Resveratrol Inhibits Herpes Simplex Virus Replication by Affecting a Host Factor

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Resveratrol Inhibits Herpes Simplex Virus Replication by Affecting a Host Factor Seth A. Faith, Ph.D. and John J. Docherty, Ph.D. Northeastern Ohio Universities College of Medicine Dept. of Microbiology, Immunology and Biochemistry

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Herpes Viruses Common human pathogens. Spectrum of diseases - cold sores, keratoconjunctivitis, genital infections, chicken pox, shingles, encephalitis, mononucleosis and congenital defects. Some members associated with B-cell lymphomas and sarcomas. Spread by direct contact with lesions, aerosols, blood products and transplantations. Effective therapies for some infections. Only one vaccine available, Varicella-zoster virus. http://pathmicro.med.sc.edu/virol/herpes.htm http://kidshealth.org/image/infections Herpes labialis (cold sore) Varicella (chicken pox)

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Herpes Simplex Virus (HSV) HSV-1 Oral herpes lesions (cold sores). Most common herpes simplex virus and is typically acquired in early childhood. By adulthood, up to 90% of individuals will have antibodies to HSV-1. (www.nlm.nih.gov/medlineplus) HSV-2 Genital ulcers and sores. Sexually transmitted. >86 million cases worldwide. 1 out of 5 Americans are HSV-2 positive. (www.cdc.gov) High risk populations Immunocompromised - disseminated life-threatening disease. Vaginally delivered neonates of HSV-2 positive mothers – fatal encephalitis.

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HSV-1,2 characteristics Enveloped virion diameter ~ 150-200 nm dsDNA genome ~ 152,000 bp Two unique regions, UL and US > 80 genes Chronological order of gene expression. Immediate-early (IE) – viral transcription Early (E) – DNA synthesis and encapsidation Late (L) – structural proteins, assembly and egress Establishes latency in neurons. Source of recurrent infections. http://137.187.88.127:80/ImageStore/Test/Images/LSBR/LSBR1a.jpg

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Current therapies for herpes simplex virus (HSV) 1) Target the virus. Nucleoside analogues (acyclovir, valacyclovir, famciclovir and vidarabine). Oral and topical formulations. Target DNA synthesis machinery. Emerging resistant strains. 2) Target the host. Pharmacological cyclin dependent kinase inhibitors (roscovitine, olomoucine, flavopiridol). Inhibit HSV replication at non-toxic doses. Inhibit viral gene transcription and DNA replication. 3) Vaccines. No approved vaccines. Impetus to discover novel treatments!

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Resveratrol 3,4’,5-trihydroxystilbene - a polyphenol phytoalexin produced by some spermatophytes, such as grapes and peanuts, in response to bacterial or fungal infection. First isolated and characterized in 1940 and has shown cancer chemopreventative, anti-inflammatory, anti-bacterial and anti-viral properties. Inhibits replication of HSV-1, HSV-2 and acyclovir resistant HSV-1 in vitro. Topical application reduced morbidity and mortality rate of mice cutaneously or vaginally infected with HSV with equal effectiveness as acyclovir.

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In vitro effectiveness of resveratrol against HSV-1

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Resveratrol affects a host target, rather than the virus Previously reported that resveratrol induces a G2/M cell cycle arrest in uninfected Vero cells. Resveratrol treatment of HSV infected cells does not yield resistant viruses.

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Resveratrol and HSV cellular targets Aggarwal et al: Anticancer Research 24 (2004)

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Probing for cellular targets of resveratrol treatment in HSV-1 infection Investigated G2/M interphase proteins known to benefit HSV. cyclin dependent kinase 1 (cdc2) cyclin A cyclin B p53 p21 Mdm2 Western blot, immunoprecipitation and phosphorylation studies yielded no significant findings. Investigate next target. Host transcriptional factor, NF-kB.

