MYH9 has been identified as an indispensable cellular protein for porcine reproductive and respiratory syndrome virus (PRRSV) entry into permissive cells using the monoclonal anti-idiotypic antibody (Mab2-5G2) recognizing an antibody that specifically interacts with PRRSV glycoprotein 5 (GP5). More recently, we found that Mab2-5G2 interacted with the MYH9 C-terminal domain, designated PRA, which is required for PRRSV internalization. In this study, we demonstrate that blocking of MYH9 with Mab2-5G2 significantly diminished PRRSV internalization by porcine alveolar macrophage (PAM) via interruption of direct interaction between GP5 and MYH9, and thus remarkably inhibited subsequent infection of PAMs by PRRSV-2 isolates. Moreover, the three-dimensional structure of the Mab2-5G2 Fab-PRA complex determined via homology modeling predicted potential docking sites required for PRRSV internalization. Further analysis of Mab2-5G2-binding sites within PRA highlighted that the amino acids E1670, K1673, E1679, and I1683 in PRA are the key Mab2-5G2-binding residues. Notably, recombinant PRA protein blocked the interaction between PRRSV GP5 and cellular MYH9 by preventing translocation of MYH9 from the cytoplasm to the cell membrane, an essential step for PRRSV virion internalization. Meanwhile, porcine cell line permissive for PRRSV bearing point mutation of E1670A in MYH9 demonstrated reduced susceptibility for PRRSV infection. In conclusion, this work increases understanding of both PRRSV pathogenesis and the mechanistic role played by MYH9 in PRRSV infection.

Porcine reproductive and respiratory syndrome virus (PRRSV) is an important pathogen that causes huge losses economically to the pig industry worldwide. Previous research suggested that receptor dependence is necessary for PRRSV infection. MYH9 and CD163 are indispensable for PRRSV entry into a porcine alveolar macrophage. In the present study, human MYH9 (hMYH9) and mouse MYH9 (mMYH9), similar to swine MYH9, could also accelerate PRRSV infection in pCD163-mediated cell lines. Knockdown of MYH9 activity using the specific small interfering RNA or inhibitor (blebbistatin) concomitantly decreased PRRSV infection. C-terminal fragment of MYH9 (PRA) proteins from different mammalian species contains a conserved binding domain (aa1676-1791) for PRRSV binding, since the recombinant MYH91676−1791protein could inhibit the PRRSV infection significantly. Furthermore, the specific polyclonal antibody of MYH91676−1791 could block PRRSV infection in host cells. These data strongly supported that MYH9, a very important cofactor, participated in PRRSV entry into target cells, which may facilitate the development of a new therapeutic agent to control PRRSV infection.