Equine herpesvirus type 1 (EHV-1) utilizes microtubules, dynein, ROCK1, and FAK to productively infect cells
Presentation
Overview
Overview
Description
Recently, we showed that equine herpesvirus type 1 (EHV-1) infection activates the cellular kinase ROCK1 and that this activation is essential for productive infection to occur. In the current study, we expanded upon this finding by investigating the downstream effectors of ROCK1, the timing of ROCK1 activation, the mechanism by which ROCK1 contributes to EHV-1 infection, and the involvement of cytoskeletal components in EHV-1 infection. Using phospho-specific antibodies, we showed that focal adhesion kinase (FAK) is activated as early as 15 minutes post-infection and that this activation is blocked in the presence of the ROCK1 inhibitor Y27632. The inhibition of FAK phosphorylation by Y27632 indicates that FAK is downstream of ROCK1. ROCK1 inhibition by Y27632 also led to a marked reduction in peri-nuclear capsid accumulation as shown by confocal microscopy. These data suggest that ROCK1 activation must occur for EHV-1 to efficiently traffic to the nucleus. The decrease in nuclear-associated EHV-1 capsids in the presence of Y27632 led us to investigate the mechanism by which virus particles travel to the nucleus. Incubation of cells with the microtubule inhibitors, nocadazole, vinblastine, and colchicine significantly reduced EHV-1 infection on both equine dermal (ED) and Chinese hamster ovary (CHO-K1) cells. In addition, infection was also significantly reduced in cells treated with inhibitors of the microtubule motor protein dynein but not kinesin. These data suggest that EHV-1 utilizes dynein and the microtubule network to efficiently traffic intracellularly. Further studies revealed that EHV-1 induces the stabilization of microtubules as early as 15 minutes post-infection. The observed microtubule stabilization was independent of ROCK1 activation as treatment of cells with Y27632 did not reduce this event. The identification of FAK as a ROCK1 substrate that is activated upon EHV-1 infection provides a reliable readout of ROCK1 activation and thus enables the investigation of upstream activators of ROCK1.
