Equine herpesvirus 1 enters cells by two different pathways, and infection requires the activation of the cellular kinase ROCK1.
Equine herpesvirus type 1 (EHV-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 EHV-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 EHV-1 entry into CHO-K1 cells does not require clathrin or caveolae. We also show that EHV-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 EHV-1 infection. These results show that EHV-1 can enter disparate cell types by at least two distinct mechanisms and that productive infection is dependent upon the activation of ROCK1.
Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, E1246 Biomedical Science Tower, Pittsburgh, PA 15261, USA.
Arthur R
AR
Frampton
Donna B
DB
Stolz
Hiroaki
H
Uchida
William F
WF
Goins
Justus B
JB
Cohen
Joseph C
JC
Glorioso
D000818:Animals; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D004705:Endocytosis; D004789:Enzyme Activation; D006566:Herpesviridae Infections / Q000209:etiology*; D004861:Herpesvirus 1, Equid / Q000472:pathogenicity*; D006736:Horses; D008854:Microscopy, Electron; D010587:Phagocytosis; D011817:Rabbits; D015398:Signal Transduction; D053586:Virus Internalization; D054460:rho-Associated Kinases / Q000378:metabolism*
C515851:ROCK1 protein, human; D054460:rho-Associated Kinases