BOVINE HERPESVIRUS 1 ENTRY BY A LOW PH-DEPENDENT, SPHINGOMYELIN-INDEPENDENT, ENDOCYTIC PATHWAY

Gabrielle Pastenkos

Bovine herpesvirus (BoHV-1) is an alphaherpesvirus that poses a significant challenge to health and welfare in the cattle industry. We investigated the cellular entry route utilized by BoHV-1. We report that BoHV-1 enters Madin Darby bovine kidney (MDBK) cells, bovine turbinate cells, and African green monkey kidney (Vero) cells via a low pH-mediated endocytosis pathway that is independent of host cell sphingomyelin. Treatment of MDBK cells with hypertonic medium, which inhibits receptor-mediated endocytosis, prevented infection as measured by a beta-galactosidase reporter assay. Treatment of cells with noncytotoxic concentrations of the lysosomotropic agents ammonium chloride and monensin, which block the acidification of endosomes, inhibited BoHV-1 entry in a concentration-dependent fashion. The kinetics of endocytic uptake of BoHV-1 from the cell surface was rapid (t1/2 of ~ 5 min). Time-of-addition experiments indicated that the lysosomotropic agents acted at early times post-infection, consistent with entry. Inactivation of virions by pretreatment with mildly acidic pH is a hallmark characteristic of viruses that utilize a low pH-activated entry pathway. When BoHV-1 particles were exposed to pH 5.0 in the absence of target membrane, infectivity was markedly reduced. Treatment of MDBK cells with noncytotoxic concentrations of staphylococcus aureus-derived sphingomyelinase successfully depleted cellular surface-exposed sphingomyelin, but did not significantly inhibit BoHV-1 infection as measured by beta-galactosidase reporter assay. Treatment of MDBK cells with noncytotoxic concentrations of the functional inhibitors of acid sphingomyelinase imipramine and amitriptyline, which induce degradation of the cellular enzyme, also did not significantly inhibit BoHV-1 beta-galactosidase production. Depletion of viral envelope sphingomyelin significantly inhibited viral beta-galactosidase production. Together, these results support a model of BoHV-1 infection in which low endosomal pH is a critical host trigger for fusion of the viral envelope with an endocytic membrane, and necessary for successful infection of the target cell, and in which the virus does not require cellular sphingomyelin or acid sphingomyelinase, but does require viral envelope sphingomyelin.

BOVINE HERPESVIRUS 1 ENTRY BY A LOW PH-DEPENDENT, SPHINGOMYELIN-INDEPENDENT, ENDOCYTIC PATHWAY

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