Date of Award
Doctor of Philosophy
Veterinary Microbiology and Preventive Medicine
Kay S. Faaberg
Cathy L. Miller
Porcine reproductive and respiratory syndrome virus (PRRSV) is a rapidly mutating pathogen eliciting respiratory and reproductive disease of high economic consequence. The PRRSV non-structural protein 2 (nsp2) is a large multifunctional protein encoded by the most genetically diverse region of the genome - the selective pressure potentiating mutation within this region is unknown. Here we report the identification of a unique function of nsp2 as a structural component of the PRRSV virion; the first PRRSV structural protein identified which is not expressed from a sub-genomic RNA or regulated via the discontinuous transcription pathway. Through the use of a set of custom á-nsp2 antibodies nsp2 was identified on the surface of the PRRSV virion by immunoelectron microscopy. Further, a class of nsp2 isoforms was defined to be packaged within or upon the PRRSV virion. Nsp2 packaging was found to be conserved across a panel of highly divergent stains of PRRSV including the genotype 1 and genotype 2 prototype strains as well as contemporary and highly-pathogenic isolates.
Next the hydrophobic domain of nsp2 was characterized as a putative multi-pass transmembrane domain predicted to facilitate nsp2 packaging through association with the viral envelope. Within an in vitro cell-free translation system nsp2 was found to strongly associate with canine microsomal membranes. Through high-speed ultracentrifugation, protease protection assay, and immunoprecipitation nsp2 was defined as an integral membrane protein and additionally identified to display an unexpected N-terminal cytoplasmic / C-terminal luminal topological orientation. Finally, membrane isolation demonstrated two sub-dominant nsp2 isoforms of approximately 117 and 106 kDa in size and of unknown composition or function were enriched within membranes.
Together, these results define previously unknown attributes of nsp2. Identification of nsp2 as a structural protein implicates it in previously unpredicted functions related to attachment, entry, or early replication events and further provides rationale for the high mutation rate and robust adaptive immune response targeting the nsp2 protein. Characterization of the nsp2 transmembrane domain demonstrates its role as an integral membrane protein and additionally raises new questions related to the unexpected topological orientation or enrichment of select isoforms within the membrane fraction.
Matthew Allan Kappes
Kappes, Matthew Allan, "Identification and characterization of a novel structural protein of porcine reproductive and respiratory syndrome virus, the replicase nonstructural protein 2" (2014). Graduate Theses and Dissertations. 14184.