the first ORF and that these ribosomes reinitiate on AUG codons 5' to the point of translation termination.     

Unusual organization associated to a tandem of IS231 may yield two peculiar cloverleaf secondary structures.

A 5.7-kb EcoRI fragment was cloned from plasmid DNA of Bacillus thuringiensis strain M15. It contains two insertion sequences (IS), IS231M2 and -M1 in the 5'-3' order, arranged in tandem, in same orientation, separated by a 540-bp region. The primary structure is typical of a composite transposon, here of 3847 bp in length, for which the name Tn231M is proposed. Each IS is delimited by 18-bp inverted repeats (IR), and flanked by 11-bp direct repeats (DR). Both IS share 99.3% nucleotide identities. IS231M1 has a single open reading frame (ORF) which encodes a putative 477-amino-acid transposase. IS231M2 has two smaller ORFs: ORF1 and ORF2, which could code for polypeptides of 329 and 118 amino acids in length, respectively. Further analysis reveals that the regions upstream of IS231M2, and downstream of -M1, and the 540-bp region, contain additional pairs of IR and DR. Interestingly, potential annealing between all pairs of IR and DR could generate two unusual cloverleaf secondary structures.