Delta-9 tetrahydrocannabinol (THC) inhibits lytic replication of gamma oncogenic herpesviruses in vitro

Abstract

Background: The major psychoactive cannabinoid compound of marijuana, delta-9 tetrahydrocannabinol (THC), has been shown to modulate immune responses and lymphocyte function. After primary infection the viral DNA genome of gamma herpesviruses persists in lymphoid cell nuclei in a latent episomal circular form. In response to extracellular signals, the latent virus can be activated, which leads to production of infectious virus progeny. Therefore, we evaluated the potential effects of THC on gamma herpesvirus replication.

Methods: Tissue cultures infected with various gamma herpesviruses were cultured in the presence of increasing concentrations of THC and the amount of viral DNA or infectious virus yield was compared to those of control cultures. The effect of THC on Kaposi's Sarcoma Associated Herpesvirus (KSHV) and Epstein-Barr virus (EBV) replication was measured by the Gardella method and replication of herpesvirus saimiri (HVS) of monkeys, murine gamma herpesvirus 68 (MHV 68), and herpes simplex type 1 (HSV-1) was measured by yield reduction assays. Inhibition of the immediate early ORF 50 gene promoter activity was measured by the dual luciferase method.

Results: Micromolar concentrations of THC inhibit KSHV and EBV reactivation in virus infected/immortalized B cells. THC also strongly inhibits lytic replication of MHV 68 and HVS in vitro. Importantly, concentrations of THC that inhibit virus replication of gamma herpesviruses have no effect on cell growth or HSV-1 replication, indicating selectivity. THC was shown to selectively inhibit the immediate early ORF 50 gene promoter of KSHV and MHV 68.

Conclusions: THC specifically targets viral and/or cellular mechanisms required for replication and possibly shared by these gamma herpesviruses, and the endocannabinoid system is possibly involved in regulating gamma herpesvirus latency and lytic replication. The immediate early gene ORF 50 promoter activity was specifically inhibited by THC. These studies may also provide the foundation for the development of antiviral strategies utilizing non-psychoactive derivatives of THC.

Figure 1

THC Inhibits of KSHV and EBV linear DNA replication. BCBL-1 or P3HR1 cell cultures were grown in standard RPMI/fetal calf serum medium. Cultures were supplemented with either various concentrations of THC dissolved in DMSO (as indicated) or with equivalent volumes of DMSO. After three days, cells were analyzed for latent episomal and lytic linear viral genomes as described [31,32]. Briefly, 10⁶ cells were loaded in wells of a vertical agarose gel then overlaid with a lysis solution containing SDS and pronase, resulting in gentle cell lysis and liberation of cellular and viral DNA. The gel was subjected to electrophoresis. After electrophoresis, Southern blotting was performed and DNA was transferred to nitrocellulose followed by hybridization with radiolabeled overlapping cosmid clone probes of KSHV, representing the entire genome, as described [32] (left panel) or with radiolabeled cloned Bam W fragment of EBV (right panel). Latent episomal DNA migrates much more slowly in these gels than linear replicating DNA [31,32]. Arrows on the autoradiograms indicate the position of both species of DNA.

Figure 2

THC inhibits MHV 68 cytopathic effect in NIH3T12 cells. Photograph of NIH3T12 infected with MHV 68 at a multiplicity of 2. Plates were cultured for 48 h in the presence or absence of various concentrations of THC (0.625–10 μg/ml) or equivalent volumes of DMSO.

Figure 3

THC inhibits MHV 68 virus yield in NIH3T12 cells. NIH3T12 monolayer cultures propagated in 24-well plates were infected and cultured in the presence of various concentrations of THC or equivalent DMSO. Forty-eight hours after infection, control cultures showed complete cytopathic effect and destruction of cells. To liberate cell-associated virus, cultures were subjected to a cycle of freeze-thawing; cells were homogenized and virus was titrated by end point dilution in 96-well plates.

Figure 4

THC is not toxic to NIH3T12 cells. NIH3T12 cells seeded at about 1/4th of confluency were cultured for 48 h in the presence of various concentrations of THC (0.625–10 μg/ml) and photographed.

Figure 5

THC has no significant effect on plaque formation of HSV-1 in NIH3T12 cells. NIH3T12 monolayer cultures propagated in 6-well plates were infected with about 100 infectious units of HSV-1 per plate and cultured in the presence of various concentrations of THC or equivalent DMSO solvent. Photographs were taken four days after infection.

Figure 6

THC has no significant effect on virus yield of HSV-1 in NIH3T12 cells. NIH3T12 monolayer cultures propagated in 24-well plates were infected with HSV-1 at a multiplicity of 2 and cultured in the presence of various concentrations of THC or equivalent DMSO. Twenty-four hours after infection, all cultures showed complete cytopathic effect and destruction of cells. To liberate cell-associated virus, cultures were subjected to a cycle of freeze-thawing; cells were homogenized and virus was titrated.

Figure 7

Effect of receptor antagonists on THC mediated suppression of KSHV reactivation. BCBL-1 cells were cultured in the presence of 1.25 μg/ml THC (lane THC), 1.25 μg/ml THC plus 5× molar excess of SR141716A and SR144528 for 72 h (lane Anti1+2), or in medium containing equivalent amount of solvent (DMSO). Cells were analyzed for episomal control and linear replicating DNA as described in Figure 1.