Genome Packaging And NSvc2-Mediated Small Brown Planthopper Transmission Mechanism Of Rice Stripe Tenuivirus

Rice stripe tenuivirus(RSV)is the pathogen that causes rice stripe disease.The disease has been widespread in rice crops in China and Southeast Asian countries.Earlier studies have reported that RSV was a filamentous,non-enveloped morphology and relied on nucleocapsid protein to package viral genomic RNA.However,the biological process of the propagation of the virus within the host was still unknown.That is,how the happened of the nucleocapsid protein self-interaction and effectively packaging viral genomes without being degraded by nuclease in the host.In addition,although RSV was already known to be transmitted by small brown planthopper in a persistent-propagative manner,the molecular mechanism of RSV infects the vector planthopper was still unclear.Analysis of these biological processes provides strong technical support for disease control and comprehensive management.In order to explore these important biological doubts,the present study mainly focused on the following two aspects:1.Genome packaging mechanism of Rice stripe tenuivirusRice stripe tenuivirus is a filamentous,multipartite negative-strand RNA virus.The viral particle is composed predominantly of a nucleocapsid protein(NP)and genomic RNA.However,the molecular details of how the RSV NP interacts with genomic RNA during particle assembly remain largely unknown.We used homology models and biochemical experiments to analysis the structural and functional characterization of NP protein.We found that there was a predicted RNA-binding groove in the structural model of NP protein and polar amino acids within the groove were critical for RNA binding and protecting the?enome RNA from RNase digestion.Biochemical experiment was shown that RSV NP formed pentamers,hexamers,heptamers,and octamers by self-interaction,and each oligomer bound genomic RNA.By modeling the predicted higher-order structures combined with biochemical experiments,we found that oligomer formation was driven by the N-terminal amino arm of the NP.Deletion of this arm abolished oligomerization and was less able to interact with RNA and protect RNA than was the wild type.These findings afford valuable new insights into molecular mechanism of RSV NPs interacting with genomic RNA.2.The mechanism of NSvc2-mediated Rice stripe tenuivirus into small brown planthopper midgut cellInsect vectors play critical roles in transmitting both plant and animal viruses.Viral helper components have been shown as molecular bridges between virus particle and insect receptor(s)during non-circulative virus transmissions.In contrast,whether circulative-transmitted viruses also utilize this strategy during their transmission remains elusive.In this study,we developed a new reverse genetics technology and demonstrated for the first time that the non-structural glycoprotein NSvc2 of Rice stripe tenuivirus(RSV),a nonenveloped circulative plant virus,functioned like a helper component to mediate RSV into its vector small brown planthopper midgut cells.Although NSvc2 is not found in purified RSV virion samples,one of its mature protein,NSvc2-N,can interact with RSV virion and directly binds to midgut to retain in midgut lumen surface via its N-glycosylation sites.Upon recognition by midgut receptor(s),midgut cells undergo endocytosis followed by compartmentalizing RSV virion and NSvc2 in early and then late endosomes.The acidic condition inside the late endosome triggers another of its mature protein NSvc2-C to go conformation change and cause membrane fusion via its highly conserved fusion loop motifs.However,the mutant defected in membrane fusion did not cause release of RSV virion from endosome into cytosol.Taken together,our results show for the first time that the circulative-transmitted plant virus also encodes a helper component-like factor as a critical molecular bridge to allow the interaction between virus particle and insect midgut during vector transmission.