Identification and characterization of a novel structural protein of porcine reproductive and respiratory syndrome virus, the replicase nonstructural protein 2
| Autor: | Matthew A. Kappes |
|---|---|
| Rok vydání: | 2018 |
| Předmět: |
biology
Immunoprecipitation viruses Immunoelectron microscopy virus diseases RNA-dependent RNA polymerase biochemical phenomena metabolism and nutrition Porcine reproductive and respiratory syndrome virus biology.organism_classification Virology Transmembrane domain Viral envelope Transcription (biology) Integral membrane protein |
| DOI: | 10.31274/etd-180810-3740 |
| Popis: | 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 α-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 |
| Databáze: | OpenAIRE |
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