Equine herpesvirus 1 enters cells by two different pathways, and infection requires the activation of the cellular kinase ROCK1

Equine herpesvirus type 1 (ERV-1), a member of the Alphaherpesviridae, displays a broad host range in vitro, allowing for detailed study of the mechanisms of productive infection, including attachment and entry, in various cell culture systems. Previously, we showed that ERV-1 infects Chinese hamster ovary (CHO-K1) cells even though these cells do not express a known alphaherpesvirus entry receptor. In this report, we show by electron microscopy and an infectious recovery assay that entry into CHO-K1 cells occurs via an endocytic or phagocytic mechanism, while entry into equine dermal (ED) or rabbit kidney (RK13) cells occurs by direct fusion at the cell surface. In both cases (endocytic/phagocytic or direct fusion), entry leads to productive infection. Using drugs that inhibit clathrin-dependent or caveola-dependent endocytosis, we showed that ERV-1 entry into CHO-K1 cells does not require clathrin or caveolae. We also show that ERV-1 infection requires the activation of cell signaling molecules. In particular, we demonstrate that activation of the serine/threonine Rho kinase ROCK1 is critical for infection. Inhibition of this kinase by drugs or overexpression of a negative regulator of ROCK1 significantly blocked ERV-1 infection. These results show that ERV-1 can enter disparate cell types by at least two distinct mechanisms and that productive infection is dependent upon the activation of ROCK1.