Dissecting The Novel Functions Of Barley Stripe Mosaic Virus ?b Protein In Viral Replication And Movement

Viruses are obligate intracellular parasitism,their genome are relatively small and with lower encoding capacity.Viral life cycle,such as infection,genome replication,movement,and pathogenicity,needs direct involvement of viral one to several proteins.During the long-term co-evolution of hosts and viruses,viral proteins always evolved proteins exerting multifunctional roles.The Barley stripe mosaic virus(BSMV)is the type member of genus Hordeivirus,which have rigid helical particles that encapsidate tripartite gRNAs designated RNA?,RNA?,and RNAy.The ?a and ?a replicase subunits are encoded by RNA? and RNA? respectively,and they mediate the whole replicase enzyme formation through physical interactions for each other.Products that responsible for virion assembly and virus movement are encoded by RNA?.?b protein is a cysteine-rich protein with 17 kDa molecular weight,and play multifunctional roles in virus infection,such as pathogenesis determinants,seed transmission factor,systemic determinants in Nicotiana benthamiana,and RNA silencing suppressor.However,Interactions between yb and other BSMV encoded proteins and they coordinately to regulate viral replication and movement have not been reported yet.Previous studies showed that yb mainly localize to cytoplasm,and small amounts could specific localize to chloroplast in BSMV infection,but the biological functions of yb for chloroplast localization is still unknown.Our studies show that viral replicase subunits ?a and ?a proteins,viral plus-and minus-strand RNAs,and viral replicative double-strand RNA intermediate intimately associate with chloroplasts observed under confocal laser scanning microscopy observation,together with previous electron microscopic studies of BSMV-infected plant tissues,our results substantiate that chloroplasts are the major sites for BSMV replication.In addition,the physical interactions between ?b and replicase subunit?a were verified by the yeast two-hybrid assay(Y2H),bimolecular fluorescence complementation(BiFC),and co-immunoprecipitation(Co-IP).Y2H analysis of different functional truncation mutants of ?a and?a proteins further identified that amino acid residues 86-127 of ?b are required for interacting with methyltransferase domain of replicase subunit aa.Genetic analysis indicated that ?a mediated the recruitment of yb to the viral replication sites on the chloroplast membranes.Mutation or deletion of ?b from BSMV resulted in reduced positive strand(+)RNAa accumulation,whereas yb mutations abolishing viral suppressor of RNA silencing(VSR)activity did not completely eliminate genomic RNA replication.Furthermore,cis-or trans-expression of the Tomato bushy stunt virus(TBSV)p19 suppressor protein protein failed to complement the ?b replication functions,indicating that the direct involvement of ?b in BSMV RNA replication is independent of VSR functions.At last,an in vitro cell-free RNA unwinding assay showed that ?b alone does not possess helicase activity.Instead yb greatly enhances ?a unwinding of dsRNA duplexes,and the helicase enhancer of yb required the presence of its single-strand(ss)RNA binding activity.These data support a model whereby two BSMV-encoded RNA-binding proteins act coordinately to regulate viral genome replication and provide new insights into strategies whereby double-stranded viral RNA unwinding is regulated,as well as formation of viral replication complexes.The infection of ?b null mutants of BSMV in N.benthamiana and barley was also examined,Results showed that yb protein is essential for the systemic infection of BSMV in N.benthamiana.In contrast,the yb null mutants of BSMV maintains the ability of infecting barley systemically,despite the relatively low efficiency.These data suggested the requirements of ?b protein in viral trafficking is host-dependent.In addition,the interactions between ?b protein and viral movement protein TGB2 were validated by Y2H and BiFC assays.Furthermore,Co-IP assays showed that TGB1 protein could co-precipitated with ?b in the context of BSMV infection,implying the involvement of yb protein in the formation of viral movement ribonucleoprotein complex(RNP).Additional mutant BSMV that replaces the ?b ORF with GFP impaired cell-to-cell movement of BSMV in N.benthamiana leaves.Taken together,?b was probably recruited to viral movement RNPs through interactions with TGB2 protein,thus playing a role in viral intra-or intercellular movement.Further studies should be performed to illustrate the molecular mechanisms and biological significance of ?b-TGB2 interactions during BSMV infection.The novel roles of ?b promoting viral replication and movement will conducive to our deeper understanding of virus-encoded suppressors of RNA silencing and viral small cysteine-rich proteins.