This dissertation focused on understanding the biology of the nonstructural protein 2 (nsp2) of porcine reproductive and respiratory syndrome virus (PRRSV), the etiological agent of porcine reproductive and respiratory syndrome (PRRS). PRRSV nsp2 is a multidomain protein, containing a putative N-terminal cysteine protease PL2 domain, a 500-700aa middle region of unknown function, a transmembrane domain and a C-terminal tail with uncertainty. In this dissertation, we report the following. (i) PRRSV nsp2 is undergoing rapid evolution in field strains exemplified by viral isolates MN184A and B. (ii) We showed that PRRSV nsp2 hypervariable regions aa12-35 and aa324-813 were not essential for viral replication in MARC-145 cells by using a reverse genetics system based on strain VR-2332. In contrast, deletion of the cysteine protease PL2 core domain, the PL2 downstream flanking sequence (aa181-323), the predicted transmembrane domain and the C-terminal domain were lethal to the virus. (iii) We provided evidence that the nsp2 protein encodes an active PL2 protease and mediates nsp2/3 processing in CHO cells with a substrate preference for the dipeptide G1196|G1197. The PL2 protease possessed both trans- and cis-cleavage activities, which could be distinguished by point mutations. Site-directed mutagenesis studies revealed that mutations that caused a specific loss of trans function of the PL2 protease, but not cis activity, were detrimental to the virus. In addition, we showed that the conserved aspartic acid residues (e.g., Asp89) played an important role in the PL2 trans-cleavage activity. (iv) We investigated the proteolytic processing of nsp2 in MARC-145 cells using recombinant PRRSV expressing foreign epitope-tagged nsp2 protein. We showed the presence of the nsp2 protein as different isoforms in PRRSV-infected cells, which appeared to share the same N terminus but differed in their respective C-termini. The nsp2 species emerged almost simultaneously in the early stage of PRRSV infection, were stable and had low turnover rates. Deletion mutagenesis suggested that the smaller nsp2 species (e.g. nsp2d, e and f) were not essential for viral replication in cell culture. Lastly, a cellular protein, heat shock 70kDa protein 5 (HSPA5), was identified as a coimmunoprecipitate of nsp2.
University of Minnesota Ph.D. dissertation. December 2008. Major: Veterinary Medicine. Advisors: Kay S. Faaberg, Ph.D.,Mark S. Rutherford, Ph.D. 1 computer file (PDF); viii, 163 pages. Ill. (some col.)
Characterization of the multidomain Nsp2 Replicase protein of porcine reproductive and respiratory syndrome virus..
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