anti-HSV Activity Research Articles

Therapeutic Approaches Using Host Defence Peptides to Tackle Herpes Virus Infections

One of the most common viral infections in humans is caused by herpes simplex virus (HSV). It can easily be treated with nucleoside analogues (e.g., acyclovir), but resistant strains are on the rise. Naturally occurring antimicrobial peptides have been demonstrated to possess antiviral activity against HSV. New evidence has also indicated that these host defence peptides are able to selectively stimulate the innate immune system to fight of infections. This review will focus on the anti-HSV activity of such peptides (both natural and synthetic), describe their mode of action and their clinical potential.

Synthesis, anti-virus and anti-tumour activities of dibenzylbutyrolactone lignans and their analogues

An efficient synthesis of dibenzylbutyrolactone lignans and their analogues has been developed. Based on a Stobbe condensation of piperonal or veratraldehyde with diethylsuccinate and alkylation with 3,4-methylenedioxybenzyl bromide to give the skeleton of the lignan. The (±)-diacid was resolved with quinine and the functional groups were transformed to obtain three benzylbutyrolactone lignans and seven analogues. Four natural lignans were prepared by this method, and five lignans were synthesised for the first time. The synthesised compounds were evaluated for anti-HIV, anti-HSV, and anti-tumour activities. Results showed that the dibenzylbutyrolactone lignans and their analogues were inactive against HIV Tat transactivation and HSV-1 in vitro, but some compounds displayed significant activity against MDA-MB-435 human breast cancer cell.

Medicinal plants of Tamil Nadu (Southern India) are a rich source of antiviral activities

In order to evaluate the potential of medicinal plants of Tamil Nadu as sources of antiviral activities, we used seven different viruses to evaluate the methanol extracts of 30 plants, derived from 22 families and recognized for their local medical applications. Antiviral activity was the minimum concentration of extracts required to completely inhibit viral cytopathic effects (CPE), i.e., MIC100 values. Many extracts showed strong activities against Herpes simplex virus (HSV) and mouse corona virus (MCV, the surrogate for human SARS virus). Some extracts were also active against influenza virus and Sindbis virus (SINV, surrogate for hepatitis C virus), but fewer were active against the non-membrane viruses feline calicivirus (FCV, the surrogate for Norovirus), rhinovirus (common cold virus), and poliovirus. The most potent extracts (low MIC100 and broad spectrum of activity) were obtained from Gymnema sylvestre R. Br. (Asclepiadaceae), Pergularia daemia (Forsskal) Chiov. (Asclepiadaceae), Sphaeranthus indicus L. (Asteraceae), Cassia alata L. (Caesalpiniaceae), Evolvulus alsinoides L. (Convolvulaceae), Clitoria ternatea L. (Fabaceae), Indigofera tinctoria L. (Euphorbiaceae), Abutilon indicum G. Don. (Malvaceae), Vitex trifolia L. (Verbenaceae), Clerodendrum inerme (L.) Gaertn (Verbenaceae), and Leucas aspera Spr. (Lamiaceae), which showed anti-MCV and anti-HSV activities at a concentration as low as 0.4 μg/mL. In some cases the activities were enhanced by light, suggesting the presence of photosensitizers. Some of these antiviral activities could contribute to the medicinal properties of the plants, and also provide more support for the concept of scientific validation of traditional plant medicines in the fight against infectious diseases.

Broad Spectrum Antiviral Fractions from the Lichen Ramalina farinacea (L.) Ach.

