Heterodimerization of PRRSV replicase membrane proteins nsp2 and nsp3 regulates their cytoplasmic tail binding to...

Recruitment of viral RNA polymerase to replicase membrane proteins is a critical step for assembly of the replication and transcription complex (RTC) during the replication of positive-stranded RNA viruses. In this study, we report that the efficient recruitment of porcine reproductive and respiratory syndrome virus (PRRSV) RNA polymerase RdRp domain requires conformational rearrangement of replicase membrane proteins nsp2 and nsp3 via formation of heterodimeric complex (nsp2/3). Specifically, nsp2 or nsp3 interacts poorly with the nsp9 RdRp core domain (aa.500-685) in pairwise transfection, but the interaction became efficient when the two membrane proteins were co-expressed. Further analysis mapped the nsp9 RdRp-binding region to the cytoplasmic tails of both nsp2 and nsp3 and revealed that reciprocal co-expression of the transmembrane domain (TM) was sufficient to activate the binding of nsp2 or nsp3 to the nsp9 RdRp domain, whereas specific amino acid mutations within the cytoplasmic tails (nsp2 P1073A/G, N1074A/G, N1081A/G, and nsp3 R210E) could disable this interaction. The significance of nsp2/3-nsp9 RdRp interactions was revealed in studies of viral mutants, which showed that PRRSV mutants carrying the above corresponding mutations were either non-viable or severely crippled in replication and exhibited a defect particularly in viral subgenomic RNA (sgRNA) synthesis. Together, our findings highlight the importance of structure change-induced engagement of PRRSV membrane proteins (nsp2 and nsp3) with viral polymerase core domain and provide insight into the orchestrated RTC assembly in PRRSV RNA synthesis.IMPORTANCEPRRSV represents a major threat to the global pork production, but there are no effective vaccines or antiviral drugs yet available. This report concerns the viral RTC assembly. We show that PRRSV replicase membrane proteins nsp2/3 heterodimerization induces a conformational rearrangement of their cytoplasmic tails to allow efficient interaction with nsp9 RdRp core domain. Mutations within the cytoplasmic tails that block the interactions lead to a defect in viral sgRNA synthesis. These findings add insights into the mechanisms of orderly assembly of PRRSV RTC and regulation of viral sgRNA synthesis and provide potential vulnerable targets for drug interventions.