Identification of Nonstructural Protein 8 as the N-Terminus of the RNA-Dependent RNA Polymerase of Porcine Reproductive and Respiratory Syndrome Virus

Abstract

Porcine reproductive and respiratory syndrome virus (PRRSV) is a member within the family Arteriviridae of the order Nidovirales. Replication of this positive-stranded RNA virus within the host cell involves expression of viral replicase proteins encoded by two ORFs, namely ORF1a and ORF1b. In particular, translation of ORF1b depends on a -1-ribosomal frameshift strategy. Thus, nonstructural protein 9 (nsp9), the first protein within ORF1b that specifies the function of the viral RNA-dependent RNA polymerase, is expressed as the C-terminal extension of nsp8, a small nsp that is encoded by ORF1a. However, it has remained unclear whether the mature form of nsp9 in virus-infected cells still retains nsp8, addressing which is clearly critical to understand the biological function of nsp9. By taking advantage of specific antibodies to both nsp8 and nsp9, we report the following findings. (1) In infected cells, PRRSV nsp9 was identified as a major product with a size between 72 and 95 kDa (72-95 KDa form), which exhibited the similar mobility on the gel to the in vitro expressed nsp8-9 ORF1b , but not the ORF1b-coded portion (nsp9 ORF1b ). (2) The antibodies to nsp8, but not to nsp7 or nsp10, could detect a major product that had the similar mobility to the 72-95 KDa form of nsp9. Moreover, nsp9 could be co-immunoprecipitated by antibodies to nsp8, and vice versa. (3) Neither nsp4 nor nsp2 PLP2 was able to cleave nsp8-nsp9 in vitro. Together, our studies provide experimental evidence to suggest that nsp8 is an N-terminal extension of nsp9. Our findings here paves way for further charactering the biological function of PRRSV nsp9.

Research Progress on Porcine Reproductive and Respiratory Syndrome Virus NSP7 Protein

“…NSP5-8 may be the major long-lived intermediate in HP-PRRSV-infected cells. These proteins play an important role in the PRRSV life cycle; however, their specific biological functions require further study [28].…”

Research Progress on the NSP9 Protein of Porcine Reproductive and Respiratory Syndrome Virus

“…The migration rate of NSP8-9 expressed in vitro is similar to natural NSP9, and NSP9 can co-immunoprecipitate with the NSP8 antibody. NSP4 and NSP2 PLP2 cannot cleave NSP8-9 in vitro , which indicates that NSP8 is the N-terminal extension of NSP9 ( 7 ). The RdRp domain is located at the C-terminal of the replicase subunit of NSP9, which contains an upstream domain with an unknown function ( 8 ).…”