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 (TLR) 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 2 hours prior to infection. A greater than 90% reduction in the yield of infectious virus was achieved at oligonucleotide concentrations of 10 to 20 micromolar. 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.

Evasion of Early Antiviral Responses by Herpes Simplex Viruses

“…Additionally, treatment with CpG-ODN containing unmethylated CpG provides protection against lethal vaginal challenge with HSV that was probably mediated by an intricate crosstalk between plasmacytoid DCs (pDCs) and vaginal stromal cells, as well as type-I IFNs [ 104 – 106 ]. Similar to the findings described with TLR3 and TLR7, these results suggest that modulating TLR9 signaling could be a promising strategy for limiting HSV infection in the host, although results from another group suggest that the antiviral effects mediated by TLR9 antagonists might not necessarily be mediated uniquely by intracellular events linked to TLR9 signaling [ 107 ]. Nevertheless, the results obtained with TLR9 agonists suggest that activating this TLR might be a useful strategy for controlling pathological responses induced by HSVs [ 108 ].…”

Pathological processes activated by herpes simplex virus-1 (HSV-1) infection in the cornea

“…3) [4,43,77]. In vitro studies have shown that inhibition of TLR9 using oligonucleotides prevented expression of essential immediate early herpes gene products, and as a result also reduced NFκB activity in nuclear extracts [90]. In the absence of TLR9, other compensatory mechanisms are involved in producing both type I interferon and cytokines, showing that TLR9 only partially contributes to the response against the virus [46,55].…”

Toll‐like receptors as novel therapeutic targets for herpes simplex virus infection

“…For example, treatment with a five adjacent guanosine residues (G‐ODN) at a concentration of 10 to 20 μM 2 hours before infection inhibited TLR9 signaling, NF‐κB activity and substantially reduced the yield of lytic virus (90%) in herpes‐susceptible cells. Also, the TLR9 inhibitory effect of CpG oligonucleotide was associated with downregulation of crucial immediate early HSV proteins, impaired viral attachment and entry, virucide activity and mitigated virus replication . Equally, an additional study using both agonists (TLR 3/9) and inhibitors (TLR9) of TLRs demonstrated an increased survival rate of mice when agonists of TLR3 polyinosinic: polycytidylic acid (PIC) and TLR9 (type B ODN 1826) were administered intranasally prior to HSV1 infection.…”