Inhibit Herpes Simplex Research Articles

Small interfering RNA targeting for infected‐cell polypeptide 4 inhibits herpes simplex virus type 1 replication in retinal pigment epithelial cells

Background: This study sought to inhibit herpes simplex virus type 1 replication using small interfering RNA which targeting infected‐cell polypeptide 4 genes to mediate transcription of early and late viral genes in herpes simplex virus type 1 lytic (productive) infection in retina epithelial cells. Methods: After pre‐ or post‐infecting with herpes simplex virus type 1, small interfering RNAs were transfected into retina epithelial cells. The antiviral effects of small interfering RNA were evaluated by Western blot, plaque assays, indirect immunofluorescence and reverse transcription polymerase chain reaction. The viral titre was detected by the 50% tissue culture infective dose method. Results: Small interfering RNA decreased infected‐cell polypeptide 4 expression in retina epithelial cells that were infected with herpes simplex virus type 1 before or after small interfering RNA transfection. Compared with herpes simplex virus type 1 infection alone or transfection with negative control small interfering RNA, the viral titre and the retina epithelial cell cytopathic effect were significantly decreased in retina epithelial cells transfected with infected‐cell polypeptide 4‐targeting small interfering RNA (50 and 100 nM) (P < 0.05). The small interfering RNA effectively silenced herpes simplex virus type 1 infected‐cell polypeptide 4 expression on both mRNA and the protein levels (P < 0.05). The inhibition of infected‐cell polypeptide 4‐targeting small interfering RNA on infected‐cell polypeptide 4 protein expression was also verified by Western blot in herpes simplex virus type 1 infected human cornea epithelial cell, human trabecular meshwork cells and Vero cells. Conclusions: Infected‐cell polypeptide 4‐targeting small interfering RNA can inhibit herpes simplex virus type 1 replication in retina epithelial cells, providing a foundation for development of RNA interference as an antiviral therapy.

Topical tenofovir, a microbicide effective against HIV, inhibits herpes simplex virus-2 replication.

The HIV reverse-transcriptase inhibitor, tenofovir, was recently formulated into a vaginal gel for use as a microbicide. In human trials, a 1% tenofovir gel inhibited HIV sexual transmission by 39% and, surprisingly, herpes simplex virus-2 (HSV-2) transmission by 51%. We demonstrate that the concentration achieved intravaginally with a 1% tenofovir topical gel has direct antiherpetic activity. Tenofovir inhibits the replication of HSV clinical isolates in human embryonic fibroblasts, keratinocytes, and organotypic epithelial 3D rafts, decreases HSV replication in human lymphoid and cervicovaginal tissues ex vivo, and delays HSV-induced lesions and death in topically treated HSV-infected mice. The active tenofovir metabolite inhibits HSV DNA-polymerase and HIV reverse-transcriptase. To exert dual antiviral effects, tenofovir requires topical administration to achieve a drug concentration higher than systemic levels achieved by oral treatment. These findings indicate that a single topical treatment, like tenofovir, can inhibit the transmission of HIV and its copathogens.

Chemotherapeutic Effects of Bioassay-Guided Extracts of the American Cockroach,Periplaneta americana

The organic extract of Periplaneta americana L. (Dictyoptera; Blattidae) has been traditionally used in southwestern China as an alternative medicine against disorders such as hepatitis, trauma, gastric ulcers, burns, and heart disease. The present study describes bioassay-guided purification and chemotherapeutic evaluation of the 60% ethanolic fraction of P americana organic extracts (PAE60). The most effective cytotoxic fraction was determined by way of repeated in vitro screenings against 12 distinct cultured human carcinoma cell lines: Eca 109, BGC823, HO8910, LS174T, CNE, HeLa, K562, PC-3, A549, BEL 7404, HL-60, and KB, followed by in vivo antitumor assays of the lead fraction (PAE60). The complexity of enriched active fraction was qualitatively evaluated using thin layer chromatography. Reconstituted PAE60 was effective at inhibiting HL-60, KB, CNE, and BGC823 cell growth with IC(50) values <20 µg mL-(1). PAE60 reduced tumor growth in S180-bearing immunocompetent mice by 72.62% after 10 days following oral doses of 500 mg kg d-(1) compared with 78.75% inhibition following 40 mg kg d-(1) of cyclophosphamide (CTX). Thymus and spleen indices of S180-bearing mice treated with PAE60 were significantly greater (P < .05) than CTX treatment groups, suggesting potential immunomodulation of antitumor host defenses by PAE60. Antiviral activity was also investigated and PAE60 inhibited herpes simplex type-2 replication (IC(50) = 4.11 ± 0.64 µg mL-(1)) with a selectivity index (CC(50) to IC(50) ratio) of 64.84 in Vero cells but was less effective on type-1 virus (IC(50) of 25.6 ± 3.16 µg mL-(1)). These results support future clinical trials on P. americana as an alternative or complementary medicinal agent.

