Submitted to: Journal of Virology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/16/2010
Publication Date: 10/1/2010
Citation: Han, J., Rutherford, M.S., Faaberg, K.S. 2010. Proteolytic products of the porcine reproductive and respiratory syndrome virus nsp2 replicase protein. Journal of Virology. 84(19):10102-10112. Interpretive Summary: Porcine reproductive and respiratory syndrome virus (PRRSV) is the most costly infectious disease of swine in the United States costing producers in excess of $560 million annually. Although vaccines exist, this pig disease remains very difficult to control and eradicate because the virus constantly changes to avoid protection afforded by vaccines. Moreover, the virus replicates and sheds for several months from pigs. The mechanism this virus uses to replicate and cause disease involves several enzymes the virus makes, one of which is called a replicase. In order for this enzyme to reach its mature functional ability, another group of proteins made by the virus, called proteases, must first process the replicase for it to be become functional. In this study we identified several unique versions a key viral protein (nsp2) involved in making this replicase functional. We were surprised to discover a cellular protein named HSPA5 was interacting with this key viral protein (nsp2) and could possibly play a role in how PRRSV infects cells and causes disease. Results of studies reported here provide additional information to scientists and vaccine companies who seek to rationally design improved vaccines against PRRSV.
Technical Abstract: The nsp2 replicase protein of porcine reproductive and respiratory syndrome virus (PRRSV) was recently demonstrated to be processed from its precursor by the PL2 protease at or near the G1196|G1197 dipeptide in transfected CHO cells. Here, the proteolytic cleavage of PRRSV nsp2 was further investigated in virally infected MARC-145 cells by using two recombinant PRRSV expressing epitope-tagged nsp2. The data revealed that nsp2 exists as different isoforms during PRRSV infection, termed nsp2a, b, c, d, e and f. Moreover, these nsp2 species appeared to share the same N-terminus but differed in their respective C-termini based on deletion mutagenesis and antibody probing. The largest protein, nsp2a, corresponded to the nsp2 product identified in transfected CHO cells. Nsp2b and c were processed within or near the TM region, presumably at or near the conserved sites G981|G982 and G828|G829|G830, respectively. The C-termini for nsp2d, e and f were mapped within the nsp2 middle hypervariable region, but no conserved cleavage sites could be predicted, suggesting likely involvement of host cell proteases. The larger nsp2 species emerged almost simultaneously in the early stage of PRRSV infection. Pulse-chase analysis revealed that all six nsp2 species were relatively stable and had low turnover rates. Deletion mutagenesis revealed that the smaller nsp2 species (e.g. nsp2d, e and f) were not essential for viral replication in cell culture. Lastly, we identified a cellular chaperone, heat shock 70kDa protein 5 (HSPA5), that was strongly associated with nsp2 and which may have important implications for PRRSV replication. Overall, these findings indicate that the PRRSV nsp2 is increasingly emerging as a multifunctional protein and may have a profound impact on PRRSV replication and viral pathogenesis.