Effect of agents that inhibit endocytosis and membrane trafficking on Herpes Simplex Virus‐ Type 2 infection in WB (rat liver epithelial) cells.

Disruption of gap junctional intercellular communication (GJIC) following Herpes simplex virus‐2 (HSV‐2) infection indicates loss of functional gap junctions from the plasma membrane. Previous studies using indirect immunofluorescence demonstrated that Cx43 was lost from the membrane early after infection. To determine whether the loss of gap junctions was associated with endocytosis or membrane trafficking following infection, we evaluated several agents that block the endocytic pathway or membrane trafficking on infectivity and Cx43 distribution. Monensin and ammonium chloride were used to neutralize the pH‐dependent endocytic pathway and effectively prevented viral infection with a therapeutic index (T.I.) of 1,250 and 6.7, respectively. Using dual‐labeled indirect immunofluorescence, both agents acted to increase intracellular accumulation of Cx43 and decreased the spread of viral protein to adjacent cells. Nocodazole, a drug that causes depolymerization of microtubules, was an effective antiviral agent (T.I > 500), and increased cytoplasmic levels of Cx43. In contrast, cytochalasin D, which disrupts actin filaments, and brefeldin A, which inhibits protein trafficking from the Golgi, had antiviral activity only at cytotoxic doses (T.I. 1.2 and 2.5, respectively). These results demonstrate that agents that prevent acidification of endocytic vesicles, particularly monensin, are effective antiviral agents in WB cells. These agents also cause increased intracellular accumulation of the gap junction protein, Cx43. We speculate that the loss of GJIC in infected cells is due to the concomitant endocytosis of gap junctions with the viral particles and that monensin prevents intracellular degradation of connexin. Supported by NIH AREA to GM, RW, and MK and WCU CAS student grant to RB.