Herpes Simplex Virus Replication Research Articles

Roles of p53 in Herpes Simplex Virus 1 Replication

p53 is a critical factor in the cellular response to a broad range of stress factors through its ability to regulate various cellular pathways. In this study, tandem affinity purification of transiently expressed herpes simplex virus 1 (HSV-1) regulatory protein ICP22 coupled with mass spectrometry-based proteomics technology and subsequent analyses showed that ICP22 interacted with p53 in HSV-1-infected cells. In p53(-/-) cells, replication of wild-type HSV-1 was reduced compared to that in parental p53(+/+) cells, indicating that p53 had a positive effect on HSV-1 replication. In contrast, the levels of viral replication of an ICP22-null mutant virus were similar in both p53(-/-) and p53(+/+) cells. At 2 h postinfection, the level of expression of ICP27, an essential viral regulatory protein, in p53(-/-) cells infected with wild-type HSV-1 or the ICP22-null mutant virus was lower than in p53(+/+) cells. In contrast, at 18 h postinfection, the level of expression of ICP0, a critical viral regulatory protein, in p53(-/-) cells infected with the ICP22-null mutant virus was higher than in p53(+/+) cells, although the levels of ICP0 expression in p53(-/-) and p53(+/+) cells infected with wild-type HSV-1 were almost identical. These results suggested that p53 overall promoted HSV-1 replication and that p53 played both positive and negative roles in HSV-1 replication: upregulating ICP27 expression very early in infection and downregulating ICP0 expression later in infection, which was antagonized by ICP22.

Role of Immediate Early Protein ICP27 in the Differential Sensitivity of Herpes Simplex Viruses 1 and 2 to Leptomycin B

Leptomycin B (LMB) is a highly specific inhibitor of CRM1, a cellular karyopherin-β that transports nuclear export signal-containing proteins from the nucleus to the cytoplasm. Previous work has shown that LMB blocks herpes simplex virus 1 (HSV-1) replication in Vero cells and that certain mutations in viral immediate early protein ICP27 can confer LMB resistance. However, little is known of the molecular mechanisms involved. Here we report that HSV-2, a close relative of HSV-1, is naturally resistant to LMB. To see whether the ICP27 gene determines this phenotypic difference, we generated an HSV-1 mutant that expresses the HSV-2 ICP27 instead of the HSV-1 protein. This recombinant was fully sensitive to LMB, indicating that one or more other viral genes must be important in determining HSV-2's LMB-resistant phenotype. In additional work, we report several findings that shed light on how HSV-1 ICP27 mutations can confer LMB resistance. First, we show that LMB treatment of HSV-1-infected cells leads to suppression of late viral protein synthesis and a block to progeny virion release. Second, we identify a novel type of ICP27 mutation that can confer LMB resistance, that being the addition of a 100-residue amino-terminal affinity purification tag. Third, by studying infections where both LMB-sensitive and LMB-resistant forms of ICP27 are present, we show that HSV-1's sensitivity to LMB is dominant to its resistance. Together, our results suggest a model in which the N-terminal portion of ICP27 mediates a nonessential activity that interferes with HSV-1 replication when CRM1 is inactive. We suggest that LMB resistance mutations weaken or abrogate this activity.

Pyrrolidine Dithiocarbamate Inhibits Herpes Simplex Virus 1 and 2 Replication, and Its Activity May Be Mediated through Dysregulation of the Ubiquitin-Proteasome System

Pyrrolidine dithiocarbamate (PDTC) is widely used as an antioxidant or an NF-κB inhibitor. It has been reported to inhibit the replication of human rhinoviruses, poliovirus, coxsackievirus, and influenza virus. In this paper, we report that PDTC could inhibit the replication of herpes simplex virus 1 and 2 (HSV-1 and HSV-2). PDTC suppressed the expression of HSV-1 and HSV-2 viral immediate early (IE) and late (membrane protein gD) genes and the production of viral progeny. This antiviral property was mediated by the dithiocarbamate moiety of PDTC and required the presence of Zn(2+). Although PDTC could potently block reactive oxygen species (ROS) generation, it was found that this property did not contribute to its anti-HSV activity. PDTC showed no activity in disrupting the mitogen-activated protein kinase (MAPK) pathway activation induced by viral infection that was vital for the virus's propagation. We found that PDTC modulated cellular ubiquitination and, furthermore, influenced HSV-2-induced IκB-α degradation to inhibit NF-κB activation and enhanced PML stability in the nucleus, resulting in the inhibition of viral gene expression. These results suggested that the antiviral activity of PDTC might be mediated by its dysregulation of the cellular ubiquitin-proteasome system (UPS).