Inhibition of Herpes Simplex Virus Type 1 and Type 2 Infections by Peptide-Derivatized Dendrimers

In response to the need for new antiviral agents, dendrimer-based molecules have been recognized as having a large number of potential therapeutic applications. They include peptide-derivatized dendrimers, which are hyperbranched synthetic well-defined molecules which consist of a peptidyl branching core and covalently attached surface functional peptides. However, few studies have addressed their applications as direct-acting antiviral agents. Here, we report on the ability of the peptide dendrimer SB105 and its derivative, SB105_A10, to directly inhibit herpes simplex virus 1 (HSV-1) and HSV-2 in vitro replication, with favorable selective indexes discerned for both compounds. An analysis of their mode of action revealed that SB105 and SB105_A10 prevent HSV-1 and HSV-2 attachment to target cells, whereas SB104, a dendrimer with a different amino acid sequence within the functional group and minimal antiviral activity, was ineffective in blocking HSV attachment. Moreover, both SB105 and SB105_A10 retained their ability to inhibit HSV adsorption at pH 3.0 and 4.0 and in the presence of 10% human serum proteins, conditions mimicking the physiological properties of the vagina, a potential therapeutic location for such compounds. The inhibition of HSV adsorption is likely to stem from the ability of SB105_A10 to bind to the glycosaminoglycan moiety of cell surface heparan sulfate proteoglycans, thereby blocking virion attachment to target cells. Finally, when combined with acyclovir in checkerboard experiments SB105_A10 exhibited highly synergistic activity. Taken together, these findings suggest that SB105 and SB105_A10 are promising candidates for the development of novel topical microbicides for the prevention of HSV infections.

In vitro Anti‐Herpes Simplex Virus Activity of 1,2,4,6‐Tetra‐O‐galloyl‐β‐d‐glucose from Phyllanthus emblica L. (Euphorbiaceae)

In this study, 1,2,4,6-tetra-O-galloyl-β-D-glucose (1246TGG), a polyphenolic compound isolated from traditional Chinese medicine Phyllanthus emblica L. (Euphorbiaceae), was found to inhibit herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) infection at different magnitudes of activity in vitro. Further studies revealed that 1246TGG directly inactivated HSV-1 particles, leading to the failure of early infection, including viral attachment and penetration. 1246TGG also suppressed the intracellular growth of HSV-1 within a long period post-infection (from 0 h p.i. to 12 h p.i.), while it might exert an antiviral effect mainly before 3 h p.i. It inhibited HSV-1 E and L gene expressions as well as viral DNA replication but did not affect the RNA synthesis of IE gene in our study. Also, in the presence of 1246TGG, the synthesis of viral protein was reduced. Taken together, it was suggested that 1246TGG might exert anti-HSV activity both by inactivating extracellular viral particles and by inhibiting viral biosynthesis in host cells. These results warrant further studies on the antiviral mechanisms of 1246TGG and suggest that it might be a candidate for HSV therapy.

