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United States Department of Agriculture

Agricultural Research Service

Research Project: INTERVENTION STRATEGIES TO CONTROL VIRAL DISEASES OF SWINE Title: Highly divergent strains of porcine reproductive and respiratory syndrome virus incorporate multiple isoforms of nonstructural protein 2 into virions

Authors
item Kappes, Matthew
item Miller, Cathy -
item Faaberg, Kay

Submitted to: Journal of Virology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: September 26, 2013
Publication Date: December 1, 2013
Citation: Kappes, M.A., Miller, C.L., Faaberg, K.S. 2013. Highly divergent strains of porcine reproductive and respiratory syndrome virus incorporate multiple isoforms of nonstructural protein 2 into virions. Journal of Virology. 87(24):13456-13465.

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 variability among strains due to a rapid rate of evolution. This rapid rate of change results in suboptimal immunological protection within the population to PRRSV. While vaccines currently exist for PRRSV, they are largely ineffective against the broader class of circulating strains; it is estimated that PRRSV costs the U.S. pork industry greater than $560 million annually, or $1.5 million per day (Neumann et al. 2005, Holtkamp 2011), within the context of current vaccination strategies. Proteins which make up the virion are critical for viral replication, and are key determinants to immune response generated against infection or vaccination. In this report, we describe the discovery of a novel component of the virus particle (virion), a viral protein where significant diversity is known to exist. Incorporation was shown to be a wholly conserved feature across a diverse set of strains, whereas the protein composition is complex. Identification of a novel component of the viral particle implies the composition of the PRRSV virion is more complicated than originally predicted, yielding new insights into viral particle composition. Inclusion of the nonstructural protein 2 (nsp2) with the PRRSV virus particle demonstrates a previously unknown localization for this protein, which have important implications on interaction with the corresponding immune surveillance systems and impart a novel downstream vaccine target of yet unknown importance. These data show yet unexplored roles for viral proteins which may be significant determinants to productive infection, host responses, and future vaccination strategies.

Technical Abstract: Viral structural proteins formulate the critical intermediary between viral infection cycles within and between hosts, function to initiate entry, participate in immediate-early viral replication steps, and are major targets for the host adaptive immune response. We report the identification of nonstructural protein 2 (nsp2) as a novel structural component of the porcine reproductive and respiratory syndrome virus (PRRSV) particle. A set of custom a-nsp2 antibodies targeting conserved epitopes within four distinct regions of nsp2 (PLP2 protease domain (OTU)), hypervariable domain (HV), putative transmembrane domain (TM) and the C-terminal region (C) were obtained commercially and validated in PRRSV infected cells. Highly purified cell-free virions of several PRRSV strains were isolated through multiple rounds of differential density gradient centrifugation and analyzed by immunoelectron microscopy (IEM) and western blot assays using the alpha-nsp2 antibodies. Purified viral preparations were found to contain pleomorphic, predominantly spherical, virions of uniform size (57.9nm ± 8.1nm diameter, n=50), consistent with the expected size of PRRSV particles. Analysis by IEM indicated the presence of nsp2 associated with the viral particle of diverse strains of PRRSV. Western blot analysis confirmed the presence of nsp2 in purified viral samples and revealed that multiple nsp2 isoforms were associated with the virion. Finally, a recombinant PRRSV genome containing a myc-tagged nsp2 was used to generate purified virus, and these particles were also shown to harbor myc-tagged nsp2 isoforms. Together, these data identify nsp2 as a virion-associated structural PRRSV protein, and reveal that nsp2 exists in/on viral particles as multiple isoforms.

Last Modified: 12/22/2014
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