Mapping the Key Residues within the Porcine Reproductive and Respiratory Syndrome Virus nsp1 alpha Replicase Protein Required for Degradation of Swine Leukocyte Antigen Class I Molecules

The nonstructural protein 1 alpha (nsp1 alpha) of the porcine reproductive and respiratory syndrome virus (PRRSV) has been shown to target swine leukocyte antigen class I (SLA-I) for degradation, but the molecular details remain unclear. In this report, we further mapped the critical residues within nsp1 alpha by site-directed mutagenesis. We identified a cluster of residues (i.e., Phe17, Ile81, Phe82, Arg86, Thr88, Gly90, Asn91, Phe94, Arg97, Thr160, and Asn161) necessary for this function. Interestingly, they are all located in a structurally relatively concentrated region. Further analysis by reverse genetics led to the generation of two viable viral mutants, namely, nsp1 alpha-G90A and nsp1 alpha-T160A. Compared to WT, nsp1 alpha-G90A failed to co-localize with either chain of SLA-I within infected cells, whereas nsp1 alpha-T160A exhibited a partial co-localization relationship. Consequently, the mutant nsp1 alpha-G90A exhibited an impaired ability to downregulate SLA-I in infected macrophages as demonstrated by Western blot, indirect immunofluorescence, and flow cytometry analysis. Consistently, the ubiquitination level of SLA-I was significantly reduced in the conditions of both infection and transfection. Together, our results provide further insights into the mechanism underlying PRRSV subversion of host immunity and have important implications in vaccine development.