Background: An ethylacetate-soluble fraction (ET4) from the lichen Ramalina farinacea has previously been shown to inhibit the infectivity of lentiviral and adenoviral vectors, as well as wild-type HIV-1. We now determined the antiviral activity of ET4 against other wild-type viruses, including the herpes simplex virus type 1 (HSV-1) and the respiratory syncytial virus (RSV). Methods: Wild-type HIV-1, HSV-1 or RSV were pre-incubated with various concentrations of ET4 for 30 min at 37°C before adding to P4CCR5 indicator cell line (HIV-1), ELVIS TM indicator cell line (HSV-1) or HEp2 cell line (RSV) in 96-well microtitre plates. Controls contain virus alone without ET4. The anti-HIV and anti-HSV activities were quantified by estimating beta-galactosidase expression of the respective indicator cell lines while the anti-RSV activity was determined via an immunofluorescent technique, employing monoclonal mouse antibody against the P-protein of RSV. Toxicity of ET4 to cell lines was evaluated in parallel using either the BrdU incorporation method or the MTT method. The effect of ET4 on the enzymatic activity of HIV-1 reverse transcriptase was also evaluated using a chemiluminescent reverse transcriptase assay. Bioassay-guided fractionation of the whole methanol extract of R. farinacea involved sequential screening of HPLC fractions using a vector-based assay technique. Results:ET4 inhibited HSV-1 and RSV potently (IC₅₀ = 6.09 and 3.65 μg/ml, respectively). Time-of-addition studies suggest that both entry and post-entry steps of the HIV-1 replication cycle and the entry step of the RSV replication cycle are targeted. Furthermore, ET4 inhibited HIV-1 reverse transcriptase with an IC₅₀ of 0.022μg/ml. Bioassay-guided fractionation of ET4 led to the identification sub-fraction rfO, with activity against lentiviral vector and HIV-1 (RNA viruses) but not against HSV-1 (DNA virus) and sub-fraction rfM, with activity against HSV-1 but not against the lentiviral vector. Conclusions:ET4 represents a novel fraction from the lichen R. farinacea with broad spectrum antiviral activity against DNA viruses (adenovirus and HSV-1) and RNA viruses (HIV-1 and RSV). The effect against DNA and RNA viruses is mediated by different sub-fractions within R. farinacea.

Human lactoferrin but not lysozyme neutralizes HSV-1 and inhibits HSV-1 replication and cell-to-cell spread

The frequent oral shedding of herpes simplex virus type 1 (HSV-1) in the absence of clinical disease suggests that symptomatic HSV-1 recurrences may be inhibited by the mucosal environment. Indeed, saliva has been shown to contain substances with anti-HSV activity. In the current study, we investigated the anti-HSV-1 activity of human lactoferrin (hLf) and lysozyme (hLz), two highly cationic polypeptides of the mucosal innate defence system.HLf blocked HSV-1 infection at multiple steps of the viral replication cycle, whereas lysozyme displayed no anti-HSV-1 activity. Preincubation of HSV-1 virions and presence of hLf during or after viral absorption period or for the entire HSV-1 infection cycle inhibited HSV-1 infection by reducing both the plaque count and plaque size in a dose- and virus strain-dependent manner. Cell-to-cell spread of wild-type HSV-1 and the strain gC-39, deleted of glycoprotein C, was dramatically reduced, but the cell-to-cell spread of HSV-1 Rid1, harboring a mutated gD and thus unable to react with the cellular HVEM receptor, remained unchanged. This suggests that the inhibition of cell-to-cell spread is mediated by effects on gD or its cellular counterparts.Our results show that the cationic nature is not a major determinant in the anti-HSV action of mucosal innate cationic polypeptides, since whereas hLf inhibited HSV-1 infection efficiently, hLz had no HSV-1 inhibiting activity. Our results show that in addition to inhibiting the adsorption and post-attachment events of HSV-1 infection, hLf is also able to neutralize HSV-1 and that the inhibition of cell-to-cell spread involves viral gD. These results suggest that Lf may have a significant role in the modulation of HSV-1 infection in the oral cavity as well as in the genital mucosa, the major sites of HSV-1 infection.

Isolation of Acridone Alkaloids and N-[(4-Monoterpenyloxy)phenylethyl]-Substituted Sulfur-Containing Propanamide Derivatives from Glycosmis parva and Their Anti-herpes Simplex Virus Activity

Six acridone alkaloids including a new glycosparvarine (1), three limonoids, and four N-[(4-monoterpenyloxy)phenylethyl]-substituted sulfur-containing propanamide derivatives including two new species, (+)-S-deoxydihydroglyparvin (10) and (+)-S-deoxytetrahydroglyparvin (11), were isolated from the branches and the leaves of Glycosmis parva CRAIB collected in the east of Thailand. Antiviral activity evaluation of isolates against herpes simplex virus (HSV) type 1 and 2 disclosed that two acridone alkaloids, glycosparvarine (1) and glycofolinine (4), showed moderate inhibitory activities with 50% effective concentration (EC50) of 348 microM and 151 microM, respectively; as well, (+)-S-deoxydihydroglyparvin (10) exhibited anti-HSV activity at the lower concentration.