Sulfated polysaccharide of Caesalpinia ferrea inhibits herpes simplex virus and poliovirus

Herpes simplex virus (HSV) is one of the most regular human pathogens, being a public health problem, and causal agent of several diseases. Poliovirus (PV) is an enteric virus and about 1% of infected individuals develop paralytic poliomyelitis due to viral invasion of the central nervous system and destruction of motor neurons. This work evaluated the activity of a sulfated polysaccharide of Caesalpinia ferrea (SPLCf) in HSV and PV replication. The antiviral effect of SPLCf at varying concentrations was tested by plaque assay under several protocols, such as time-of-addition, adsorption and penetration inhibition and virucidal. Syntheses of viral protein and nucleic acid were also monitored by the immunofluorescence assay and PCR. The SPLCf inhibited virus adsorption and steps after penetration, and inhibited the synthesis of viral protein. Virucidal effect was also shown and nucleic acid synthesis was concurrent with positive results. Our findings suggested that the substance with low toxicity represent a potential viral inhibitor.

Synthesis and antiviral activities of methylenecyclopropane analogs with 6-alkoxy and 6-alkylthio substitutions that exhibit broad-spectrum antiviral activity against human herpesviruses.

Methylenecyclopropane nucleosides have been reported to be active against many of the human herpesviruses. The most active compound of this class is cyclopropavir (CPV), which exhibits good antiviral activity against human cytomegalovirus (HCMV), Epstein-Barr virus, both variants of human herpesvirus 6, and human herpesvirus 8. CPV has two hydroxymethyl groups on the methylenecyclopropane ring, but analogs with a single hydroxymethyl group, such as the prototypical (S)-synguanol, are also active and exhibit a broader spectrum of antiviral activity that also includes hepatitis B virus and human immunodeficiency virus. Here, a large set of monohydroxymethyl compounds with ether and thioether substituents at the 6 position of the purine was synthesized and evaluated for antiviral activity against a range of human herpesviruses. Some of these analogs had a broader spectrum of antiviral activity than CPV, in that they also inhibited the replication of herpes simplex viruses 1 and 2 and varicella-zoster virus. Interestingly, the antiviral activity of these compounds appeared to be dependent on the activity of the HCMV UL97 kinase but was relatively unaffected by the absence of thymidine kinase activity in HSV. These data taken together indicate that the mechanism of action of these analogs is distinct from that of CPV. They also suggest that they might be useful as broad-spectrum antiherpesvirus agents and may be effective in the treatment of resistant virus infections.

Antiviral Activity of Trappin-2 and Elafin In Vitro and In Vivo against Genital Herpes

Serine protease inhibitor elafin (E) and its precursor, trappin-2 (Tr), have been associated with mucosal resistance to HIV-1 infection. We recently showed that Tr/E are among principal anti-HIV-1 molecules in cervicovaginal lavage (CVL) fluid, that E is ∼130 times more potent than Tr against HIV-1, and that Tr/E inhibited HIV-1 attachment and transcytosis across human genital epithelial cells (ECs). Since herpes simplex virus 2 (HSV-2) is a major sexually transmitted infection and risk factor for HIV-1 infection and transmission, we assessed Tr/E contribution to defense against HSV-2. Our in vitro studies demonstrated that pretreatment of endometrial (HEC-1A) and endocervical (End1/E6E7) ECs with human Tr-expressing adenovirus (Ad/Tr) or recombinant Tr/E proteins before or after HSV-2 infection resulted in significantly reduced virus titers compared to those of controls. Interestingly, E was ∼7 times more potent against HSV-2 infection than Tr. Conversely, knockdown of endogenous Tr/E by small interfering RNA (siRNA) significantly increased HSV-2 replication in genital ECs. Recombinant Tr and E reduced viral attachment to genital ECs by acting indirectly on cells. Further, lower viral replication was associated with reduced secretion of proinflammatory interleukin 8 (IL-8) and tumor necrosis factor alpha (TNF-α) and decreased NF-κB nuclear translocation. Additionally, protected Ad/Tr-treated ECs demonstrated enhanced interferon regulatory factor 3 (IRF3) nuclear translocation and increased antiviral IFN-β in response to HSV-2. Lastly, in vivo studies of intravaginal HSV-2 infection in Tr-transgenic mice (Etg) showed that despite similar virus replication in the genital tract, Etg mice had reduced viral load and TNF-α in the central nervous system compared to controls. Collectively, this is the first experimental evidence highlighting anti-HSV-2 activity of Tr/E in female genital mucosa.