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0 hr Vero control Mock 4 hr HSV-1 4 hr Mock 12 hr HSV-1 12 hr Mock 4 hr HSV-1 4 hr Mock 12 hr HSV-1 12 hr Cdc2 protein levels B-actin cdc2 Less exposure time 10 %SDS-PAGE 3 bands of phosphorylation 0 hr Vero control Mock 20 hr HSV-1 20 hr Mock 28 hr HSV-1 28 hr Mock 20 hr HSV-1 20 hr Mock 28 hr HSV-1 28 hr 0.2% DMSO 219 uM Resv 0.2% DMSO 219 uM Resv

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cdc2 effects (cont.) B-actin cdc2 No Trt Vero 12 hpi No Trt Vero 24 hpi 0.2% DMSO 12 hpi 0.2% DMSO 24 hpi 219 uM Piceatannol 12 hpi 219 uM Piceatannol 24 hpi 219 uM Resv 12 hpi 219 uM Resv 24 hpi 100 uM Rosco 12 hpi 100 uM Rosco 24 hpi 0 hr Vero control 0.2% DMSO 24 hr 219 uM Resv 24 hr 0.2% DMSO 48 hr 219 uM Resv 48 hr Resv 24 hr / DMSO 24hr cdc2 recovery Effects piceatannol and rosco compared to resveratrol

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Phosphorylation of cdc2 p-cdc2 Y15 p-cdc2 T14Y15 0 hr Vero Mock + DMSO Mock + Resv HSV-1 + DMSO HSV-1 + Resv Mock + DMSO Mock + Resv HSV-1 + DMSO HSV-1 + Resv 4 hpi 12 hpi

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Cyclin Partners Cyclin A Cyclin B1 0 hr Vero Mock + DMSO Mock + Resv HSV-1 + DMSO HSV-1 + Resv Mock + DMSO Mock + Resv HSV-1 + DMSO HSV-1 + Resv 4 hpi 12 hpi

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Pulldown assay IB: cyclin A IB: cyclin B1 0 hr Vero Mock + DMSO Mock + Resv HSV-1 + DMSO HSV-1 + Resv Mock + DMSO Mock + Resv HSV-1 + DMSO HSV-1 + Resv 4 hpi 12 hpi IP: cdc2

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NF-kB Santoro GM, The EMBO Journal, Vol. 22, No. 11 pp. 2552-2560, 2003

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Viruses activate NF-kB Santoro GM, The EMBO Journal, Vol. 22, No. 11 pp. 2552-2560, 2003

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Resveratrol inactivates NF-kB in HSV-1 infected cells Electromobility shift assay EMSA

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Resveratrol blocks NF-kB activation in HSV-2 and Acyclovir-Resistant HSV-1 Mock Mock + Resv HSV-2 HSV-2 + Resv ACV-R HSV-1 ACV-R HSV-1 + Resv 0 3 6 12 24 hpi

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Dose dependent inactivation of NF-kB EMSA 12 hpi Mock HSV-1 0 0CM 219 0 55 110 219 uM Resv %

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0 219 110 10 1 0.1 uM Resv NF-kB (cont.) Direct Inactivation Super Shift 200x Cold Probe No Ab Anti-p50 Anti-p65 24 hpi HSV-1 Nuclear extracts

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Therapeutic Indices – in vitro

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Piceatannol vs. Resveratrol

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IkB-alpha is degraded during HSV-1 infection Control Media 219 uM Resv Mock HSV-1 0 3 6 12 24 3 6 12 24 hpi

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Localization of NF-kB IHC for p65, 6 hpi Control Media 219 uM Resv Mock HSV-1 40x

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Determination of apoptosis – caspase-3 activity assay

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COX activity- PGE2 synthesis

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Real-time gene expression of HSV-1 Western Blot

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Hypothetical Mechanism ICP4 Apoptosis IE E L ICP0,22,27,47 ICP4 DNA replication Apoptosis Prevention Structural Proteins Assembly Egress NF-kB Resveratrol ? X

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Summary HSV-1, HSV-2 and acyclovir resistant HSV-1 activate NF-kB. Resveratrol blocks this activation in a dose dependent manner parallel to its anti-herpetic concentrations. NF-kB translocates to the nucleus of infected cells regardless of treatment. Resveratrol induced caspase-3 in uninfected cells, but not in HSV-1 infected cells. ICP4 and gC gene activation is suppressed by resveratrol.

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