Antiviral activities from hydrolyzable tannins are well documented and are generally thought to target viral adsorption to the host cell membrane (for HSV and HIV), as well as reverse transcriptase activity of HIV (reviewed in references and ). We have previously identified several tannins of various plant sources that exhibit potent antiviral activities against HSV-1 and HSV-2. These include 1,3,4,6-tetra-O-galloyl-β--glucose (), casuarinin (), ent-epiafzelechin-(4α→R8)-epiafzelechin (), excoecarianin (), geraniin (), hippomanin A (), prodelphinidin B-2 3′-O-gallate (), prodelphinidin B-2 3,3′-di-O-gallate (), pterocarnin A (), and putranjivain A (). Studies from other laboratories have also reported a series of tannins and related compounds capable of inhibiting HSV infections (, , , , ). These earlier reports provide strong precedent for our studies and suggest that the tannins constitute an excellent focus for antiviral discovery, particularly in the field of HSV therapeutics.
Identification of multiple drugs that can act on different phases of the viral life cycle can be particularly useful in managing HSV-1 infection or reactivation in either immunocompromised individuals or cases of ACV resistance. To pursue this goal, we extended our previous studies and concentrated our efforts on four chemically defined hydrolyzable tannins (), including chebulagic acid (CHLA), chebulinic acid (CHLI), punicalagin (PUG), and punicalin (PUN), which are present in T. chebula (, , ). Although an effect against HSV-1 has been previously reported for CHLA, the mechanism of its activity was not elucidated (). In the present study we report that two of the tannins tested, specifically CHLA and PUG, were found to be most effective against HSV-1. Detailed studies into their inhibitory action revealed that both drugs specifically target HSV-1 particles, block virus entry into the cell, inhibit cell-to-cell spread of the virus, and reduce secondary infection from released virions. The antiviral mechanism is attributed to the binding of CHLA and PUG to viral glycoproteins that interact with cell surface GAGs. Their ability to effectively control viral entry and spread, underscore the potential of these two hydrolyzable tannins for treating HSV-1 infection and/or recurrence.
There is currently no cure that completely resolves latent infections caused by alphaherpesviruses. Therefore, the development of small molecules capable of inhibiting infection by reactivated virus represents an attractive therapeutic strategy, particularly in immunocompromised individuals who are often at risk of generating ACV-resistant HSV-1 strains. In a search for such molecules, we report that CHLA and PUG, two hydrolyzable tannins isolated from the fruits of T. chebula, effectively inhibited HSV-1 infection in A549 cells without significantly reducing cell viability. In addition, our results suggest that CHLA and PUG specifically targeted HSV-1 particles by binding to viral glycoproteins that interact with cellular GAGs, rendering the virus incapable of adsorbing, penetrating, and spreading throughout the cell monolayer. These features underscore the potential of tannins as HSV-1 entry inhibitors.
Our data show that entry events, including primary and secondary infection, viral attachment and/or penetration, and cell-to-cell spread are inhibited only when the tannins and HSV-1 glycoproteins are in contact with each other. Pretreatment of host cells with the tannins, followed by washes to remove unadsorbed compounds, had no effects upon HSV-1 replication. This indicated that masking cell surface receptors or entry factors for HSV-1 by the tannins is unlikely. Viral binding assays using ELISA and flow cytometry analyses revealed that the tannins blocked viral attachment to the host cell. While CHLA and PUG could inactivate the HSV-1 particles, we do not believe that a direct lysis effect of the viral membrane is responsible for their effects, since HSV-1 infection of GAG-deficient mutant cell lines was still observed, even in the presence of these compounds (). Given their large molecular weights (CHLA, 954; and PUG, 1,084) and high affinity for proteins and sugars, the two hydrolyzable tannins are thought to bind to HSV-1 glycoproteins on the infectious virions making them inert, impairing glycoprotein function, and preventing successful attachment and entry of the host cell. These tannins could also bind to viral glycoproteins on the infected-cell surface, rendering them unavailable to mediate the cell-to-cell spread of virus.
Taken together, the data indicate that CHLA and PUG function as GAG competitors to inhibit the initial events of HSV-1 infection (adsorption and penetration) and the cell-to-cell spread of virus. The interaction of HSV-1 glycoproteins with cellular GAGs plays a critical role in viral infections, and the hydrolyzable tannins could offer a primary means of defense against HSV-1 infections.
To obtain a more accurate dose-response curve for these two hydrolyzable tannins, A549 cells were infected with HSV-1-GFP (MOI = 1) in the presence of the tannins, and fluorescent signals were quantified. The HSV-1-GFP was susceptible to the antiviral effects of the tannins. Both CHLA and PUG displayed anti-HSV-1 activity in a dose-dependent manner (), and the concentrations of CHLA at 60 μM and PUG at 40 μM, which provided near complete protection against the virus infection at an MOI of 1, were chosen for all subsequent experiments.
