Multiplication Of Rice Dwarf Virus In Insect Vector Of Leafhopper

Rice dwarf virus(RDV)is a member of Phytoreovirus in Reoviridae family.It causes rice dwarf disease,and is transmitted by leafhopper in a persistent-propagative manner.Recently,the life cycle of RDV in cultural cells of insect vector,including infection,replication,assembly,spread and release,is investigated;the property of viral transmission by leafhopper and the sequential infection of RDV in the internal organs of its insect vector after ingestion of virus are clarified.However,the propagative mechanism of RDV in leafhopper is still unknown yet,especially the mechanisms of viral replication and spread have not been reported.Thus,the mechanisms of RDV replication and spread in insect vector,as well as the mechanism underlying vector specificity of RDV were investigated in this thesis.The genome of RDV consists of 12 dsRNA segments,encoding at least seven structure proteins(P1,P2,P3,P5,P7,P8 and P9)and five non-structure proteins(Pns4,Pns6,Pns10,Pns11 and Pns12).The major outer capsid protein P8,minor outer capsid protein P2 and P9 assemble into the outer capsid shell of virion.The viral core particle is constituted by PI as RNA-dependent-RNA-polymerase(RdRp),P5 as a guanylyl transferase,and P7 with RNA-binding activity,packaged by inner core protein P3.In cells of insect vector,non-structure protein Pns4 forms a minitubule,Pns10 assembles into tubules which package virions to facilitate viral intercellular spread.Pns6,Pns11 and Pns12 compose the matrix of viral factory,termed as viroplasm.Here,the function of Pns6,Pns11 and Pns12 in leafhopper vector cells in monolayers(VCMs)was firstly investigated.It was found that the knockdown of Pns6,Pns11 and Pns12 expression due to RNA interference(RNAi)induced by synthesized dsRNA from Pns6,Pns11 and Pns12 genes,via simultaneously reducing the relative expression level of genes and the expression of three proteins,caused the function and number of viroplasm to be significantly affected,and the synthesis of viral genomic dsRNA was strongly inhibited,finally the viral infection was blocked.These results revealed that the effect of RNAi significantly impact on the viral replication cycle,including expression of viral protein,synthesis of viral genome,formation of viroplasm and production of infectious progeny virions.The mechanism for this effect was associated the roles Pns6,Pns11 and Pns12 played in viroplasm.Thus,Pns6,Pns11 and Pns12 were likely to closely relate to viral replication.Lacking of any one of these three proteins could cause RDV infection to be blocked.In order to verify the result in VCMs,dsRNAs were introduced into leafhopper by microinjection.The result showed that the knockdown Pns6,Pns11 and Pns12 by microinjection of leafhopper with dsRNA,affect the function of viroplasm via decreasing the relative expression level of corresponding gene,leading to viral accumulation reduce,result in the viruliferous rate decreased and viral spread in leafhopper slowed.These results confirmed that Pns6,Pns1 1 and Pns12 contributed to viral replication in the process of RDV infection.These three proteins were the key replication factors of virus.Absence of any one of them could affect viral replication.Previous study showed that Pns12 has the ability to self-associate to form the viroplasm-like inclusion in the absence of viral infection,suggesting that Pns12,serving as the essential protein,might initiate the viroplasm formation.For the purpose of understanding the mechanism of the genesis and maturation of viroplasm induced by RDV infection in VCMs and insect vector,the yeast two-hybrid and GST pull-down assays were used to determine the interaction network of viroplasm proteins.The result showed that Pns12 directly interacted with Pns11 and core protein P3,Pns11 directly interacted with Pns6.Thus,it was assumed that Pns12 may serve as a scaffold for matrix of viroplasm,and is a principal regulator of the viroplasm formation,recruitment of viral RNA and proteins into the viroplasms,and genome replication,and we proposed a model for the genesis and maturation of viroplasm.In order to be efficiently transmitted by leafhopper,following the replication of persistent-propagative RDV,progeny virions should spread from epithelia cells in filter chamber of initial infection to other tissue for expansion,and finally infect salivary glands.Thus,the spread in insect vector is also an important process for viral multiplication.Previous cytologic and genetic data revealed that tubular structures,constructed of the nonstructural viral protein Pns10,contain viral particles and are directly involved in the intercellular spread of RDV among cultured leafhopper cells.Here,we demonstrated that RDV exploited these virus-containing tubules to move along actin-based microvilli of the epithelial cells in the alimentary canal,including filter chamber,midgut and hindgut,facilitating the intercellular spread,even passed through the microvilli to extent to lumen.After RDV virions crossed through