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

The membrane insertion and topology of nonstructural protein 2 (nsp2) of porcine reproductive and respiratory syndrome virus (PRRSV) strain VR-2332 was assessed using a cell free translation system in the presence or absence of artificial membranes. Expression of PRRSV nsp2 in the absence of all other viral factors resulted in the genesis of both full-length nsp2 as well as a select number of C-terminal nsp2 isoforms. Addition of membranes to the translation stabilized the translation reaction, resulting in predominantly full-length nsp2 as assessed by immunoprecipitation. Analysis further showed full-length nsp2 strongly associates with membranes, along with two additional large nsp2 isoforms. Membrane integration of full-length nsp2 was confirmed 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 amino (N)-terminal (cytoplasmic) and C-terminal (luminal) domains were orientated on opposite sides of the membrane surface.