Excoecarianin, Isolated from Phyllanthus urinaria Linnea, Inhibits Herpes Simplex Virus Type 2 Infection through Inactivation of Viral Particles

Phyllanthus urinaria Linnea (Euphorbiaceae) is one of the traditional medicinal plants widely used by oriental people to treat various diseases. We have previously demonstrated that the acetone extract of P. urinaria inhibits herpes simplex virus type 2 (HSV-2) but not HSV-1 infection. In a continuing effort to clarify the antiviral mechanisms of P. urinaria, we isolated the pure compound excoecarianin from the whole plant of P. urinaria through acetone extraction, and investigated its anti-HSV-1 and HSV-2 activities. Our results indicated that excoecarianin protected Vero cells from HSV-2 but not HSV-1 infection, and its 50% inhibitory concentration (IC50) was 1.4 ± 0.1 μM. The antiviral effective concentration of excoecarianin did not affect the viability or the morphology of Vero cells. Although excoecarianin inhibited HSV-2 infection, the inhibitory effect, however, was most prominent when excoecarianin was concurrently added with the virus. Pretreatment of Vero cells with excoecarianin with removal of the drug prior to infection did not yield any antiviral effects, and the same observation was made for post viral entry treatment. Subsequent studies revealed that excoecarianin inactivated HSV-2 virus particles to prevent viral infection. A synergistic antiviral effect against HSV-2 was also observed when Vero cells were treated with a combination of acyclovir (ACV) and excoecarianin. These results suggested that excoecarianin merits to be further explored as an entry inhibitor against HSV-2 and could potentially be investigated for combinatorial drug treatment with nucleoside analogues such as ACV in therapeutic management of HSV-2 infection.

Inhibition of Anti-HIV MicroRNA Expression: A Mechanism for Opioid-Mediated Enhancement of HIV Infection of Monocytes

Inhibition of Anti-HIV MicroRNA Expression: A Mechanism for Opioid-Mediated Enhancement of HIV Infection of Monocytes

Antiviral drug resistance and helicase–primase inhibitors of herpes simplex virus

A new class of chemical inhibitors has been discovered that interferes with the process of herpesvirus DNA replication. To date, the majority of useful herpesvirus antivirals are nucleoside analogues that block herpesvirus DNA replication by targeting the DNA polymerase. The new helicase-primase inhibitors (HPI) target a different enzyme complex that is also essential for herpesvirus DNA replication. This review will place the HPI in the context of previous work on the nucleoside analogues. Several promising highly potent HPI will be described with a particular focus on the identification of drug-resistance mutations. Several HPI have good pharmacological profiles and are now at the outset of phase II clinical trials. Provided there are no safety issues to stop their progress, this new class of compound will be a major advance in the herpesvirus antiviral field. Furthermore, HPI are likely to have a major impact on the therapy and prevention of herpes simplex virus and varicella zoster in both immunocompetent and immunocompromised patients alone or in combination with current nucleoside analogues. The possibility of acquired drug-resistance to HPI will then become an issue of great practical importance.

A Cationic TAT Peptide Inhibits Herpes Simplex Virus Type 1 Infection of Human Corneal Epithelial Cells

Abstract Purpose: To determine if a peptide, TAT-Cd(0), inhibits Herpes simplex virus type 1 infection of human corneal epithelial cells. TAT-Cd(0) and a control peptide, E(50,51)TAT-Cd(0), were added at various times throughout infection with the lacz-expressing hrR3 virus, and viral replication was measured by β-galactosidase activity. Toxicity was assessed using a dye reduction assay. The CC(50) value for TAT-Cd(0) was ∼100 μM. In assays with peptide present at all times, TAT-Cd(0) was 150-fold more active than E(50,51)TAT-Cd(0) (EC(50) 0.2 vs. 30.0 μM). The EC(50) values of TAT-Cd(0) for entry inhibition, cell protection, virus inactivation, and inhibition of attachment were 0.1, 0.4, 9.5, and 3.0 μM, respectively. TAT-Cd(0) was less effective when added 1 h postinfection (EC(50) = 30.0 μM). TAT-Cd(0) is an effective inhibitor of Herpes simplex virus type 1 infection in human corneal epithelial cells and affects multiple steps before, or very early, in infection. The peptide has potential as an antiviral and further studies are warranted.