Antiviral activities of extracts from Hong Kong seaweeds

We extracted six Hong Kong brown seaweed species with hot water for their antiviral properties. The cytotoxicity and antiviral activity of these extracts were tested by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenlytetrezolium bromide] method, cytopathic effect reduction assay, and plaque reduction assay. The antiviral effect was further determined by flow cytometric analysis. The results showed that most of these extracts inhibited the propagation of herpes simplex virus types 1 and 2 (HSV-1 and HSV-2) standard strains with very low cytotoxicity to the host cells. The extracts of Hydroclathrus clathratus and Lobophora variegata showed more potential anti-HSV activities than the extracts of the other four seaweeds. They also had moderate anti- respiratory syncytial virus (RSV) activities but could not inhibit influenza A virus. Hydroclathrus clathratus was further extracted by diluted acid and alkali and the antiviral effects of the extracts were also detected. The result showed that the hot water extract contained the main carbohydrate components that exhibited the antiviral activities against various strains of HSV, including the acyclovir-resistant strain. HI-3, a compound fractionated from this hot water extract, showed a dose-dependent anti-HSV activity in flow cytometric analysis and plaque reduction assay.

Polysaccharides from Gracilaria corticata: Sulfation, chemical characterization and anti-HSV activities

In this study, we have analyzed water-extracted polysaccharides of Gracilaria corticata. The water extract (WE), a galactan-containing sub-fraction (F3) and their hyper sulfated derivatives (WES1, WES2, F3S1 and F3S2) had anti-HSV activity with inhibitory concentration 50% (IC 50) from 1.1 to 27.4 μg/ml. Sub-fraction F3, which has a molecular mass of 30 kDa, consists of a backbone of β-(1 → 3) and α-(1 → 4)-linked-galactopyranosyl residues. This linear galactan contained Gal 2Xyl 1, Gal 2AnGal 2, Gal 4 and Me-Gal 3AnGal 2 as oligomeric building subunits. Sulfate group was located at C-4 of (1 → 3)-linked galactopyranosyl residues of the native galactan, and appeared to be very important for the anti-herpetic activity.

Marchantiophyta and inedible mushrooms: marvelous sources of bioactive molecules

The biological activity ascribed to the Marchantiophyta is due mainly to sesqui- and diterpenoids and phenolic compounds. Novel terpenoids, phenolics and acetogenins showing characteristic scent, hot-taste, allergenic contact dermatitis, cytotoxicity, insect antifeedant, anti-HIV, superoxide radical release inhibitory, antimicrobial, antifungal, muscle relaxing, apoptosis inducing activity against HL-60, NO production inhibitory activity, liver X receptors agonist/antagonist activity have been isolated from the Marchantiophyta (Blasiaceae, Frullaniaceae, Jungermanniaceae, Lejeuneaceae, Marchantiaceae, Pelliaceae, Plagiochialceae, Porellaceae and Aytoniaceae) species. The chemical constituents of 24 inedible mushroom species belonging to five families, Scutigeraceae, Polypolaceae, Xylariaceae, Thelephoraceae and Paxillaceae were studied. Various type of chemical substances including p-terphenyls, cytochalacines, pregnanes, azaphilones, binaphthyls, lanostanes, 3(R)-hydroxybutyric acid, its dimmer and trimer which showed a broad activity of in many biological systems, such as antimicrobial, antinematicidal, NO production and TNF-α release inhibitory activity, anti-HIV, anti-HSV and antioxidant activities were isolated and their structures characterized.

