To investigate how acyclovir-resistant (ACVr) herpes simplex virus (HSV) evades drug therapy and causes disease, HSV-1 isolates from a bone marrow transplant (BMT) patient were studied. The patient developed ACVr disease after an initial BMT and, following a second BMT, reactivated ACVr HSV despite high-dose acyclovir prophylaxis. ACVr isolates from each episode contained the same point mutation in the viral thymidine kinase (tk) gene, documenting the emergence, latency, and reactivation of this mutant. The mutants were exceedingly impaired for TK activity in sensitive enzyme, plaque autoradiography, and drug-susceptibility assays. Nevertheless, these mutants and a tk deletion mutant constructed in the same genetic background reactivated from latency in mouse trigeminal ganglia, in contrast to similar mutants from laboratory strains. It is hypothesized that alleles in the clinical isolate compensate for the loss of TK in this animal model. Such genetic variability may be important for ACVr disease in humans.

A human cytomegalovirus mutant that was isolated for resistance (10-fold) to the antisense oligonucleotide fomivirsen (ISIS 2922) exhibited cross-resistance to a modified derivative of fomivirsen with an identical base sequence but little or no resistance to an oligonucleotide with an unrelated sequence. No changes in the mutant’s DNA corresponding to the fomivirsen target sequence were found.

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...

SummaryPenciclovir is the active form of the orally available prodrug famciclovir, which is entering clinical use for herpesvirus infections. Like aciclovir, penciclovir is an acyclic guanosine analogue that is phosphorylated by viral thymidine kinase and whose triphosphate can inhibit viral DNA polymerase. We tested several well-characterized herpes simplex virus mutants with aciclovir-resistance mutations in the viral DNA polymerase gene for altered sensitivity to penciclovir. The mutants varied in their susceptibilities to penciclovir with one exhibiting 2-fold hypersensitivity, one marginal resistance and three about 3-fold resistance. Marker rescue and DNA sequencing analyses mapped the penciclovir-resistance mutation of one mutant, AraA r7, to a single base change that alters a glycine to a cysteine at residue 841 within conserved region III of a-like DNA polymerases. The results have implications for the mechanism of selective action of penciclovir, for the potential for development of resistance in the clinic, and for the substrate recognition properties of herpes simplex virus DNA polymerase.

Herpes simplex virus encodes proteins, such as DNA polymerase, that are essential for its replication and proteins, such as thymidine kinase, that are not essential for replication in cell culture, but are important for pathogenesis in animal models. However, certain mutations affecting these proteins exert little or no effect on replication or pathogenesis. We tested the effects of combining two such mutations--one that alters DNA polymerase and one that decreases but does not abolish thymidine kinase activity--on replication in cultured cells and on acute and latent infections in mice. The double mutant replicated similarly to the single mutants and wild-type virus both in cell culture and acutely in the mouse eye. However, it was severely impaired for acute replication in trigeminal ganglia and for reactivatable latent infections. This impairment depended upon the polymerase mutation. Similarly, although Ro 31-5140, a thymidine kinase inhibitor, did not potentiate the antiviral effects of phosphonoacetic acid, a polymerase inhibitor, in cell culture, the two drugs in combination substantially inhibited viral reactivation from latency at concentrations that had little or no effect when used singly. These synergistic effects may have implications for viral functions during pathogenesis and for antiviral chemotherapy.