Terminalia chebula Retz. (T. chebula), a member of the Combretaceae family, is a traditional medicinal plant that is native to India and Southeast Asian countries. The dried ripened fruit of T. chebula (Fructus Chebulae), often referred to as “myrobalans,” contains antioxidants () and is commonly used as a broad-spectrum medicinal agent for the treatment of dysentery, asthma, cough, sore throat, bloody stools, and diseases of the heart and bladder (). T. chebula is rich in tannins, which are polyphenolic secondary metabolites found in higher plants (, , ). Tannins are characterized by their relatively high molecular mass (500 to 20,000 Da) and the unique ability to form insoluble complexes with proteins, carbohydrates, nucleic acids, or alkaloids (, , ). The hydrolyzable class of tannins possesses structures that generally consist of gallic or ellagic acid esters conjugated to a sugar moiety (, ). These polyphenols have high affinity for proteins and polysaccharides and are thought to be the major bioactive compounds found in the leaves and the fruit of T. chebula.
HSV-1 viral glycoproteins are known to mediate HSV-1 binding, internalization, and cell-to-cell spread. From the preceding data, it appears that the hydrolyzable tannins CHLA and PUG target viral glycoprotein(s), which would explain the need for virus to be present during inhibition and their effect on virus entry and spread. In an attempt to elucidate the underlying mechanism, we checked whether the two tannins interacted with HSV-1 glycoproteins in order to block entry-associated events. Using a virus-free system, we overexpressed HSV-1 glycoproteins that have been shown to mediate cell fusion (occurs during entry and cell-to-cell spread) by transfecting the individual gB, gD, gH, and gL genes into A549 cells, followed by treatment with the tannins. Expression of all four genes induced cell fusion resulting in polykaryocyte formation (>10 nuclei), which is absent after transfection with the empty vector control. The two tannins and heparin each blocked polykaryocyte formation, suggesting that CHLA and PUG interact with HSV-1 glycoproteins to prevent virus attachment, entry, and cell-to-cell spread ().
Herpes simplex virus 1 (HSV-1) is a common human pathogen that causes lifelong latent infection of sensory neurons. Non-nucleoside inhibitors that can limit HSV-1 recurrence are particularly useful in treating immunocompromised individuals or cases of emerging acyclovir-resistant strains of herpesvirus. We report that chebulagic acid (CHLA) and punicalagin (PUG), two hydrolyzable tannins isolated from the dried fruits of Terminalia chebula Retz. (Combretaceae), inhibit HSV-1 entry at noncytotoxic doses in A549 human lung cells. Experiments revealed that both tannins targeted and inactivated HSV-1 viral particles and could prevent binding, penetration, and cell-to-cell spread, as well as secondary infection. The antiviral effect from either of the tannins was not associated with induction of type I interferon-mediated responses, nor was pretreatment of the host cell protective against HSV-1. Their inhibitory activities targeted HSV-1 glycoproteins since both natural compounds were able to block polykaryocyte formation mediated by expression of recombinant viral glycoproteins involved in attachment and membrane fusion. Our results indicated that CHLA and PUG blocked interactions between cell surface glycosaminoglycans and HSV-1 glycoproteins. Furthermore, the antiviral activities from the two tannins were significantly diminished in mutant cell lines unable to produce heparan sulfate and chondroitin sulfate and could be rescued upon reconstitution of heparan sulfate biosynthesis. We suggest that the hydrolyzable tannins CHLA and PUG may be useful as competitors for glycosaminoglycans in the management of HSV-1 infections and that they may help reduce the risk for development of viral drug resistance during therapy with nucleoside analogues.
Several HSV-1 glycoproteins are known to interact with cell surface GAGs. To further explore the virus-host interactions that are being targeted by the tannins, we used a series of cell lines known to possess defects in surface HS and CS synthesis. The relative infectivities of HSV-1 (KOS) are ca. 10% for HS-deficient gro2C cells and 0.5% for HS/CS-deficient sog9 cells compared to parental mouse L cells (). Stable expression of the EXT1 gene in sog9 cells (sog9-EXT1) restores HS biosynthesis and susceptibility to HSV-1 infection (). To evaluate the effects of the drugs in the presence or absence of GAG expression, each cell line was infected with different dilutions of HSV-1 sufficient to achieve 200 PFU/well (MOI = 0.0004) in the presence of the tannins. CHLA and PUG effectively protected the parental mouse L cells and sog9-EXT1 cells from infection, but antiviral effects were diminished in HS-deficient gro2C cells, and almost completely abolished in HS/CS-deficient sog9 cells (). Similar results were obtained in experiments using different MOIs (data not shown). These observations strongly suggest that CHLA and PUG target interactions between HSV-1 glycoproteins and GAGs. CHLA inhibition also appeared to be more sensitive to cell surface deficiency in GAGs compared to that of PUG.