Anti-herpetic activity of a sulfated xylomannan from Scinaia hatei

Many viruses display affinity for cell surface heparan sulfate proteoglycans with biological relevance in virus entry. This raises the possibility of the application of sulfated polysaccharides in antiviral therapy. In this study we have analyzed polysaccharide fractions isolated from Scinaia hatei. The crude water extract (ShWE) as well as one fraction (F1) obtained by size exclusion chromatography had potent anti-HSV activity. Their inhibitory concentration 50% (IC50) values ranging from 0.5 to 4.6μg/ml were much lower than the cytotoxic concentration 50% (CC50) values (⩾1000μg/ml). These fractions had very low anticoagulant activity. Furthermore, they had a weak inactivating effect on virions in a virucidal assay at concentrations in the range of 60–100μg/ml. Chemical, chromatographic and spectroscopic methods showed that the major polysaccharide, which had 0.4 sulfate group per monomer unit and an apparent molecular mass of 160kDa, contained a backbone of α-(1→3)-linked d-mannopyranosyl residues substituted at C-6, C-4 and C-2 with single stub of β-d-xylopyranosyl residues. Sulfate groups, when present, are located at C-4 of α-(1→3)-linked d-mannopyranosyl units, and appeared to be very important for the anti-herpetic activity of this polymer.

New Advances in Anti-HSV Chemotherapy

Treatment of human herpes simplex virus (HSV) diseases represents an important goal, as herpetic infections are not controlled by vaccination. Many therapeutic agents have been developed and used for HSV infections and several alternative natural compounds are under investigation. Most of the drugs clinically employed against HSV types 1 and 2 are represented by guanosine nucleoside analogues, such as aciclovir and aciclovir-like drugs. The emergence of aciclovir-resistant virus strains provided a stimulus for increased search of new effective agents. Alternative drugs are other nucleoside analogues, such as the vidarabine, brivudin, and cidofovir, or pyrophosphate analogues such as foscarnet, that showed efficacy for HSV infections refractory to aciclovir. However, the risk of adverse effects reported for available anti-herpetic compounds and the frequent development of drug-resistant strains of HSV following therapeutic treatment generate the need for new antiviral agents. In the last years, several studies have been carried out on the anti-HSV activity of different components of innate host defences such as cationic antimicrobial peptides. The antiviral activity of these peptides often appears to be related to the viral adsorption and entry process or is a result of a direct effect on the viral envelope. Other natural compounds, extracts from medicinal plants employed in ethnomedicine and displaying marked anti-herpetic activity, are at present under investigation to determine the scientific evidence and rationale for their clinical use. This review discusses the anti-HSV activity of compounds licensed for clinical use and promising natural molecules.

The in vitro Anti-Herpes Simplex Virus Type-1 and Type-2 Activity of Long Dan Xie Gan Tan, a Prescription of Traditional Chinese Medicine

Background: Long Dan Xie Gan Tan (LDXGT), a decoction of radix gentianae for purging the pathogenic inflammation of the liver, is a widely used prescription among many in traditional Chinese medicine. The prescription is primarily used to treat the disorders induced by damp-heat in the liver and the gall bladder. Methods: In this study, the in vitro anti-herpes simplex virus type-1 (HSV-1) and type-2 (HSV-2) activity of the water extract of LDXGT was investigated. Results: LDXGT water extract was shown to exhibit anti-HSV activity. The IC₅₀ values of LDXGT against HSV-1 and HSV-2 infections were 257.5 ± 12.2 and 494.6 ± 1.8 µg/ml, respectively. It had a CC₅₀ value of 4,077.2 ± 2.4 µg/ml towards Vero cells and showed no cytotoxic effect at a concentration of 2,000 µg/ml or below. The prescription was also found to inactivate HSV-2 infectivity in a dose-, time- and temperature-dependent manner. Conclusions: In summary, the water extract of LDXGT was concluded to inhibit HSV-1 and HSV-2 infection at different magnitudes of potency, and our observations also suggested that the effect was likely mediated by directly inactivating the virus infectivity.

Anti-HSV activity of digitoxin and its possible mechanisms

Herpes simplex virus type 1 (HSV-1) can establish latent infection in the nervous system and usually leads to life-threatening diseases in immunocompromised individuals upon reactivation. Treatment with conventional nucleoside analogue such as acyclovir is effective in most cases, but drug-resistance may arise due to prolonged treatment in immunocompromised individuals. In this study, we identified an in-use medication, digitoxin, which actively inhibited HSV-1 replication with a 50% effective concentration (EC 50) of 0.05 μM. The 50% cytotoxicity concentration (CC 50) of digitoxin is 10.66 μM and the derived selective index is 213. Several structural analogues of digitoxin such as digoxin, ouabain octahydrate and G-strophanthin also showed anti-HSV activity. The inhibitory effects of digitoxin are likely to be introduced at the early stage of HSV-1 replication and the virus release stage. The observation that digitoxin can inhibit acyclovir-resistant viruses further implicates that digitoxin represents a novel drug class with distinct antiviral mechanisms from traditional drugs.

