Canid herpesvirus 1 (CHV-1) is a Varicellovirus that causes self-limiting infections in adult dogs but morbidity and mortality in puppies. Using a multipronged approach, we discovered the CHV-1 entry pathway into Madin-Darby canine kidney (MDCK) epithelial cells. We found that CHV-1 triggered extensive host cell membrane lamellipodial ruffling and rapid internalization of virions in large, uncoated vacuoles, suggestive of macropinocytosis. Treatment with inhibitors targeting key macropinocytosis factors, including inhibitors of Na+/H+ exchangers, F-actin, myosin light-chain kinase, protein kinase C, p21-activated kinase, phosphatidylinositol-3-kinase, and focal adhesion kinase, significantly reduced viral replication. Moreover, the effect was restricted to exposure to the inhibitors early in infection, confirming a role for the macropinocytic machinery during entry. The profile of inhibitors also suggested a role for signaling via integrins and receptor tyrosine kinases in viral entry. In contrast, inhibitors of clathrin, caveolin, microtubules, and endosomal acidification did not affect CHV-1 entry into MDCK cells. We found that the virus colocalized with the fluid phase uptake marker dextran; however, surprisingly, CHV-1 infection did not enhance the uptake of dextran. Thus, our results indicate that CHV-1 uses a macropinocytosis-like, pH-independent entry pathway into MDCK cells, which nevertheless is not based on stimulation of fluid uptake.

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