VP16‐HCF interaction is required for herpes simplex virus exit from latency in neurons in vivo

A mechanistic understanding of viral and host functions regulating herpes simplex virus (HSV) latency and reactivation remains a major goal. We have shown that the interaction of VP16 and Oct‐1 is required very early during entry into the lytic cycle from the latent state and have proposed a VP16‐dependent model of reactivation in vivo. HCF‐1 has important roles in promoting HSV replication and a model has been proposed in which stress induces translocation of HCF‐1 into the neuronal nucleus and reactivation is regulated by direct recruitment of HCF to viral IE genes. To investigate whether HCF‐1 can precipitate viral gene expression in latently infected neurons in the absence of binding to VP16, a mutation in VP16 at Y364A, shown previously to disrupt VP16‐ HCF interaction (but not interaction with Oct‐1) was engineered into HSV‐1. Three independent isolates and a genomically restored virus were examined in vitro and in the mouse ocular model. Our findings demonstrate that the interaction of VP16 with HCF‐1 is required for efficient infection at low moi, efficient viral replication in vivo, and exit from latency/entry into the lytic cycle in trigeminal ganglion neurons in mice following hyperthermic stress. In the absence of VP16 binding, nuclear translocation of HCF is not sufficient to initiate the viral lytic cycle, demonstrating the key role of VP16 in regulating in vivo reactivation.

A microRNA encoded by HSV-1 inhibits a cellular transcriptional repressor of viral immediate early and early genes

Viral microRNAs are one component of the RNA interference phenomenon generated during viral infection. They were first identified in the Herpesviridae family, where they were found to regulate viral mRNA translation. In addition, prior work has suggested that Kaposi's sarcoma-associated herpesvirus (KSHV) is capable of regulating cellular gene transcription by miRNA. We demonstrate that a miRNA, hsv1-mir-H27, encoded within the genome of herpes simplex virus 1 (HSV-1), targets the mRNA of the cellular transcriptional repressor Kelch-like 24 (KLHL24) that inhibits transcriptional efficiency of viral immediate-early and early genes. The viral miRNA is able to block the expression of KLHL24 in cells infected by HSV-1. Our discovery reveals an effective viral strategy for evading host cell defenses and supporting the efficient replication and proliferation of HSV-1.

Concurrent chemotherapy inhibits herpes simplex virus-1 replication and oncolysis

Herpes simplex virus-1 (HSV-1) replication in cancer cells leads to their destruction (viral oncolysis) and has been under investigation as an experimental cancer therapy in clinical trials as single agents, and as combinations with chemotherapy. Cellular responses to chemotherapy modulate viral replication, but these interactions are poorly understood. To investigate the effect of chemotherapy on HSV-1 oncolysis, viral replication in cells exposed to 5-fluorouracil (5-FU), irinotecan (CPT-11), methotrexate (MTX) or a cytokine (tumor necrosis factor-α (TNF-α)) was examined. Exposure of colon and pancreatic cancer cells to 5-FU, CPT-11 or MTX in vitro significantly antagonizes both HSV-1 replication and lytic oncolysis. Nuclear factor-κB (NF-κB) activation is required for efficient viral replication, and experimental inhibition of this response with an IκBα dominant-negative repressor significantly antagonizes HSV-1 replication. Nonetheless, cells exposed to 5-FU, CPT-11, TNF-α or HSV-1 activate NF-κB. Cells exposed to MTX do not activate NF-κB, suggesting a possible role for NF-κB inhibition in the decreased viral replication observed following exposure to MTX. The role of eukaryotic initiation factor 2α (eIF-2α) dephosphorylation was examined; HSV-1-mediated eIF-2α dephosphorylation proceeds normally in HT29 cells exposed to 5-FU, CPT-11 or MTX. This report demonstrates that cellular responses to chemotherapeutic agents provide an unfavorable environment for HSV-1-mediated oncolysis, and these observations are relevant to the design of both preclinical and clinical studies of HSV-1 oncolysis.

