Characterization Of The Complete Genome Of Barley Yellow Striate Mosaic Virus And Functional Analysis Of Phosphorylation Of Its Phosphoprotein

Rhabdovirus consists of diverse members of negative-sense, single-stranded RNA viruses with broad host ranges that collectively encompass vertebrates, invertebrates and plants causing negative effect on human health, agriculture, and wild life ecology. The transcription and replication of rhabdoviruses requires core proteins, nucleoprotein (N), phosphoprotein (P), and the large polymerase (L), as well as their non-segmented, negative-strand RNA genomes. Although many factors are identified to involved in the regulation of transcription and replication process during animal rhabdovirus infections, specific mechanisms responsible for viral transcription and replication switches remain elusive. In addition, the functions of the plant rhabdovirus P proteins are largely unknown due to the lack of infectious cDNA clones of plant rhabdviruses.Barley yellow striate mosaic virus (BYSMV) is a world-wide cytorhabdovirus,and was recently reported to cause disease in the wheat field of North China. Here, the complete genome of BYSMV was obtained using the methods of assembly of virus-derived siRNAs, RT-PCR and RACE(rapid-amplification of cDNA ends). The genome consists of 12,706 nucleotides and contains nine transcripts encoding ten proteins including five structural proteins and five accessory proteins.Interestingly, the second mRNA behind P mRNA contains two open reading frames (ORF4 and ORF5)in an alternative frame. A small hydrophobic protein is encoded by ORF5 via a leaky scanning mechanism, which was proved by in vitro translation experiments. The sequence analyses revealed that BYSMV and NCMV have a similar genome organization and high sequence identities of their major structural proteins indicating that the two viruses are more closely related to each other.Based on the full-length genomic sequence of BYSMV, the genomic-sence minireplicon (gMR) and the antigenomic-sence minireplicon (agMR) of the BYSMV successfully developed through agrobacterium-mediated co-expression of the viral N, P,L, MR derivatives and viral silencing suppressors (VSR). The transcription and replication efficiency of gMR is lower than those of agMR.The MR systems facilitate functional studies of viral and/or host effectors involving in viral transcription and replication.In the MR systems, BYSMV P proteins migrated as two apparent molecular masses of 42- (P42) and 44-kDa (P44) in SDS-PAGE gels, which represent a basic phosphorylation form (P42) and a hyperphosphorylation form (P44) in phosphatase treatment assays, respectively. Seventeen phosphorylated serine, threonine, and tyrosine residues were determined in BYSMV P protein by mass spectrometry. Notably, a serine-rich region (SR) including five phosphorylated serine residues (S189,S191, S194, S195, and S198) locate in the C-terminal disordered region of the BYSMV P protein.Compared with the wild-type P (PWT), an alanine-substituted mutant mimicking nonphosphorylated serines (PS5A) in the SR region only produced the P42 form, and supported enhanced minigenome replication accompanied with reduced transcription. While, the aspartate-substituted mutant mimicking phosphorylated serines (PS5D) migrated as the P44 form and facilitated the switch from viral replication to transcription. The results indicate that the phosphorylation of the BYSMV P protein SR region regulates the switch between viral transcription and replication. Results from protein interaction assays showed that the mutants PS5A and PS5D exhibited severely weakened interactions with L and N-RNA viral nucleoprotein core complexes, respectively, but had similar self-interactions and binding affinity to nascent RNA-free N protein. The results demonstrate that P protein phosphorylation affects the function of the polymerase complexes during transcription and replication.Taken together, our results demonstrate that the reversible phosphorylation and dephosphorylation within the C-terminal SR region of the BYSMV P protein drives conformational changes that form different complexes with L and N-RNA templates modulating viral transcription and replication switches. These findings provide new evidence for the regulation of P proteins in transcription and replication switches during rhabdovirus infection.