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ARS Home » Midwest Area » Ames, Iowa » National Animal Disease Center » Virus and Prion Research » Research » Publications at this Location » Publication #309691

Research Project: Defining a Novel Structural Component of Porcine Reproductive and Respiratory Syndrome Virus and its Role in Disease Pathogenesis

Location: Virus and Prion Research

Title: Porcine reproductive and respiratory syndrome virus nonstructural protein 2 (nsp2) topology and selective isoform integration in artificial membranes

Author
item Kappes, Matthew
item Miller, Cathy - Iowa State University
item Faaberg, Kay

Submitted to: Virology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/30/2015
Publication Date: 7/1/2015
Citation: Kappes, M.A., Miller, C.L., Faaberg, K.S. 2015. Porcine reproductive and respiratory syndrome virus nonstructural protein 2 (nsp2) topology and selective isoform integration in artificial membranes. Virology. 481:51-62.

Interpretive Summary: Porcine reproductive and respiratory syndrome virus (PRRSV) is the foremost disease of swine in the United States. Control and eradication of PRRSV is largely hindered by the significant genetic and antigenic variability noted between strains, which is maintained by a rapid rate of evolution. In this report, nonstructural protein 2 (nsp2) of PRRSV, shown previously to be a novel structural component of the PRRSV virion that exists as several different sized species, was examined for its ability to interact with artificial membranes. Nsp2 was found to traverse the membrane in an unexpected way, and the two ends of the protein were shown to be on opposite sides of the membranes. In addition, when the membrane fraction was separated from the aqueous fraction, only three protein species were identified as membrane bound.

Technical Abstract: Membrane modification of host subcellular compartments is critical to the replication of many RNA viruses. Enveloped viruses additionally require the ability to requisition cellular membranes during egress for the development of infectious progeny. Porcine reproductive and respiratory syndrome virus (PRRSV) is an enveloped virus encoded by a positive-sense single-stranded RNA genome. PRRSV nsp2 is a predicted transmembrane protein with a recognized C-terminal hydrophobic region; however, the functionality of the putative nsp2 transmembrane domain has not been defined. In this report, the insertion and topology of nsp2 was assessed using cell free translation in the presence or absence of membranes. Nsp2 was found to strongly associate with membranes and, surprisingly, two additional large nsp2 isoforms of approximately 117 and 106 kDa were enriched within the membrane fraction. Membrane integration was further defined for full-length nsp2 through high-speed density fractionation, protection from protease digestion, and immunoprecipitation. The results demonstrated that nsp2 integrated into the membranes with an unexpected topology, where the N-terminal (cytoplasmic) and C-terminal (luminal) domains were orientated on opposite sides of the membrane surface.