DNA sensing-independent inhibition of herpes simplex virus 1 replication by DAI/ZBP1.

DNA-dependent activator of interferon regulatory factor (DAI) acts as a cytosolic B-form DNA sensor that induces type I interferons. However, DAI is not required for DNA sensing in certain cell types due to redundancy of the DNA sensing system. Here, we investigated the effect of DAI on herpes simplex virus 1 (HSV-1) infection in HepG2 hepatocellular carcinoma cells. DAI transcription was induced after gamma interferon (IFN-γ) treatment or HSV-1 infection. HSV-1 replication was enhanced by DAI knockdown, and ectopic DAI expression repressed viral replication in a manner requiring the Zβ and D3 domains, but not the Zα domain. This activity of DAI was more prominent at low multiplicity of infection (MOI) and correlated with the reduced expression of viral immediate-early genes. Consistently, DAI repressed the activation of ICP0 promoter in reporter gene assays. DAI knockdown did not affect the B-DNA-mediated IFN-β transcription and IRF3 activation, and overexpression of DAI and RIP1 did not enhance NF-κB activation by B-DNA treatment, demonstrating that DAI is not essential for the B-DNA-mediated IFN production in HepG2 cells. DAI colocalized with ICP0 in a subset of nuclear and cytoplasmic foci in infected cells and interacted with ICP0 in coimmunoprecipitation assays. The anti-HSV-1 effect of DAI was not observed in ICP0-deleted mutant virus infection at a high MOI in HepG2 cells and mouse embryonic fibroblasts. Degradation of IFI16 and PML by ICP0 was enhanced in infection of DAI-knockdown cells. Collectively, these results demonstrate that DAI can suppress HSV-1 growth independent of DNA sensing through mechanisms involving suppression of viral genomes and regulation of ICP0.

Mucosal HSV-2 Specific CD8+ T-Cells Represent Containment of Prior Viral Shedding Rather than a Correlate of Future Protection

It is largely unknown why certain infected hosts shed Herpes Simplex Virus-2 (HSV-2) more frequently and have more severe disease manifestations than others. One idea is that different density or functional capacity of tissue resident effector memory CD8+ T-cells between infected persons may explain phenotypic variability. To generate hypotheses for contrasting shedding patterns in different infected hosts, a spatial mathematical model was employed to evaluate the effects of variability in tissue resident effector memory CD8+ T-cell response, and HSV-2 replication and spread, on viral shedding rate. Model simulations suggest that high levels of CD8+ T-cells in the mucosa do not necessarily indicate a protective phenotype but rather an effective response to recent shedding. Moreover, higher CD8+ T-cell expansion rate and lower viral replication rate, which correlate with better short-term control, may have only a minor impact on long-term shedding rates. Breakthrough shedding occurs under all sets of model parameter assumptions, because CD8+ T-cell levels only surpass a protective threshold in a minority of genital tract mucosal micro-regions. If CD8+ T-cell levels are artificially increased using an immunotherapeutic approach, better control of shedding is predicted to occur for at least a year. These results highlight the complex co-dependent relationship between HSV-2 and tissue resident CD8+ lymphocytes during the course of natural infection.

A Cohort Historical Analysis of the Relationship between Thyroid Hormone Malady and Alpha-Human Herpesvirus Activation.

