Functional analysis of non-muscle myosin heavy chain IIA (amino acids 1831–1882) in facilitating pseudorabies virus entry via interaction with glycoproteins gH and gL

Pseudorabies virus (PRV) is the etiological agent responsible for porcine pseudorabies, causing substantial financial losses in the global swine industry. Non-muscle myosin heavy chain IIA (NMHC IIA), as an actin-binding motor protein, has been recognized as a functional factor in herpesvirus entry. However, its precise mechanism in PRV invasion remains poorly defined. Our investigation demonstrated that targeted inhibition of NMHC IIA, using a specific chemical inhibitor, siRNA-mediated knockdown, C-terminus of NMHC IIA (sPRA, amino acids [aa] 1672-1981), or rabbit anti-sPRA serum, significantly decreases PRV infection in PK-15 cells in a dose-dependent manner. During the early stage of PRV infection, NMHC IIA underwent rapid and pronounced translocation from the cytoplasm to the cell surface, which is beneficial to the virus entry. The study identified that sPRA mediates PRV internalization by binding to viral glycoproteins gH and gL. Furthermore, we pinpointed the subdomain spanning aa 1831-1882 within sPRA as the critical domain for both mediating the interaction with gH/gL and facilitating viral internalization. These results advance our comprehension of the molecular pathways through which NMHC IIA supports PRV infection and highlight its potential as a therapeutic target for PRV intervention. IMPORTANCE: subfamily, not only poses an ongoing threat to the global swine industry but also presents a potential zoonotic risk. Although the host protein NMHC IIA has been identified as a critical factor mediating virus entry, its precise molecular mechanism during PRV infection is still unclear. Herein, we demonstrated that NMHC IIA participates in PRV internalization through binding to viral glycoproteins gH and gL. Moreover, we precisely mapped the critical functional region, spanning amino acids 1831-1882 in the C-terminus of NMHC IIA, responsible for both the viral entry process and the interaction with viral proteins. These findings could deepen the understanding of the interaction mechanism between PRV and the host, and provide a promising broad-spectrum antiviral target.