BrYV Infectious Clones Construction,and RTD-host Protein Screening,and Mammalian Virus-derived SiRNAs Characterization Analyzation

Poleroviruses belong to the Luteoviridae family and have phloem limitation.In the past decades,lots of efforts have been devoted to try to elucidate the mechanism of the phloem limitation and systemic movement of poleoroviruses.The readthrough protein(RTP)is reported to be responsible for systemic movement,symptom,and phloem limitation.The readthrough domain(RTD)of RTP was predicted on the surface of the virion and probably assisted the virus movement through potential interactions with host and viral factors.However,the detailed mechanisms are still unknown.Brassica yellows virus(BrYV),which could infect several cruciferous crops,is a new species of the Polerovirus genus.Based on sequence analysis,BrYV has at least three genotypes designated as A,B,and C,respectively.In this work,the full-length cDNA infectious clones of BrYV A and B were constructed and used for the biological characterization study.The candidate host protein,which could interact with BrYV RTD,has been determined and the relationship between this host protein and BrYV infection has been primarily analyzed.The infectious clones of the two genotypes could systemically infect Nicothiana benthamiana by agroinfiltration,and induced necrosis on inoculated leaves but no obvious symptom on upper ones.Since the sequence differences are found at the 5' terminal between A and B,the 5' terminal of these two clones were substituted with each other to generate two mutant viruses to test whether symptom and infectivity would be effected.Compared to their original ones,no obvious differences of the symptoms and infectivity of these mutants were observed.By yeast two hybridization selection of Arabidopsis thaliana cDNA library and using BrYV RTD as a bait,S-adenosylmethionine synthetase4(SAMS4)was selected as a candidate host partner.In N.benthamiana,the full-length AtSAMS4 was located in both cytoplasm and nucleus and RTD was located in nucleus,and their interaction was demonstrated by BiFC.The full-length mRNA sequence of NbSAMS was cloned.NbSAMS mRNA expression level was decreased to 50%by BrYV infection,which suggested that BrYV infection was probabaly closely related to the expression of SAMS.BrYV infectious clones would be a useful tool for poleroviruses study and the interaction between RTD and host SAMS could give us useful clues to understand virus-host relationships.RNA silencing is the primary antiviral defense in plants and invertebrates.The production of viral siRNA(vsiRNAs)is the molecular marker and key factor for this antiviral reaction.The typical vsiRNAs were diced from double-stranded RNAs by Dicer and have equal ratio for positive and negative strand,and could be enriched in AGO complex.In mammals,RNA silencing is also called RNA interference(RNAi).Although RNAi pathway is conserved in mammals,whether it has antiviral function is controversial.One important reason is that,in the past decade,vsiRNAs have not been detected in mammals or mammalian cells infected with diverse RNA viruses.Nodamura virus(NoV),a member of Alphanodavirus,has lethal infectivity for suckling mice.It has been reported that,without expressing its suppressor protein B2,the mutant virus NoV?B2 replicated to much lower level in suckling mice compared to NoV,and produced abundant 22 nt viral small RNAs showed Dicer-product characterization.In this work,co-immunoprecipitation(co-IP)combined with deep sequencing analysis were used for further analyzation of NoV?B2-dereived small RNAs.Besides of the known Dicer-dependent characterization,AGO-bound siRNAs has equal ratio for strands and were enriched with preference for U as the first nucleotide in 5' terminal(1U),which means the typical vsiRNAs were detected in NoV?B2-infected suckling mice.B2-bound and NoV-derived small RNAs were highly enriched and exhibited approximately equal strand ratios for all sizes with a peak of 22 nt,but exhibited no preference for 1U.Notably,NoV small RNAs obtained by AGO-IP were extremely low abundant compared to NoV?B2.Using this strategy,small RNAs from human cells infected with two strains of Influenza virus A(IAV)and their suppressor protein NS 1-deficient mutant virus IAVdelNS1 were analyzed.The known properties of mouse vsiRNAs were detectable in small RNAs obtained by AGO-IP from human 293T cells infected with IAVdelNS1.However,without using NS 1-deletion mutant virus nor AGO-IP,no such vsiRNAs could be detected.This is the first time to show that mammalian virus could produce siRNAs bound to AGO complex.A new strategy for detection of siRNAs produced by mammalian virus was developed that is combining suppressor-deficient virus with AGO-IP and deep sequencing analysis.It will facilliate the functional and mechanistic characterization of antiviral RNAi in mammals.