BackgroundA number of physiological factors have been suggested to participate in the alpha- Human Herpesvirus (αHHV) reactivation, such as hormonal aberration. Thyroid hormone (TH) was shown to play a suppressive role in Herpes Simplex Virus Type-1 (HSV-1) gene expression and replication in cell culture and animal models. We hypothesize that reactivation of αHHV in humans may be due to, at least in part, by TH status.MethodsPrior to implementing a full-scale population-based prospective inquiry into this hypothesis, a pilot study using a medical claims data base and a case-controlled, retrospective cohort investigation was conducted to develop a hypothetical link between TH complication and αHHV reactivation. Using diagnostic codes for treating thyroid disorders and αHHV infections as proxies for biologic/clinic outcomes, we queried a large, comprehensive hospital data base to construct two patient cohorts: Cohort 1 was comprised of patients receiving TH diagnoses over a twelve-year period, and Cohort 2 was composed of patients not receiving TH diagnoses during this period. Diagnoses of αHHV were recorded for each cohort and the difference in the frequency was examined for statistical significance. Demographic analyses such as age, gender, etc were also performed.ResultsUsing 2×2 contingency table analyses and Statistical Analysis Software (SAS), an Odds Ratio (OR) of 2.83 was observed for the total population of 21 years old and above with a chi-square of 61.55 and p < 0.001, confirming that a severe significant difference was found between these two cohorts. This result suggested that patients with αHHV diagnosis have higher chances to have TH disorders. Additional investigation revealed that female were at higher/significant probability to have both TH and αHHV diagnosis, indicating a link of αHHV reactivation to a complex hormonal profile difference between genders. Our observation indicated that female patients of 21 years of age and above exhibited a very high incidence (OR of 3.40, p < 0.001) compared to the male groups (OR of 1.91, p < 0.05), indicating the possibility that hormonal alteration in females maybe transient but robust and can lead to αHHV reactivation more often than the males.ConclusionThese results indicated that TH dysfunction may have implication in αHHV pathogenesis and females exhibited much higher probability to suffer αHHV reactivation due to TH disruption. Although the results from this pilot study have limitations and require additional controlled clinical examination such as more detailed patient records, lab data, therapeutic outcome, etc, it provides a tool to assess the effects of hormone imbalance on virus reactivation by retrospective analyses using existing large scale data base.

Beta-amyloidogenic peptides protect against Herpes Simplex Virus-1 (HSV-1) infections

Event Abstract Back to Event Beta-amyloidogenic peptides protect against Herpes Simplex Virus-1 (HSV-1) infections Karine Bourgade-Navarro1*, Geneviève Giroux2, Christian Bocti3, Aurélie Le Page1, Gilles Dupuis4, Eric H. Frost2 and Tamàs Fülöp1 1 University of Sherbrooke, Research Center on Aging, Canada 2 University of Sherbrooke, Microbiology and Infectious Diseases, Canada 3 University of Sherbrooke, Division of Neurology, Canada 4 University of Sherbrooke, Biochemistry, Canada The causes of Alzheimer’s disease (AD) are debated. Experimental data and epidemiological studies suggest that Herpes Simplex Virus-1 (HSV-1) infection is a risk factor. HSV-1 stays latent in trigeminal neurons and can be reactivated under low immune status of the host. AD is characterized by deposits of neurofibrillar tangles and senile plaques which lead to cortical neurodegenerescence. Senile plaques found extracellularly in brains of patients contain the fibrillar form of beta-amyloidogenic Aβ1-40 and Aβ1-42 peptides generated by presenelin cleavage of amyloid precursor protein (APP). Interestingly, herpetic encephalitis caused by Herpes viruses affects the same brain regions as AD. The question is open whether beta-amyloidogenic peptides also possess a physiological role. In this connection, they display anti-bacterial and anti-yeast activities in vitro. We cultured fibroblast (MRC5) and epithelial (A549) cell lines in the presence of amyloidogenic peptides Aβ1-40 and Aβ1-42 added before, simultaneously or after challenge with HSV-1. DNA was extracted and viral replication quantified by real-time PCR 24 h later. Controls were scrambled peptides and antimicrobial peptide LL-37. Results showed that peptides Aβ1-40 and Aβ1-42 inhibited HSV-1 replication, depending on their sequence of addition. For instance, they showed efficiency when added 2 h before or simultaneously to virus challenge. However, they were inefficient when added 2 h or 6 h after HSV-1. In contrast, LL-37 inhibited viral replication under all conditions. Scrambled peptides had no activity. Our data suggest that amyloidogenic peptides Aβ1-40 and Aβ1-42 may belong to a novel class of antimicrobial peptides (AMP) that possess antiviral activity. Keywords: Beta-amyloidogenic peptides, Herpes Simplex Virus, anti-viral replication, antimicrobial peptides, Alzheimer’s disease Conference: 15th International Congress of Immunology (ICI), Milan, Italy, 22 Aug - 27 Aug, 2013. Presentation Type: Abstract Topic: Host-pathogen interactions Citation: Bourgade-Navarro K, Giroux G, Bocti C, Le Page A, Dupuis G, Frost EH and Fülöp T (2013). Beta-amyloidogenic peptides protect against Herpes Simplex Virus-1 (HSV-1) infections. Front. Immunol. Conference Abstract: 15th International Congress of Immunology (ICI). doi: 10.3389/conf.fimmu.2013.02.00259 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 13 Mar 2013; Published Online: 22 Aug 2013. * Correspondence: Mrs. Karine Bourgade-Navarro, University of Sherbrooke, Research Center on Aging, Sherbrooke, Québec, J1H 5N4, Canada, karine.n.bourgade@usherbrooke.ca Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Karine Bourgade-Navarro Geneviève Giroux Christian Bocti Aurélie Le Page Gilles Dupuis Eric H Frost Tamàs Fülöp Google Karine Bourgade-Navarro Geneviève Giroux Christian Bocti Aurélie Le Page Gilles Dupuis Eric H Frost Tamàs Fülöp Google Scholar Karine Bourgade-Navarro Geneviève Giroux Christian Bocti Aurélie Le Page Gilles Dupuis Eric H Frost Tamàs Fülöp PubMed Karine Bourgade-Navarro Geneviève Giroux Christian Bocti Aurélie Le Page Gilles Dupuis Eric H Frost Tamàs Fülöp Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Ataxia telangiectasia patients cannot cause severe Herpes Simplex because of disturbed Herpes simplex virus replication by ATM mutation