Interferon lambda inhibits herpes simplex virus type I infection of human astrocytes and neurons

Herpes simplex virus Type I (HSV-1) is a neurotropic virus that is capable of infecting not only neurons, but also microglia and astrocytes and can establish latent infection in the central nervous system (CNS). We investigated whether IFN lambda (IFN-λ), a newly identified member of IFN family, has the ability to inhibit HSV-1 infection of primary human astrocytes and neurons. Both astrocytes and neurons were found to be highly susceptible to HSV-1 infection. However, upon IFN-λ treatment, HSV-1 replication in both astrocytes and neurons was significantly suppressed, which was evidenced by the reduced expression of HSV-1 DNA and proteins. This IFN-λ-mediated action on HSV-1 could be partially neutralized by antibody to IFN-λ receptor. Investigation of the mechanisms showed that IFN-λ treatment of astrocytes and neurons resulted in the upregulation of endogenous IFN-α/β and several IFN-stimulated genes (ISGs). To block IFN-α/β receptor by a specific antibody could compromise the IFN-λ actions on HSV-1 inhibition and ISG induction. In addition, IFN-λ treatment induced the expression of IFN regulatory factors (IRFs) in astrocytes and neurons. Furthermore, IFN-λ treatment of astrocytes and neurons resulted in the suppression of suppressor of cytokine signaling 1 (SOCS-1), a key negative regulator of IFN pathway. These data suggest that IFN-λ possesses the anti-HSV-1 function by promoting Type I IFN-mediated innate antiviral immune response in the CNS cells.

Herpes Simplex Virus (HSV) Immediate-Early (IE) Promoter-Directed Reporter System for the Screening of Antiherpetics Targeting the Early Stage of HSV Infection

Most of the current antiherpetics target viral DNA polymerase, but with the emergence of drug-resistant viruses, antiherpetics with different targets have become necessary. Inhibition of herpes simplex virus (HSV) replication at the early stages of infection minimizes cytotoxicity and immune suppression induced by HSV infection. In this report, quantitative reporter systems that use recombinant HSV and a stably transfected cell line were developed for the screening of agents targeting the early stages of HSV infection. The reporter genes in both systems were directed by HSV immediate-early (IE) promoters, so considerably less time was required for the quantification of HSV infection than the traditional plaque reduction assay. The results show that both reporter assays were sensitive to antiherpetic screening. Both assays were quantitative, rapid, easy to perform, and highly adaptable for automatic high-throughput screening. Exploiting the flexibility of these 2 assays, modified assays were also proposed for the detailed analysis of antiherpetic mechanisms.

High efficiency preparation of bioactive human α-defensin 6 in Escherichia coli Origami(DE3)pLysS by soluble fusion expression

Human alpha-defensin 6 (HD(6)), a small cysteine-rich cationic peptide specially expressed in epithelial cells of digestive tract, may play a crucial role in mucosal immunity. This is the first report on efficient production of bioactive HD(6) through a gene-engineering approach in Escherichia coli. The recombinant plasmid pET32a-omHD(6) was primarily constructed by inserting a PCR fragment encoding mature HD(6) peptide (mHD(6)) preceded by an enterokinase recognition sequence into the expression vector pET32a(+), in frame with the upstream thioredoxin (TrxA) gene. Under optimized expression conditions, a high percentage (>60%) of soluble TrxA-omHD(6) fusion protein was obtained with a yield of about 1.69 g/l, and the theoretical productivity of recombinant mHD(6) (rmHD(6)) reached 0.38 g/l. A feasible three-step purification strategy involving nickel-sepharose chromatography, enterokinase-cleavage and cation exchange chromatography was developed to purify rmHD(6), followed by characteristic identifications by Western blot, mass spectrometry and sequencing. About 102 mg/l of rmHD(6) with its intact N-terminal amino acid sequence was finally achieved. The in vitro experiments showed that rmHD(6) possesses high potency to inhibit herpes simplex virus-2 infection. This work settles substantial foundation for further functional study of HD(6).

