title: Oligonucleotides designed to inhibit TLR9 block Herpes simplex virus type 1 infection at multiple steps.
abstract: Herpes simplex virus type 1 (HSV-1) is an important human pathogen which requires activation of nuclear factor-kappa B (NFκB) during its replication cycle. The persistent nature of HSV-1 infection, and the emergence of drug-resistant strains, highlights the importance of research to develop new antiviral agents. Toll-like receptors (TLRs) play a prominent role during the early antiviral response by recognizing viral nucleic acid and gene products, activating NFκB, and stimulating the production of inflammatory cytokines. We demonstrate a significant effect on HSV-1 replication in ARPE-19 and Vero cells when oligonucleotides designed to inhibit TLR9 are added 2h prior to infection. A greater than 90% reduction in the yield of infectious virus was achieved at oligonucleotide concentrations of 10-20 μM. TLR9 inhibitory oligonucleotides prevented expression of essential immediate early herpes gene products as determined by immunofluorescence microscopy and Western blotting. TLR9 oligonucleotides also interfered with viral attachment and entry. A TLR9 inhibitory oligonucleotide containing five adjacent guanosine residues (G-ODN) exhibited virucidal activity and inhibited HSV-1 replication when added post-infection. The antiviral effect of the TLR9 inhibitory oligonucleotides did not depend on the presence of TLR9 protein, suggesting a mechanism of inhibition that is not TLR9 specific. TLR9 inhibitory oligonucleotides also reduced NFκB activity in nuclear extracts. Studies using these TLR inhibitors in the context of viral infection should be interpreted with caution.
authors.affiliation: Department of Ophthalmology and Visual Sciences, University of Wisconsin-Madison, Madison, WI 53706, United States. Electronic address: msauter@wisc.edu.
authors.forename: Monica M
authors.initials: MM
authors.lastname: Sauter
authors.affiliation: Department of Medical Microbiology and Immunology, University of Wisconsin-Madison, Madison, WI 53706, United States. Electronic address: Joshua.gauger@gmail.com.
authors.forename: Joshua J L
authors.initials: JJ
authors.lastname: Gauger
authors.affiliation: Department of Ophthalmology and Visual Sciences, University of Wisconsin-Madison, Madison, WI 53706, United States; Department of Medical Microbiology and Immunology, University of Wisconsin-Madison, Madison, WI 53706, United States; McPherson Eye Research Institute, University of Wisconsin-Madison, Madison, WI 53706, United States. Electronic address: crbrant@wisc.edu.
authors.forename: Curtis R
authors.initials: CR
authors.lastname: Brandt
mesh_terms: D000818:Animals; D000998:Antiviral Agents / Q000494:pharmacology*; D002522:Chlorocebus aethiops; D015967:Gene Expression Regulation, Viral / Q000187:drug effects; D006561:Herpes Simplex / Q000235:genetics / Q000276:immunology / Q000821:virology*; D018259:Herpesvirus 1, Human / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D006801:Humans; D016328:NF-kappa B / Q000235:genetics / Q000276:immunology; D009841:Oligonucleotides / Q000494:pharmacology*; D051217:Toll-Like Receptor 9 / Q000235:genetics* / Q000276:immunology; D014709:Vero Cells; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism; D053585:Virus Attachment / Q000187:drug effects
chemical_list: D000998:Antiviral Agents; D016328:NF-kappa B; D009841:Oligonucleotides; D051217:Toll-Like Receptor 9; D014764:Viral Proteins
keywords: ARPE-19; Herpes simplex virus; ICP4; NFκB; Retinal pigment epithelium; TLR9