Epigallocatechin Gallate Inactivates Clinical Isolates of Herpes Simplex Virus

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.

New targets for new anti-herpes drugs

Although infections are often subclinical, herpes simplex virus (HSV) can cause mild to severe diseases, especially in immunocompromised patients. There are few drugs licensed for the treatment of HSV infections. Most target the viral DNA polymerase, such as acyclovir that remains the reference treatment some thirty years after its discovery! Extensive clinical use of this drug has led to the emergence of resistant strains, mainly in immunocompromised patients, these infections can be managed with only two drugs, foscarnet and cidofovir, both much more toxic than acyclovir. This highlights the crucial need for the development of new anti-herpes drugs that can inhibit infection by both wild-type viruses and drug-resistant strains. Over the last few years, significant efforts have been made to set up a range of strategies for the identification of potential new antiviral drugs. One alternative is to develop drugs with different mechanisms of action. The present article reviews potential viral and cellular targets that are now known to be involved in HSV multiplication and for which specific inhibitors with anti-HSV activity, at least in cell culture, have been identified. These drugs inhibit viral proteins involved in viral replication (DNA polymerase, ribonucleotide reductase or helicase-primase complex). Other drugs acting on cellular proteins needed for viral replication have also been described; these drugs are targetting cyclin-dependent kinases or the polyamine biosynthetic pathway.

Synthesis and Anti-HSV Activity of new N1-Acyclic C4 and C6-Disubstituted Pyrazolo[3,4-D]Pyrimidine Nucleosides

Several N 1 -(2-hydroxyethoxy)methyl, (4-hydroxybutyl) and (2,3-dihydroxy-1-propoxy)methyl-C 4 ,C 6 -disubstituted-1H-pyrozolo[3,4-d]pyrimidines were synthesized. Some of them were evaluated against herpes simplex virus 1 and 2 replications in E 6 SM cells.

Amphipathic DNA Polymers are Candidate Vaginal Microbicides and Block Herpes Simplex Virus Binding, Entry and Viral Gene Expression

Amphipathic DNA polymers are promising therapies for the prevention of HIV and genital herpes infections. Recent studies on a panel of such compounds indicated potent activity against HIV binding and entry. This current study was conducted to explore the anti-herpes simplex virus (HSV) activity of the same panel of compounds and to determine their mechanism of activity. The anti-HSV activity of a 40-nucleotide degenerate polymer (REP 9), a 40-nucleotide polycytidine amphipathic DNA polymer (REP 9C) and an analogue lacking amphipathic activity (Randomer 3) were compared in plaque reduction assays in the absence or presence of human genital tract secretions; the mechanisms of anti-HSV activity were explored. REP 9 inhibited HSV infection 10,000-fold, whereas Randomer 3 displayed no anti-HSV activity. The antiviral activity was independent of sequence but was dependent on size: the most potent activity was observed for analogues of 40 nucleotides in length. Mechanistic studies indicated that REP 9 and REP 9C blocked HSV-2 binding and entry, were active when added post-entry, inhibited viral gene expression and blocked HSV-induced apoptosis. Confocal microscopy studies showed rapid delivery of fluorescently tagged REP 9 and REP 9C into human epithelial cells, and delivery was significantly greater in infected cells as compared with uninfected cells. REP 9 exhibited no cytotoxicity and retained anti-HSV activity in the presence of cervicovaginal secretions and when virus was introduced in seminal plasma. REP 9 and REP 9C represent a novel class of antiviral agents that act by multiple mechanisms. These compounds warrant further development for systemic or topical delivery for the prevention and treatment of HIV and HSV.