Ataxia-telangiectasia (A-T) is an autosomal recessive multisystem disorder characterized by ocular and cutaneous telangiectasia, hyper sensitivity to ionizing radiation, increased incidence of malignancies, and variable immunodeficiency (high risk of bacterial and viral infection). A-T results from mutations in a single gene (ataxia-telangiectasia, mutated; ATM). ATM responds to a variety of abnormal DNA structures and initiates signaling cascades leading to the DNA damage responses (DDRs). There are no reports of severe herpes simplex in A-T patients, although A-T patients show much higher rate of lethal varicella and other viral infections than healthy individuals.

Efficient herpes simplex virus 1 replication requires cellular ATR pathway proteins.

Herpes simplex virus 1 (HSV-1) is a double-stranded DNA virus that replicates in the nucleus of the host cell and is known to interact with several components of the cellular DNA-damage-signaling machinery. We have previously reported that the DNA damage response kinase, ATR, is specifically inactivated in HSV-1-infected cells. On the other hand, we have also shown that ATR and its scaffolding protein, ATRIP, are recruited to viral replication compartments, where they play beneficial roles during HSV-1 replication. In order to better understand this apparent discrepancy, we tested the hypothesis that some of the components of the ATR pathway may exert an antiviral effect on infection. In fact, we learned that all 10 of the canonical ATR pathway proteins are stable in HSV-infected cells and are recruited to viral replication compartments; furthermore, short hairpin RNA (shRNA) knockdown shows that several, including ATRIP, RPA70, TopBP1, Claspin, and CINP, are required for efficient HSV-1 replication. We also determined that activation of the ATR kinase prior to infection did not affect virus yield but did result in reduced levels of recombination between coinfecting viruses. Together, these data suggest that ATR pathway proteins are not antiviral per se but that activation of ATR signaling may have negative consequences during viral replication, such as inhibiting recombination.

CD200R1 Supports HSV-1 Viral Replication and Licenses Pro-Inflammatory Signaling Functions of TLR2

The CD200R1:CD200 axis is traditionally considered to limit tissue inflammation by down-regulating pro-inflammatory signaling in myeloid cells bearing the receptor. We generated CD200R1−/− mice and employed them to explore both the role of CD200R1 in regulating macrophage signaling via TLR2 as well as the host response to an in vivo, TLR2-dependent model, herpes simplex virus 1 (HSV-1) infection. CD200R1−/− peritoneal macrophages demonstrated a 70–75% decrease in the generation of IL-6 and CCL5 (Rantes) in response to the TLR2 agonist Pam2CSK4 and to HSV-1. CD200R1−/− macrophages could neither up-regulate the expression of TLR2, nor assemble a functional inflammasome in response to HSV-1. CD200R1−/− mice were protected from HSV-1 infection and exhibited dysfunctional TLR2 signaling. Finally, both CD200R1−/− mice and CD200R1−/− fibroblasts and macrophages showed a markedly reduced ability to support HSV-1 replication. In summary, our data demonstrate an unanticipated and novel requirement for CD200R1 in “licensing” pro-inflammatory functions of TLR2 and in limiting viral replication that are supported by ex vivo and in vivo evidence.

