The objective of this project was to characterize the makeup of nsp2, shown to be a new structural component of the PRRSV virion. Radiolabelled nsp2 protein, in the absence or presence of synthetic enclosed membranes (microsomes), was produced to understand if nsp2 could be part of the virion membrane. After synthesis, the membrane fraction was isolated by centrifugation. Nsp2 was found to be produced as several different sized products, just as was found in PRRSV infected cells. Some nsp2 products were found in tight association with the membrane fraction, but no shift in protein size was observed, suggesting no modification by membranes, such as sugar residue addition. The nsp2 protein orientation with respect to the artificial membranes, referred to as topology, was determined. Surprisingly, a small portion of the C-terminal of the nsp2 protein, when expressed in the in vitro system used, was protected. The unexpected result indicated that this domain of the protein would be oriented towards the outer part of the virion. Additional studies must be completed using the same nsp2 proteins expressed in cell culture to confirm this result.

The novel finding that nsp2 was a structural protein (part of the infecting virus) led us to further examine the nsp2 biochemical structure, post-synthesis additions such as sugar modification, as well as to determine the origin and potential cleavage of the different sized nsp2 products. Our unexpected results have led us to postulate that additional proteins, viral or cellular, may be needed in order to achieve correct membrane orientation. Thus, the next study will involve expression of the same constructs in cultured cells.

Kay Suzanne Faaberg, Ph.D.
Research Microbiologist USDA-ARS
Virus and Prion Research Unit
National Animal Disease Center, USDA, ARS
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