Rho-associated kinase 1 (ROCK1) exerts distinct roles during the early infection versus replication phases of Minute...

Rho-associated kinase 1 (ROCK1) is a host kinase involved in the regulation of cytoskeletal dynamics, vesicle trafficking, and cell cycle control; however, its role in parvoviral infection remains largely unexplored. Building on our previous finding that minute virus of canines (MVC) activates the ROCK1/MLC2 signaling pathway to disrupt tight junction integrity and expose the Occludin coreceptor, we further investigated the role of ROCK1 signaling in MVC entry and replication. We demonstrate that ROCK1 exerts distinct, stage-specific roles during MVC infection. At the early stage of MVC infection, ROCK1 activation triggers the ROCK1/LIMK1/CFL1 pathway and NHE1-mediated intracellular alkalinization, promoting cytoskeletal remodelling and facilitating viral entry. Treatment with a ROCK inhibitor or siRNA-mediated ROCK1 knockdown disrupted cytoskeletal rearrangements, reduced NHE1 expression and CFL1 phosphorylation, and impaired viral internalisation. However, during later stages of infection, selective ROCK1 knockdown enhanced MVC replication and viral protein expression. ROCK1 was found to translocate into the nucleus and colocalize with MVC nonstructural proteins NS1 and NP1. Mechanistically, ROCK1 suppression was associated with altered G1/S regulators, including reduced p53/p21/cyclin D1 and increased cyclin E expression, leading to prolonged S-phase progression. Collectively, these findings indicate that ROCK1 exerts stage functions during MVC infection, promoting viral entry while limiting viral replication through regulation of cell-cycle progression. This dual role underscores the complex interplay between viral infection and host signaling pathways and provides mechanistic insights that may inform the development of host-directed antiviral strategies targeting ROCK1 signaling.