Rice Stripe Virus Mrna Transcription Initiation Mechanism And Subcellular Localization Of RSV NSvc2Proteins In Plant Cells

Rice stripe virus (RSV), the type member of the genus Tenuivirus, caused significant losses in rice fields in China and is known to be transmitted by Laodelphax striatellus in a persistent, circulative-propagative manner. Currently, RSV raised serious concerns and was focused on the genome structure and its coding strategy, the function of proteins, resistance breeding and other aspects, but many aspects are worthy of in-depth knowledge and research. RSV, a negative-sense strand RNA virus, has no cap structure at its RNAs5’ terminal, whereas there is a non-viral RNA sequence at its mRNAs5’terminal. It was asked where do these short, non-viral cap structures come and what rules can be found it? Another issue of concern is the morphology of RSV particles. RSV and the viruses of Bunyaviridae have a close phylogenetic relationship, but how to explain the phenomena that the morphologies of their particles are largely different. To answer these doubts, two aspects were carried out in this study as follows:1.RSV mRNA transcription initiation mechanismRNA cap structure found at the5’end of eukaryotic mRNA and the majority of the viral genomic RNA. The cap has several important biological roles, such as protecting mRNA from degradation by5’exoribonucleases and directing pre-mRNA splicing and mRNA export from the nucleus. But all segemet, negative-sense strand RNA virus have no cap structure at its RNAs5’terminal, whereas there is a non-viral RNA sequence at its mRNAs5’terminal, as well as RSV. But whether RSV acquiring cap structures from cellular mRNAs by’cap snatching’has not been determined.To ascertain the origin of these short and non-viral cap structure, in this study RSV and Cucumber mosaic virus (CMV), a sense strand RNA virus having the cap structure at its RNAs5’end, were co-infected Nicotiana benthamiana. CMV RNAs were found to serve as cap donors for rice stripe virus (RSV) transcription initiation during their co-infection of N. benthamiana. The5’end of CMV RNAs was cleaved preferentially at residues that had multiple-base complementarity to the3’end of the RSV template. The length requirement for CMV capped primers to be suitable for elongation varied between12and20nt (nucleotides), and those of12-16nt were optimal for elongation and generated more CMV-RSV chimeric mRNA transcripts. The original cap donors that were cleaved from CMV RNAs were predominantly short (10-13nt). However, the CMV capped RNA leaders that underwent long-distance elongation were found to contain up to five repetitions of additional AC dinucleotides. Sequence analysis revealed that these AC dinucleotides were used to increase the size of short cap donors in multiple prime-and-realign cycles. Each prime-andrealign cycle added an AC dinucleotide onto the capped RNA leaders; thus, the original cap donors were gradually converted to longer capped RNA leaders (of12-20nt). Interestingly, the original10nt (or11nt) cap donor cleaved from CMV RNA1/2did not undergo direct extension; only capped RNA leaders that had been increased to>12nt were used for direct elongation. These findings suggest that this repetitive priming and realignment may serve to convert short capped CMV RNA leaders into longer, more suitable sizes to render a more stabilized transcription complex for elongation during RSV transcription initiation.2-Subcellular localization of RSV NSvc2proteins in plant cellsViruses of Tenuivirus have a close phylogenetic relationship with the viruses in the family of Bunyaviridae, but Tenuiviruses adopt a long filamentous morphology while Bunyaviruses are membrane-enveloped sphere particles. For the viruses in Bunyaviridae, targeting of glycoproteins to the Golgi apparatus plays a pivotal role in the maturation of membrane-enveloped sphere particle. NSvc2glycoprotein encoded by RSV vcRNA2, exhibiting high homology with glycoproteins of Bunyaviruses, has many characteristics in common with glycoprotein of the viruses in the family Bunyaviridae. However, their viron morphologies are different. We will ask whether the characteristics of RSV glycoprotein NSvc2resulted in the difference the virion morphology between RSV and viruses in the family Bunyaviridae.To support this speculation, the amino acids sequences of RSV NSvc2were analyzed firstly. The results showed that RSV NSvc2has four transmembrane (TM) domains (6-26aa,269-291aa,362-379aa and807-827aa). And two of them (1-18aa and362-379aa) were also predicted to be a signal peptide. RSV NSvc2was cut at382aa into two mature proteins amino-terminal NSvc2(NSvc2-N,41kDa) and carboxyl-terminal NSvc2(NSvc2-C,51kDa). To further clarify the causes of RSV particle morphology, we described the characteristics of RSV NSvc2in N. benthamiana. The results supported that RSV NSvc2could be processed really into two mature proteins NSvc2-N and NSvc2-C. Meanwhile, we demonstrated that NSvc2-N glycoprotein targeted to the Golgi apparatus whereas NSvc2-C accumulated in the ER membrane in N. benthamiana cells. Upon co-expression, NSvc2-N redirected NSvc2-C from ER to Golgi apparatus. The targeted NSvc2glycoproteins moved together with Golgi stacks along the ER track. Targeting of NSvc2glycoproteins to Golgi apparatus was strictly dependent on functional anterograde traffic out of the ER to Golgi or retrograde transport route. Further analysis of expressed truncated and chimeric NSvc2proteins demonstrated that the Golgi targeting signal mapped to a region of NSvc2-N (amino-acid269-315) encompassing the transmembrane domain and the adjacent24amino-acids of the cytosolic tail. All these data determined that RSV NSvc2targeted into Golgi stack. These cytological features of RSV glycoproteins are similar with the viruses of Bunyaviridae. Our findings provide new insights into the intracellular targeting of RSV glycoproteins in plant cells, and the real causes of RSV filamentous particles will be expected to find.