Heterogenous Combination Of Cucumovirus Replicases Regulates Virus Genomic And Subgenomic RNA Replication

Cucumber mosaic virus(CMV) and Tomato aspermy virus(TAV) are both members of the Cucumovirus genus, the Bromovirus family, a group of important plant pathogen. The genome of CMV and TAV consists of three single-stranded positive-sense RNAs. RNA1 and RNA2 encodes protein 1a and protein 2a respectively, which are involved in the replication complex in association with host proteins. Protein 2b is a multi-functional protein which is encoded by the subgenomic RNA4 A of genome RNA2. The 2b protein is a virulence factor, and it can counteract host defense as a viral suppressors of RNA silencing. In the open field, mixed-infection with different viruses in plant, generally results in the changes of viral accumulation and symptoms. Interestingly, in the mixedinfection plants, intra- and intermolecular recombination occur at a low frequency, it plays an important role in evolution and diversification of plant virus.In this study, we use pseudorecombinants between CMV and TAV to discover the molecular basis of CMV 1a protein regulating synthesis of viral subgenome RNA4 A by determining 1a function domain responsible for RNA4 A synthesis, and the effects of Cucumovirus viral heterologous replicase on viral genomic and subgenomic RNA.TAV RNA1(T1) and CMV RNA2(C2) are heterogeneously combined, mix with either CMV RNA3(C3) or TAV RNA3(T3), and then inoculated onto Nicotiana benthamiana plants by agroinfiltration, the heterologous viral replicase TAV 1a /CMV 2a is unable to produce subgenomic RNA4 A. Result of fluorescence observation and Western blot analysis show that rarely fluorescence is detected in the co-expression of TAV 1a, CMV 1a and F209-EGFP. We speculate that CMV 1a protein plays an important role in synthesis of subgenomic RNA4 A. Protein 1a possesses a methyltransferase domain in its N-terminal part and a helicase motif in the C-terminal part. We find that exchange complete or part of protein domains between CMV 1a and TAV 1a, protein1 a recombinant cannot support viral subgenomic RNA4 A replication. The results suggest that replication of CMV subgenomic RNA4 A requires complete CMV1 a protein.Via the expression method of virus gene based on T-DNA to analysis the effects of exchange between CMV and TAV genome on viral replication in plant, CMV-specific and TAV-specific RNA probes are used for Northern blot analysis of viral RNAs. total RNA are extracted from the system leaves inoculated with CMV RNA1 and RNA3 with TAV RNA2 after 14 days. Results of Northern blot analysis show that heterologous replicase CMV1a/TAV2 a can support the replication of viral genome RNAs. Sequencing results of viral genomic RNAs show that the recombination event exists in CMV RNA3 which contains the 3'UTR of TAV RNA2 in the 3'-terminal part of CMV RNA3. Coexpression of CMV 1a, TAV 2a and recombinant RNA3, heterologous replicase CMV 1a/TAV 2a can replicate the recombinant RNA3. Taken together, the recombinant virus C1T2C3 can overcome defects in virus replication conferred by the heterogeneous combination of their replicases by in vivo mutation and recombination.This work finds that effectively replication of CMV subgenomic RNA4 A requires complete protein domain of CMV 1a. And in the pseudorecombinant C1T2C3, it can produce a recombinant RNA3, which can be found after several passages through N. tabacum.