Abstract
Pelargonium flower break virus (PFBV, genus Carmovirus) has a single-stranded positive-sense genomic RNA (gRNA) which contains five ORFs. The two 5′-proximal ORFs encode the replicases, two internal ORFs encode movement proteins, and the 3′-proximal ORF encodes a polypeptide (p37) which plays a dual role as capsid protein and as suppressor of RNA silencing. Like other members of family Tombusviridae, carmoviruses express ORFs that are not 5′-proximal from subgenomic RNAs. However, in one case, corresponding to Hisbiscus chlorotic ringspot virus, it has been reported that the 3′-proximal gene can be translated from the gRNA through an internal ribosome entry site (IRES). Here we show that PFBV also holds an IRES that mediates production of p37 from the gRNA, raising the question of whether this translation strategy may be conserved in the genus. The PFBV IRES was functional both in vitro and in vivo and either in the viral context or when inserted into synthetic bicistronic constructs. Through deletion and mutagenesis studies we have found that the IRES is contained within a 80 nt segment and have identified some structural traits that influence IRES function. Interestingly, mutations that diminish IRES activity strongly reduced the infectivity of the virus while the progress of the infection was favoured by mutations potentiating such activity. These results support the biological significance of the IRES-driven p37 translation and suggest that production of the silencing suppressor from the gRNA might allow the virus to early counteract the defence response of the host, thus facilitating pathogen multiplication and spread.
Highlights
Eukaryotic gene expression is highly controlled at translational level
Pelargonium flower break virus (PFBV) genomic RNA (gRNA) directs synthesis of p27, p86 and p37 To test whether PFBV gRNA serves as mRNA for translation of the two 59-proximal open reading-frames (ORFs) as expected, wild-type transcripts derived from the infectious clone of the virus pSP18-IC [30] were subjected to in vitro translation reactions with wheat germ extracts (WGE)
Northern blot analysis with a PFBV-specific probe of RNA extracted from aliquots of the translation reaction mixture taken at regular intervals, did not reveal the presence of any outstanding degradation product indicating that synthesis of p37 from the gRNA did not result from an endonucleotytic cleavage that led to a shorter RNA
Summary
Eukaryotic gene expression is highly controlled at translational level. The process of translation is composed by three phases, initiation, elongation and termination, of which initiation is considered to be the rate-limiting step and, it is the phase that is most often subjected to regulation [1,2,3]. Translational initiation usually takes place according to a mechanism of ribosome scanning that is 59-end dependent and requires a 59-cap and a 39-polyadenylated tail [1,4], two structures that are typically present in cytoplasmic cellular mRNAs. In the scanning mechanism, the 40S small ribosome subunit is recruited to the 59-end of the mRNA, via a network of protein-protein and RNA-protein interactions, and undergoes a linear 59 to 39 migration until the first initiation codon (usually AUG) is reached. The 40S small ribosome subunit is recruited to the 59-end of the mRNA, via a network of protein-protein and RNA-protein interactions, and undergoes a linear 59 to 39 migration until the first initiation codon (usually AUG) is reached If this codon lies in an optimal sequence context, the subunit pauses, the translational initiation complex is formed and translation can proceed [5,6]. Just in some instances an mRNA whose translation is initiated by the scanning model may direct synthesis of more than one protein through mechanisms that modify ribosome behaviour as leaky scanning, reinitiation, frameshift or readthrough of leaky stop codons [7,8,9]
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