Convergent or Linear? A Challenging Question in Carbocyclic Nucleoside Chemistry

North-methanocarbathymidine (N-MCT) is a carbocyclic nucleoside analogue with potent anti-HSV and KSHV activity. The critical step in the synthesis of N-MCT and other carbocyclic nucleoside analogues is the introduction of the nucleobase into the carbocyclic moiety. For this, convergent and linear strategies were compared side by side. In the convergent approach, the base was coupled to the carbocyclic moiety by either a Mitsunobu reaction or by displacement of a mesylate. This strategy leads to the formation of various amounts of the O 2 -regioisomer, depending on the applied procedure. Additionally, by-products of the Mitsunobu reaction have to be separated from the coupling products. Although lengthier, the linear strategy leads selectively to the target compound N-MCT with overall yields comparable to the convergent approaches, making this strategy more compatible for future large-scale syntheses.

Anti‐herpes simplex virus activities of Eugenia caryophyllus (Spreng.) Bullock & S. G. Harrison and essential oil, eugenol

In this study, an extract from the flower buds of Eugenia caryophyllus (Spreng.) Bullock & S. G. Harrison and the essential oil, eugenol, were evaluated for their anti-herpes simplex virus properties on standard HSV-1(F), standard HSV-2(G) and ten HSV isolates. The plaque reduction assay showed that HSV-1(F), HSV-2(G), two HSV-1 isolates (2, 30) and four HSV-2 isolates (1, 2, 3, 21) were inhibited by E. caryophyllus. Only HSV-1 isolates 1 and 30 were inhibited by eugenol. Thus, strains or isolates of viruses may affect the range of inhibition. Moreover, particles of HSV standard strains were directly inactivated by E. caryophyllus and eugenol. The total virus yield of HSV standard strains and isolates at 30 h also declined after treatment with E. caryophyllus and eugenol. The E. caryophyllus extract exerted higher antiviral replication on HSV-2(G) than on HSV-1(F). The inhibition of the viral yield of HSV-1 isolates was higher than standard HSV-1(F) and standard HSV-2(G) was also inhibited more than most of the HSV-2 isolates. The anti-HSV activity of eugenol against HSV-1(F) and HSV isolates was stronger than with the E. caryophyllus crude extract. However, the percentage inhibition was more pronounced on HSV-1(F) than on HSV-2(G). Moreover, HSV-1(1) and HSV-2(1, 32) could not replicate when eugenol was included in the assay.

Molecular Umbrellas: a Novel Class of Candidate Topical Microbicides To Prevent Human Immunodeficiency Virus and Herpes Simplex Virus Infections

Molecular umbrella compounds may function as novel topical microbicides to prevent human immunodeficiency virus (HIV) and herpes simplex virus (HSV) infections. In a preliminary structure-activity investigation, one umbrella compound, designated Spm8CHAS, was identified which inhibited both HIV and HSV infections with no cellular toxicity. The objectives of the current studies were to define its spectrum of antiviral activity, characterize its mechanism of action, and explore the possibility of combining Spm8CHAS with HIV-specific reverse transcriptase inhibitors. Spm8CHAS inhibited infections by laboratory and clinical R5 and X4 clade B and clade C HIV strains in cell culture. Ectocervical tissue explants exposed to HIV-1(BaL) in the presence of Spm8CHAS were completely protected (50% inhibitory concentration [IC(50)], 13.6 microg/ml), and transfer of virus to target T cells via migratory cells was abolished (IC(50), 3.8 microg/ml). Spm8CHAS inhibited HSV-2 infection of epithelial cells 10,000-fold if present throughout the infection. Notably, adding Spm8CHAS to cultures following HSV entry significantly reduced viral infection, indicating that the drug also acts postentry. Subsequent studies indicated that Spm8CHAS blocks cell-to-cell spread of HSV. Confocal microscopy using a fluorescently labeled analog of Spm8CHAS demonstrated that this conjugate crosses the plasma cell membrane and is transported to the nucleus. Combinations of Spm8CHAS with UC-781 or 9-[R-2-(phosphonylmethoxy)propyl] adenine monohydrate in vitro exhibited additive anti-HIV activity with preserved anti-HSV activity. The abilities of Spm8CHAS to inhibit primary isolates of HIV, block HSV infection postentry, and cross cell membranes support the development of a combination microbicide containing Spm8CHAS with an HIV-specific reverse transcriptase inhibitor to prevent both HIV and HSV infections by multiple mechanisms.