Epigallocatechin gallate inactivates clinical isolates of herpes simplex virus

Abstract

In the absence of a fully effective herpes simplex virus (HSV) vaccine, topical microbicides represent an important strategy for preventing HSV transmission. (-)-Epigallocatechin gallate (EGCG) (molecular weight, 458.4) is the primary catechin in green tea. The present study shows that EGCG has greater anti-HSV activity than other green tea catechins and inactivates multiple clinical isolates of HSV type 1 (HSV-1) and HSV-2. EGCG reduced HSV-2 titers by >or=1,000-fold in 10 to 20 min and reduced HSV-1 titers by the same amount in 30 to 40 min. The anti-HSV activity of EGCG is due to a direct effect on the virion, and incubating Vero and CV1 cells with EGCG for 48 h prior to infection with HSV-1 and HSV-2, respectively, does not reduce HSV production. Electron microscopic (EM) studies showed that purified virions exposed to EGCG were damaged, and EM immunogold labeling of the envelope glycoproteins gB and gD was significantly reduced following EGCG treatment while capsid protein labeling was unchanged. When purified HSV-1 envelope glycoproteins gB and gD were incubated with EGCG and then examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, lower-molecular-weight gB and gD bands decreased and new higher-molecular-weight bands appeared, indicating the EGCG-dependent production of macromolecular complexes. gB and gD are essential for HSV infectivity, and these results suggest that EGCG could inactivate HSV virions by binding to gB, gD, or another envelope glycoprotein. EGCG is stable in the pH range found in the vagina and appears to be a promising candidate for use in a microbicide to reduce HSV transmission.

FIG. 1.

Inactivation of HSV virions by green tea catechins. Each catechin was used at a concentration of 100 μM and incubated with virus from clarified cell supernatant for 1 h at 37°C prior to titration with Vero cells for HSV-1 (strain F1) and CV1 cells for HSV-2 (strain 333). (A) HSV-1. (B) HSV-2. (C) Structures of EGCG, EGC, ECG, GCG, and EC. Titers are the mean ± standard deviation of three separate experiments.

FIG. 2.

EGCG inactivates clinical isolates of HSV-1 (A) and HSV-2 (B). Clinical isolates were prepared as clarified cell supernatants. HSV-1 isolates were grown and their titers were determined in Vero cells, and HSV-2 isolates were grown and their titers were determined in CV-1 cells. Titers are the mean ± standard deviation of three separate experiments. The IC₉₉s of EGCG, i.e., the concentrations that reduced viral titers by 99% from the zero concentration titer, were calculated graphically.

FIG. 3.

Times required for EGCG to inactivate HSV-1 (A) and HSV-2 (B) clinical isolates. Isolates were prepared and titers were determined as described in the legend to Fig. 2. Titers are the mean ± standard deviation of three separate experiments.

FIG. 4.

Treatment of Vero cells (A) and CV-1 cells (B) with EGCG (100 μM). Cells were treated with EGCG for the indicated periods, EGCG was removed, and then Vero cells were infected with HSV-1 (strain F1) and CV-1 cells were infected with HSV-2 (strain 333). HSV production was determined after 2 days as described in Materials and Methods.

FIG. 5.

The presence of EGCG during HSV-1 replication does not inhibit virus replication. Vero cells were infected with HSV-1 (MOI, 10) (strain F1), and 1 h later EGCG (100 μM) was added to the medium of treated cells. Cells were processed as described in Materials and Methods, and the cells were examined by transmission EM after a 24-h incubation. Scale bars on the electron micrographs are 1,160 nm. (A) Control. (B) EGCG treated. TCID₅₀, 50% tissue culture infective dose.

FIG. 6.

EGCG damages HSV-1 virions. (a) Purified virus (strain F1) was incubated in tissue culture medium for 2 h at 37°C and then prepared for EM (see Materials and Methods). (b) Purified virus was incubated with EGCG (100 μM) for 2 h and then prepared for EM. (c) Purified virions were incubated with monoclonal antibody specific for the gD envelope glycoprotein (clone 1103, 67 μg/ml) and labeled with immunogold (see Materials and Methods). (d) Purified virions were incubated with EGCG (100 μM) for 2 h and treated as for panel C. Scale bars are 240 nm. The inserts in the upper right corners of panels a and b show representative virions magnified ×1.6.

FIG. 7.

Envelope glycoproteins gB and gD form macromolecular complexes in the presence of EGCG. EGCG (545 μM) was incubated with recombinant purified gB or gD or with purified BSA for 24 h and then analyzed by SDS-PAGE, followed by Coomassie blue staining. All of the bands in lanes with gB and gD (with or without EGCG) were analyzed by tandem mass spectrometry (Mass Spectrometry Facility, New York State Institute for Basic Research). Arrows indicate the presence of endochitinase either alone in the gD lanes or mixed with gB.