ERRATUM: Female Genital Tract Secretions Inhibit Herpes Simplex Infection: Correlation with Soluble Mucosal Immune Mediators and Impact of Hormonal Contraception

ERRATUM: Female Genital Tract Secretions Inhibit Herpes Simplex Infection: Correlation with Soluble Mucosal Immune Mediators and Impact of Hormonal Contraception

Novel Synthetic, Salt-Resistant Analogs of Human Beta-Defensins 1 and 3 Endowed with Enhanced Antimicrobial Activity

Human beta-defensins (hBDs) are antimicrobial peptides of human innate immunity. The antibacterial activities of hBDs 1, 2, and 4 but not the activity of hBD3 are impaired by high salt levels. We have designed and synthesized seven novel hBD analogs, constituted by different domains of hBD1 (which is constitutively expressed in humans) and of hBD3 (which is induced by microorganisms and inflammatory factors in humans), that would maintain and potentially increase the wild-type antimicrobial activities and be salt resistant. We have compared the antibacterial, antiviral, and chemotactic activities of the analogs with those of hBD1 and hBD3. We show that the hBD1 internal region and the hBD3 C-terminal region are critical for antibacterial activity also at high salt concentrations, whereas deletion of the N-terminal region of hBD3 results in an increase in antibacterial activity. All analogs inhibited herpes simplex virus; antiviral activity was enhanced by the hBD1 internal region and the hBD3 C-terminal region. Wild-type and analog peptides were chemotactic for granulocytes and monocytes, irrespective of the salt concentrations. These new peptides may have therapeutic potential.

ORIGINAL ARTICLE: Female Genital Tract Secretions Inhibit Herpes Simplex Virus Infection: Correlation with Soluble Mucosal Immune Mediators and Impact of Hormonal Contraception

Female genital tract secretions inhibit herpes simplex virus (HSV) infection, however, the intra- and inter-subject variability, contribution of specific mediators, and impact of reproductive hormones have not been defined. of study Cervicovaginal lavage (CVL) (n = 89) obtained from nine cyclers and seven women on hormonal contraception (HC), who completed between three and eight weekly visits, were examined for anti-herpes simplex virus activity and concentrations of mediators. The CVL inhibited HSV infection by a mean value of approximately 57% during the follicular or luteal phase, but only by 36% in hormonal contraceptive users. Human neutrophil peptides 1-3 (HNP1-3) (P = 0.03), IL-8 (P = 0.003), lactoferrin (P = 0.005), lysozyme (P = 0.003), IgA (P = 0.002), and IgG (P = 0.02) correlated with antiviral activity. Intra-subject and inter-subject variability was observed, suggesting that factors other than hormones contribute to innate defense. Endogenous antimicrobial activity may provide a biomarker of healthy mucosal immunity and may be reduced in the setting of HC. However, larger prospective studies are needed.

Inhibition of Herpes Simplex Virus by Polyamines

Herpes simplex virus (HSV) establishes latent infection in humans with periodic reactivation. Acyclovir, valacyclovir and foscarnet are in medical use today against HSV type-1 (HSV-1) and type-2 (HSV-2), inhibiting the DNA synthesis of the viruses. Additional drugs that will affect the growth of these viruses by other mechanisms and also decrease the frequency of appearance of drug-resistant mutants are required. Cationic polysaccharides were synthesized by conjugation of various oligoamines to oxidized polysaccharides by reductive amination. Polycations of dextran, pullulan and arabinogalactan were grafted with oligoamines of 2-4 amino groups forming Schiff-base imine-based conjugates followed by reduction with borohydride to obtain the stable amine-based conjugate. Evaluation of toxicity to BS-C-1 cells and antiviral activity against HSV-1 and HSV-2 of the different compounds was performed in vitro by a semiquantitative assay. A quantitative study with a selected compound followed. Structure-activity relationship studies showed that the nature of the grafted oligoamine of the polycation plays an essential role in the antiviral activity against HSV-1 and HSV-2. Dextran-propan-1,3-diamine (DPD) was found to be the most potent of all the compounds examined. DPD did not decrease the infectivity of HSV upon direct exposure to the virions. The growth of HSV was significantly inhibited when DPD was added to the host cells 1 h prior to infection, thus preventing the adsorption and penetration of the virus into the cells. Our in vitro data warrant clinical investigation. DPD could have an advantage as a topical application in combination therapy of HSV lesions.

