Virucidal Activity of a GT-Rich Oligonucleotide against Herpes Simplex Virus Mediated by Glycoprotein B

Abstract

We have investigated the antiviral mechanism of a phosphorothioate oligonucleotide, ISIS 5652, which has activity against herpes simplex virus (HSV) in the low micromolar range in plaque reduction assays. We isolated a mutant that is resistant to this compound. Marker rescue and sequencing experiments showed that resistance was due to at least one of three mutations in the UL27 gene which result in amino acid changes in glycoprotein B (gB). Because gB has a role in attachment and entry of HSV, we tested the effects of ISIS 5652 at these stages of infection. The oligonucleotide potently inhibited attachment of virus to cells at 4°C; however, the resistant mutant did not exhibit resistance at this stage. Moreover, a different oligonucleotide with little activity in plaque reduction assays was as potent as ISIS 5652 in inhibiting attachment. Similarly, ISIS 5652 was able to inhibit entry of preattached virions into cells at 37°C, but the mutant did not exhibit resistance in this assay. The mutant did not attach to or enter cells more quickly than did wild-type virus. Strikingly, incubation of wild-type virus with 1 to 2 M ISIS 5652 at 37°C led to a time-dependent, irreversible loss of infectivity (virucidal activity). No virucidal activity was detected at 4°C or with an unrelated oligonucleotide at 37°C. The resistant mutant and a marker-rescued derivative containing its gB mutations exhibited substantial resistance to this virucidal activity of ISIS 5652. We hypothesize that the GT-rich oligonucleotide induces a conformational change in gB that results in inactivation of infectivity.Oligonucleotides hold considerable promise for treating viral infections. Although much recent attention has focused on small interfering RNAs, the majority of oligonucleotides that have been studied as antiviral agents to date are modified oligodeoxynucleotides (ODNs) designed to work via an antisense mechanism, such as the licensed anticytomegalovirus drug, fomivirsen (2, 3). However, other ODNs with antiviral activity are not complementary to viral nucleic acid (13,31,35). These ODNs are GT rich and have the propensity to form G-quartet structures stabilized by non-Watson-Crick guanineguanine base pairs (reviewed in references 26 and 33). Certain of these GT-rich ODNs have anti-human immunodeficiency virus (HIV) activity and, evidently, target the HIV envelope protein gp120 (12,35). Others have been reported to exhibit activity against herpes simplex virus (HSV), but their mechanism(s) of action are not known (13).HSV most commonly causes genital herpes, cold sores, and corneal keratitis and can cause more severe disease, particularly in the immunocompromised (reviewed in reference 32). The virion consists of an icosahedral nucleocapsid containing the ϳ150-kbp double-stranded DNA genome, surrounded by a less-ordered layer of proteins called the tegument and a lipid bilayer envelope that contains a number of virus-encoded glycoproteins (reviewed in reference 25). The replication cycle of HSV begins with the initial attachment of viru...

Oligonucleotides designed to inhibit TLR9 block Herpes simplex virus type 1 infection at multiple steps

“…The G-ODN oligonucleotide decreased viral titers significantly when added to ARPE-19 post infection, while the control and TLR9 inhibitory oligonucleotides did not. Similar results were obtained with the guanosine repeat containing ISIS 5652 oligonucleotide versus the ISIS 2922, which lacks the guanosine repeat, in plaque reduction assays on Vero cells (Shogan et al, 2006). These data suggest a distinct mechanism of antiviral action for oligonucleotides containing guanosine repeats.…”

“…The virucidal activity of the G-ODN oligonucleotide may be due to interaction with HSV-1 glycoproteins yielding conformational changes that affect their functionality. ISI 5652, a 20 base PS-ON, which contains 3 stretches of 4 guanosines, has been shown to exhibit virucidal activity against HSV-1 KOS (Shogan et al, 2006). The G-ODN oligonucleotide decreased viral titers significantly when added to ARPE-19 post infection, while the control and TLR9 inhibitory oligonucleotides did not.…”

“…PS-ONs can prevent viral attachment via interaction with the α helices of viral glycoproteins such as the HIV-1 fusion protein gp41 (Vaillant et al, 2006) or the HSV-1 viral glycoprotein gB (Shogan et al, 2006). Our studies into the mechanism of TLR9 oligonucleotide antiviral activity revealed that all oligonucleotides interfered with HSV-1 attachment and entry into ARPE-19, 293XLnull, and 293XLhTLr9HA cells.…”

“…The antiviral activity of PS-ONs is dependent on the length and hydrophobicity of the oligonucleotide as demonstrated by studies with HIV-1 (Vaillant et al, 2006), LCMV (Cardin et al, 2009; Lee et al, 2008), HCMV (Luganini et al, 2008), HCV (Counihan and Lindenbach, 2009), DHBV (Noordeen et al, 2013a; Noordeen et al, 2013b) and HSV (Bernstein et al, 2008; Guzman et al, 2007; Shogan et al, 2006). A GT rich 20 base PS-ON (ISIS 5652) was shown to have potent antiviral activity against HSV which is thought to be mediated by a conformational change in the viral glycoprotein gB (Shogan et al, 2006). …”

Peptides Derived from Glycoproteins H and B of Herpes Simplex Virus Type 1 and Herpes Simplex Virus Type 2 Are Capable of Blocking Herpetic Infection in vitro

“…Finally, an overlay (1% agarose in M199 supplemented with 3% FBS) was added to each well and the infection was left to proceed for 3 days or until the viral control showed more than 80% of CPE. The cells were then fixed with 80% acetone in PBS and stained with crystal violet [38,39]. …”