Antiherpetic activity of an Agaricus brasiliensis polysaccharide, its sulfated derivative and fractions

Agaricus brasiliensis is an edible mushroom, traditionally used for the treatment of several diseases. In this paper, a polysaccharide (PLS) from A. brasiliensis, its carboxymethylated (CPLS) and sulfated (SPLS) derivatives, as well as, fractions (F1–F3) obtained from the PLS were investigated for their effect in the replication of herpes simplex virus and bovine herpes virus in HEp-2 cell cultures. The PLS, SPLS and F3 inhibited both virus strains similarly, in a dose-dependent curve. F1, F2 and CPLS did not show significant effect even at higher concentrations. All the compounds showed neither virucidal or viral adsorption inhibition activities nor effect when cells were treated prior to infection. Our study demonstrated that the extracts of A. brasiliensis, can be promising for future antiviral drug design and its biotechnological production is economically feasible.

Herpes Simplex Viruses: Mechanisms of DNA Replication

Herpes simplex virus (HSV) encodes seven proteins necessary for viral DNA synthesis-UL9 (origin-binding protein), ICP8 (single-strand DNA [ssDNA]-binding protein), UL30/UL42 (polymerase), and UL5/UL8/UL52 (helicase/primase). It is our intention to provide an up-to-date analysis of our understanding of the structures of these replication proteins and how they function during HSV replication. The potential roles of host repair and recombination proteins will also be discussed.

Imiquimod Suppresses Propagation of Herpes Simplex Virus 1 by Upregulation of Cystatin A via the Adenosine Receptor A1Pathway

Imiquimod is recognized as an agonist for Toll-like receptor 7 (TLR7) in immunocompetent cells. TLR7, as well as TLR3 and TLR8, triggers the immune responses, such as the production of type I interferons (IFNs) and proinflammatory cytokines via recognition of viral nucleic acids in the infected cells. In this study, we proposed that imiquimod has an IFN-independent antiviral effect in nonimmune cells. Imiquimod, but not resiquimod, suppressed replication of human herpes simplex virus 1 (HSV-1) in FL cells. We analyzed alternation of gene expression by treatment with imiquimod using microarray analysis. Neither type I IFNs, nor TLRs, nor IFN-inducible antiviral genes were induced in imiquimod-treated FL cells. Cystatin A, a host cysteine protease inhibitor, was strongly upregulated by imiquimod and took a major part in the anti-HSV-1 activity deduced by the suppression experiment using its small interfering RNA. Upregulation of cystatin A was suggested to be mediated by antagonizing adenosine receptor A(1) and activating the protein kinase A pathway. Imiquimod, but not resiquimod, was shown to interact with adenosine receptor A(1). Imiquimod-induced anti-HSV-1 activity was observed in other cells, such as HeLa, SiHa, and CaSki cells, in a manner consistent with the cystatin A induction by imiquimod. These results indicated that imiquimod acted as an antagonist for adenosine receptor A(1) and induced a host antiviral protein, cystatin A. The process occurred independently of TLR7 and type I IFNs.

Inhibitory effect and structure–activity relationship of some Biginelli-type pyrimidines against HSV-1

Inevitable emergence of multi-drug resistant strains to current available drugs makes an impetus for finding new therapeutic agents against herpes simplex virus (HSV). In this study, a series of novel derivatives of Biginelli-type pyrimidines were evaluated as potential anti-HSV-1 compounds by plaque reduction method. The cellular toxicity was assessed by XTT proliferation assay. The time course of anti-HSV activity of the most active compound was studied to show the anti-viral effect in various intervals of replication cycle. Compounds 2, 6, 8, 11, 12, 17, 18, 20, and 40 had the highest activity for inhibition of HSV. Compound 40 inhibited HSV replication with IC50 of 0.9 μmol/l and had CC50 of up to 100 μmol/l. This compound was a noteworthy inhibitor against HSV with TI value of 111. Compound 20 also showed considerable inhibitory activity with IC50 of 1.8 μmol/l. Result of time-of-addition study showed that compound 40 inhibits HSV replication at a stage after entry of virions to the target cells. Analysis of structure of the studied compounds demonstrated clear relationships with their anti-HSV effects. Some of the compounds seem to be promising candidates for future anti-HSV drug discovery researches. Structural manipulation based on the obtained structure–activity relationships would propose some new leads for anti-HSV drug discovery programs.