Herpes Simplex Virus Infection of Endothelial Cells Is Enhanced by Thrombin.

Herpes simplex virus type 1 (HSV1) and type 2 (HSV2) are highly prevalent and form life-long infections. While acute effects are not often critical, chronic infection has been linked to vascular disease. To better understand the underlying basis for the clinical correlations, our lab has demonstrated that HSV can initiate and sustain clot formation directly on the virus surface, bypassing the normal cellular requirements. This is facilitated by anionic phospholipid and tissue factor on HSV1 and HSV2, and the participation of at least HSV1- encoded glycoprotein C (gC) in factor VIIa-dependent factor X (FX) activation, emulating tissue factor. The evolution of the HSV surface as a procoagulant agent suggests that generation of thrombin is advantageous to the viral lifecycle. The current study therefore examines the hypothesis that thrombin enhances infection. Several purified HSV1 strains were evaluated, including the wild-type lab strain, MacIntyre, and the clinical isolate, NS. These viruses were compared to gC-deficient HSV1, ns-1, and the gC-restored strain, rns. As an example of HSV2, strain G, was also examined. ns-1 has been shown previously to generate less FXa per particle than the gC-containing viruses. To restrict in situ thrombin production, human umbilical vein endothelial cells were inoculated in serum-free culture media. Consistent with our hypothesis, standard plaque-forming assays revealed that co-incubation of the virus and cells with purified thrombin enhanced infection by up to 400%. The observed half-maximal concentration was approximately 20 nM thrombin for all viruses studied. As a demonstration of specificity, hirudin attenuated the effects of purified thrombin. The involvement of the protease activated receptor 1 (PAR1) in this mechanism was shown using the functional signaling analogue, thrombin receptor activating peptide (TRAP). In the absence of serum, TRAP resulted in increased infection comparable to that of purified thrombin. Approximately half the effect of TRAP was observed at 10uM, while a PAR4-activating peptide had no effect. Enabling the production of thrombin during the infection process, plaque-forming assays were also conducted with serum in the cell culture media. The presence of serum enhanced infectivity by as much as 20-fold. At a constant hirudin concentration of 5 U/ml, infection was attenuated by approximately 50% for the gC-deficient strain, ns-1, whereas the other viruses were only marginally or insignificantly affected, supporting a role for thrombin generation in situ and the participation of gC. Collectively, these data show that thrombin production by HSV enhances infection through a mechanism involving PAR1-mediated cell modulation.