Mechanistic Studies Of Sonchus Yellow Net Rhabdovirus Movement And Superinfection Exclusion

Plant rhabdoviruses are a group of enveloped negative-stranded RNA viruses that infect various kinds of cereal and economic crops and cause severe yield losses Sonchus yellow net virus(SYNV),a member in the genus Nucleorhabdovirus,family Rhabdoviridae,is one of the most extensively studied plant rhabdoviruses.SYNV replicates in the nucleus and buds from the inner nuclear membranes to accumulate in the perinuclear spaces.Rhabdoviruses infecting plants,insects,fish and vertebrates all encode five conserved structural proteins in the order of 3’N-P-P3-M-G-L 5’ and share some common features in viral RNA replication and transcription,budding,morphology and so on.Additionally,plant rhabdoviruses encode another nonstructural protein P3 as a movement protein(MP)that is necessary for virus spreading in plant However,little is known about the mechanism of plant rhabdovirus movement due to the lack of reverse genetic systems for quite a long time.This study mainly focuses on mechanisms of SYNV cell-to-cell movement,long-distance movement,and superinfection exclusion(SIE)phenomenon based on the infectious clones of plant rhabdoviruses constructed previously in our laboratory.To determine how SYNV moves between cells,we constructed a series of SYNV deletion mutants including the nonstructural protein sc4 deletion mutant,matrix protein(M)deletion mutant,glycoprotein(G)deletion mutant and the M and G double deletion mutant to analyze the local movement ability of these mutants.The results confirmed sc4 as a movement protein of SYNV since deletion of sc4 gene abolished SYNV cell-to-cell movement;Limited localized cell-to-cell movement of rSYNV-GFP△MG suggested that uncoiled nucleocapsids(NCs)which consist of nucleoprotein(N),phosphoprotein(P),large polymerase(L)and viral genomic RNA were infectious movement entities.Transiently expressed sc4 located mainly at the cell periphery,with a minor proportion distributed in the nucleus of Nicotiana bentha.miana epidermal cells.Sc4 did not co-localization extensively with the microtubule marker MAP65-RFP,the Golgi marker ManI-mCherry,the ER network marker GFP-HDEL,or the microfilament marker ABD2-GFP.Using chemical inhibitors that block the protein secretory system and cytoskeleton activities,we found 20 μg/ml Brefeldin A(BFA)inhibited the local movement of SYNV through disrupting the ER network,whereas the microtubule inhibitor Oryzalin and the microfilaments inhibitor Latrunculin B(Lat B)had no effects on SYNV local movement.In addition,sc4 also exhibited non-specific single-stranded RNA binding activity in vitro.BiFC and yeast two hybridyzation assays showed that sc4 interacted with N and P,the two primary nucleocapsid proteinsWe successfully complemented the local movement of ToMV-GFPOMP and PVX-GFPAp25 by MPs encodec by five distinct plant rhabdoviruses,i.e.SYNV,Potato yellow dwarf virus,(PYDV),Riceyellow stunt virus(RYSV),Rice stripe mosaic viruss(RSMV)and Tomato yellow mottle-associated virus(TYMaV),demonstrating the functional similarities between MPs from both positive and negative strand RNA viruses.However,trans-complementation assay with a movement protein dificient mutant of SYNV(rSYNV-GFPOsc4)showed that the cell-to-cell movement of SYNV is only compatible with its own MP(sc4),but not with noncogante MPs of rhabdoviruses including PYDV,RYSV,RSMV and TYMaV,and MPs of Cucumber mosaic virus(CMV),Tobacco mosaic virus(TMV),Rice stripe virus(RSV),Tomato spotted wilt virus(TSWV),Tomato bushy stunt virus(TBSV),Tomato mosaic virus(ToMV).Similarly,movement of a MP-deficient cytorhabdovirus,rTYMaV-GFPAP3 is only complemented by its cognate MP(TYMaV P3),demonstrating that intercellular movement of rhabdovirus was specifically dependent on its own MP.By protein interaction assays,we found that MPs of SYNV and TYMaV interacted only with their respective cognate N proteins but not with noncogante N proteins.Based on these data,we suggested that specific MP-N interaction mediated movement specificity.Further co-expression experiments showed that sc4 facilited the SYNV N-P complex to relocate from the nuclear repication site to cell periphery.These results suggested that rhabdovirus MPs could guide inter-and intra-cellular movement of the nucleocapsid by specifically interacting with the cognate core proteins.To date,our understanding of long-distance movement of plant viruses is mainly based on studies of positive-stranded RNA viruses,and little is known about mechanisms underlying enveloped plant negative-stranded RNA viruses long distance movement.To explore the mode of enveloped rhabdovirus movement,we fused fluorescent proteins to SYNV structural proteins by taking advantage of our recently developed SYNV reverse genetics system.Freehand and ultra-thin sections of vascular tissues such as petiole,stem and root of N.benthamiana plants systemically infected by SYNV were observed under the confocal microscopy and transmission electron microscopy to track the localization of viral proteins and virions.Our results showed that SYNV can replicate and transport in phloem and virions or structural proteins were present in phloem components,xylem parenchyma cells,and even xylem vessels of N.benthamiana.However,there was no direct evidence to show SYNV virions utilized xylem vessels for long distance movementSuperinfection exclusion is a phenomenon that a primary virus infection prevents subsequent infection by the same or a related virus.It is also known as homologous interference or cross protection.Using fluorescent protein-tagged SYNV,we showed that superinfection exclusion existed between SYNV derivatives but not with unrelated PVX.Transiently expressed SYNV M inhibited the infection of SYNV whereas the M mRNA did not,indicating that the SYNV SIE was induced on the M protein level.Correspondingly,the deletion of M protein resulted in a remarkable reduction in the exclusion ability to SYNV homologous viruses.Further studies showed that M protein suppressed the SYNV replication and transcription through interacting with the N protein.The point mutant MG126L which lost the interaction ability with N protein failed to suppress the replication and transcription of SYNV MR.And we found the M NLS mutants failed to to suppress the replication and transcription of SYNV MR.These results suggested that large amount of M protein expressed by primary infected SYNV after replication suppressed the replication and transcription of viral genome,rendering it impossible for the invading homologous viruses to replicate and infect.In summary,we utilized SYNV reverse genetics system to study the characteristics of SYNV sc4,the mode of SYNV inter-and intra-cellular movement as well as the long-distance movement.We reported the SIE phenomenon on SYNV and revealed the molecular mechanism underlying the SIE.Our data facilitate our understanding of the molecular mechanisms of the movement and infection of plant negative-stranded RNA virus,and provide a foundation for the prevention and control of such virus diseases.