the basal lamina,Pns10 tubules trafficked along actin-based muscle fibers of visceral muscle tissues of alimentary system,reproductive system,and other organs and tissues,facilitating the efficient spread of virus in the body of its insect vector leafhoppers.It was confirmed that RDV could use the tools like Pns10 tubules for spread in leafhopper.Further study showed that the synthesized dsRNA targeting for Pns10 gene could induce RNAi in VCMs.When gene of Pns10 was knocked down,formation of Pns10 tubules were strongly inhibited.By using virus-neutralizing antibodies to avoid infection by free virions or not,it was found that inhibition of Pns10 tubules formation blocked viral infection,but had no impact on virus replication and accumulation.Ingestion of dsRNA targeting for Pns10 gene by leafhopper could induce RNAi.The knockdown of Pns10 gene caused formation of tubules was inhibited,leading to viral intercellular spread was blocked,and viruliferous rate reduced,but had no impact on virus multiplication.All these results demonstrated that Pns10 tubules were capable of packaging virions for viral intercellular spread,serving as viral determinants for virus transmission.It also indicated that RDV exploited virus-containing tubules to move along actin-based apparatus for spread in leafhopper,which was a novel strategy of spread in insect vector for persistent-propagative plant virus.For the purpose of revealing the mechanism of RDV exploiting Pns10 tubules to efficient spread in leafhopper,interactor proteins of vector were screened using yeast-two-hybrid system.Seven candidates,including lipophorin precursor,vigillin,apoptosis-inducing factor,vitellogenin,mitochondrial porin,tropomodulin and cytoplasmic actin,were obtained as the interactor proteins of Pns10.RT-qPCR assay showed that lipophorin precursor,vigillin,apoptosis-inducing factor and vitellogenin were down-regulated,while mitochondrial porin and tropomodulin were up-regulated,compared to non-viruliferous leafhopper.Because Pns10 associated with micorvilli,the main component of which is cytoplasmic actin,the interaction of Pns10 with cytoplasmic actin was investigated.DUAL membrane system for membrane protein interaction and GST Pull-down assay verified the specific interplay of Pns10 and cytoplasmic actin of leafhopper.The interaction of Pns10 and target protein of insect vector is likely to cause the difference of viral spread in different vectors,which determines the vector transmission specificity of virus.Here,investigation was further carried out to reveal the mechanism of vector transmission specificity of RDV in the perspective of viral spread in leafhopper species the leafhopper species Nephotettix cincticeps and Recilia dorsalis collected from different areas.The capabilities comparison of viral acquisition and transmission confirmed that RDV was mainly transmitted by N.cincticeps,but is ineffectively transmitted by R.dorsalis.Microinjection of leafhopper with viral inoculum showed similar level of virus infection and transmission in two species,indicating that midgut escape barriers regulated vector transmission specificity of RDV on histology level.Detection for expression and dissemination of Pns10 tubules in leafhopper demonstrated that virus-containing tubules composed of Pns10 associated with the intestinal microvilli of N.cincticeps,but not with those of R.dorsalis.On the molecular level,Pns10 specifically interacted with cytoplasmic actin,the main component of insect microvilli of N.cincticeps,but not with that of R.dorsalis,suggesting that the interaction of Pns10 of RDV with insect cytoplasmic actin affected virus-vector specificity.The leucine at position of 262 of cytoplasmic actin was possible to determine the interaction of Pns10 and with the cytoplasmic actin,leading to different spread of RDV in leafhopper,finally regulate the vector transmission specificity.In conclusion,this study for the first time confirmed three non-structural proteins Pns6,Pns11 and Pns12 of RDV were essential for viral replication in leafhopper,which were the key factors of RDV replication.We also clarified the interaction network of genesis and maturation of viroplasm among Pns6,Pns11,Pns 12 and core protein P3.RDV exploited virus-containing tubules composed of non-structural viral protein Pns10 to traffic along actin-based cellular machinery,allowing efficient cell-to-cell spread of the virus in leafhopper vector,revealing Pns10 was a key factor of transmission determinant.And the interaction of Pns10 with cytoplasmic actin of leafhopper regulated vector transmission specificity of virus.These results provided theoretic base for making control strategy of blocking the viral multiplication in insect vector.The RNAi system and cell culture of insect vector used in this study helped overcome the lack of a reverse genetics system for investigating the functions of RDV protein in the case of viral infection,providing useful tools investigate the molecular mechanisms enabling efficient transmission of viruses by vector insects.