Plant RNA viruses encode essential viral proteins that depend on the host translation machinery for their expression. However, genomic RNAs of most plant RNA viruses lack the classical characteristics of eukaryotic cellular mRNAs, such as mono-cistron, 5′ cap structure, and 3′ polyadenylation. To adapt and utilize the eukaryotic translation machinery, plant RNA viruses have evolved a variety of translation strategies such as cap-independent translation, translation recoding on initiation and termination sites, and post-translation processes. This review focuses on advances in cap-independent translation and translation recoding in plant viruses.

Selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) is a widely used technique for RNA structure analysis using N-methylisatoic anhydride (NMIA) treatment that has been proven to be applicable to different types of RNA templates. In this study, we performed the structural analysis of the viral satellite RNA of cucumber mosaic virus TA-Tb (satCMV TA-Tb) using the SHAPE method. In the preliminary experiment, we optimized the protocol of SHAPE method for analyzing satCMV TA-Tb by determining a suitable quantity of template RNA. This optimization effectively reduced the appearance of a large number of intense bands in the NMIA-untreated sample lane, along with a strong overall background signal that prevented the clear elucidation of the RNA structure. SHAPE analysis indicated the presence of non-structured, single-stranded flexible regions throughout satCMV TA-Tb with prominent flexible stretches located around nucleotide positions 145 to 200. The positions of these flexible regions were particularly consistent with a secondary structure of satCMV TA-Tb predicted by mfold software v.2.3, which consisted of five 5′- and 3′-proximal stem-loops and one internal large multi-branched stem-loop. Sequence alignment and secondary RNA structure prediction of other satCMV sequences that are phylogenetically the same group with satCMV TA-Tb also suggested the presence of 5′- and 3′-proximal stem-loop structures. Our data provide the structural basis for elucidating the mechanism by which satCMV TA-Tb regulates the pathogenicity and replication of its helper virus.

Cucumber mosaic virus (CMV) infection is often associated with satellite RNA (satRNA), which can sometimes interfere with the replication and symptom expression of CMV. However, the mechanism underlying symptom attenuation has remained unclear. We previously discovered a larger type (than the usual type) of satellite RNA (satRNA TA-Tb) of CMV that reduced the symptom severity of CMV. Herein, we show that satRNA TA-Tb is associated with a reduction in CMV RNA accumulation, and particularly, a strong reduction of RNA4 accumulation at later stages of infection. Deletion analysis showed that the deletion of ten nucleotides of 5′ and 3′ termini, but not the internal sequence regions proximal to the 5′- and 3′-terminal regions, abolished satRNA TA-Tb replication. The alignment of satRNA TA-Tb with usual satRNA isolates showed four internal extra sequence regions (exR1–4) in satRNA TA-Tb. A satRNA TA-Tb mutant with deletion in the exR1 region retained the ability to attenuate CMV symptoms, whereas deletion of the exR2–4 regions abolished the attenuating effect of satRNA TA-Tb, but did not affect its replication. Overall, these results suggest that some short, internal extra sequence regions are dispensable for satRNA TA-Tb replication, but important for symptom attenuation function, supporting the possibility that the RNA structure of satRNA TA-Tb is important for its function in symptom attenuation.

Plant viruses are major plant pathogens and account for nearly half of emerging plant diseases worldwide. To date, few effective management measures are available for the efficient control of plant viral diseases. Cross-protection based on attenuated vaccine is an effective strategy to prevent plant viral diseases. The primary requirement for the development of attenuated vaccines is a vector with excellent characteristics, such as low pathogenicity, genetic stability, and the capacity for stable insertion of exogenous fragments. In this study, the cucumber mosaic virus (Fny strain) was genetically modified to serve as an attenuated vaccine vector. Based on pre-termination of the 2b protein-coding region and deletion of the 3′ UTR in CMV RNA2, six RNA2 mutants, designated R2-2bPTI, R2-2bPTII, R2-2bPTIII, R2-2bPTIV, R2-2bPTV, and R2-2bPTVI, were constructed. Experiments with different lengths tobacco phytoene desaturase (PDS) fragment insertion evaluated the capacity of each mutant to accommodate exogenous fragment. Based on the R2-2bPTIV, a viral fragment insertion mutant R2-2bPTIV-TMPYTVPX targeting cucumber mosaic virus (CMV), tobacco mosaic virus (TMV), potato virus Y (PVY), tobacco vein banding mosaic virus (TVBMV), and potato virus X (PVX) was constructed. This mutant provided effective cross-protection against these targeted virulent viruses. This study developed a series of CMV-based vaccine vector, providing materials and data for the development of plant viral diseases attenuated vaccines.