Activation of Toll‐like receptors inhibits herpes simplex virus‐1 infection of human neuronal cells

Toll-like receptors (TLRs) play an essential role in initiating intracellular type I interferon (IFN)-mediated innate immunity against viral infections. We examined whether human neuronal cells (primary human neurons, NT2-N and CHP-212 cells) express TLRs and mount type I IFN-mediated innate immunity against herpes simplex virus-1 (HSV-1) infection. Human neuronal cells expressed TLR family members 1-10 and IFN-alpha/beta. The activation of TLR3 or TLR8 by double-stranded RNA (poly-I:C) or single-stranded RNA (ssRNA) induced IFN-alpha/beta expression. In addition, HSV-1 infection of human neuronal cells induced IFN-alpha expression. Investigation of the mechanisms showed that poly-I:C or ssRNA treatment enhanced the expression of several IFN regulatory factors. Importantly, the activation of TLR3 or TLR8 by poly-I:C or ssRNA prior to HSV-1 infection reduced the susceptibility of the neuronal cells to infection. These observations indicate that human neuronal cells possess intracellular TLR-mediated innate immune protection against HSV-1 infection.

Inhibition of Herpes Simplex Virus by Polyamines

Background:Herpes simplex virus (HSV) establishes latent infection in humans with periodic reactivation. Acyclovir, valacyclovir and foscarnet are in medical use today against HSV type-1 (HSV-1) an...

Studies on in vitro proteolytic sensitivity of peptides inhibiting herpes simplex virus ribonucleotide reductases lead to discovery of a stable and potent inhibitor

The nonapeptide, HSV R2-(329-337), corresponding to the subunit 2 (R2) carboxyl terminus of herpes simplex virus (HSV) ribonucleotide reductases, specifically inhibits this enzyme activity. We report here that under standard reductase assay conditions, this peptide was rapidly degraded by proteases present in the partially purified enzyme extract. The main process of proteolysis involves the successive removal of Tyr329 and Ala330, which corresponds to an aminopeptidase activity. Determination of the proteolytic susceptibility of HSV R2-(329-337) analogs showed that natural modifications which are present in the homologous varicella zoster virus (VZV) nonapeptide decreased its susceptibility to protease action 1.5-fold. Nx-acetylation, a modification known to protect peptides against aminopeptidase attacks, greatly improved the proteolytic resistance of HSV and VZV nonapeptides. Moreover, Ac-VZV R2-(298-306) exhibited a 15-fold higher potency on reductase inhibition than HSV R2-(329-337). The degradation process of HSV R2-(329-337) was partially inhibited by amastatin, bestatin, and leupeptin whereas it was completely abolished by bacitracin, suggesting a combined action of more than one aminopeptidase activity. Moreover, bacitracin protected most of these nonapeptide analogs from proteolysis, although it was less effective in preventing HSV R2-(332-337) degradation. Our results indicate that it is possible to determine, in the presence of bacitracin, the relative inhibitory potencies of HSV R2-(329-337) analogs with minimal error due to proteolytic susceptibility. Moreover, HSV R2-(329-337) modifications that were found to protect the peptide against degradation might be useful to increase its efficacy in vivo.

The Antiherpetic Drug Acyclovir Inhibits HIV Replication and Selects the V75I Reverse Transcriptase Multidrug Resistance Mutation

The antiviral drug acyclovir is a guanosine nucleoside analog that potently inhibits herpes simplex virus (HSV) replication. Acyclovir treatment in patients coinfected with HSV and human immunodeficiency virus (HIV) has been observed to alter disease course and decrease HIV viral load, a finding that has been attributed to indirect effects of HSV suppression on HIV replication. Based on this hypothesis, several clinical studies have recently investigated the use of acyclovir for treatment of patients coinfected with HSV and HIV or for prophylaxis against HIV transmission. In this report, we use a single round HIV infectivity assay to show that acyclovir directly inhibits HIV infection with an IC50 of approximately 5 microm. The target of acyclovir in HIV-infected cells is validated as HIV reverse transcriptase (RT) by the emergence of the RT variant V75I under the selective pressure of acyclovir. The V75I mutation is part of the multidrug resistance pathway that enhances viral resistance to many of the best RT inhibitors approved for the treatment of HIV. Biochemical analyses demonstrate that acyclovir triphosphate is a chain terminator substrate for HIV RT and can compete with dGTP for incorporation into DNA. Although acyclovir may prove a useful lead for development of new HIV treatments, the selection of resistant mutants raises a cautionary note to the use of acyclovir monotherapy in patients coinfected with HSV and HIV.