Clinical isolate of herpes simplex virus type 2 that induces a thymidine kinase with altered substrate specificity

Abstract

In vitro and in vivo studies were done on a berpes simplex virus type 2 strain recovered from a patient on acyclovir (ACV) which was ACV resistant but expressed thymidine (dThd) kinase (EC 2.7.1.21) activity. Plaque-purified clones derived from the original clinical sample were heterogeneous with respect to plaque size and drug susceptibility. The heterogeneity of this viral mixture was also evident from varied I251-labeled 5-iodo-2'-deoxycytidine autoradiographic patterns and from varied expression of dThd kinase-associated phosphorylating activities. Four clones from this mixture were 1-0i-D-arabinofuranosylthymine (ara-T) susceptible and ACV resistant. Extracts of cells infected with these 0lones catalyzed the phosphorylation of ara-T but little of ACV. The virus-coded dThd kinase was purified from one of these clones to determine whether its substrate specificity was altered. The amount of virus-coded dThd phosphorylating activity with the cell extracts was estimated to be sevenfold lower with the resistant clone than with the MS strain of herpes simplex virus type 2. The dThd kinase eluted from a dThd-agarose affinity column under the same conditions with extracts from both sources and substrate saturations of both enzymes by acyclic nucleoside analog phosphate acceptors were classical hyperbolic functions. However, there were significant differences in the kinetic parameters of substrates between the two enzymes. Apparent Km (Kin') values for dThd, deoxycytidine, ara-T, ACV, and the acyclic guanosine analog 9-[[2-hydroxy-1-(hydroxymethyl)ethoxy]methyl]guanine (BW B759U) were 2-to 60-fold higher with the variant enzyme than with the enzyme from laboratory strain MS. Comparing these two enzymes, relative maximal phosphorylation rates (Vm) were eightfold lower for ACV but unchanged for BW B759U. In contrast, the relative rates for deoxycytidine and ara-T were eight-and twofold higher, respectively. The surprisingly good substrate activity with BW B759U compared with that of ACV (Vm/Km' = 0.39 versus 0.01) coincided with susceptibility of the ACV-resistant virus to BW B759U. This clinical variant retained its pathogenicity for mice and was only moderately less neurovirulent than wild-type virus. Although such mutants have the potential to induce illness less responsive to therapy, the recurrence from which the isolate was obtained was typical for this patient in severity and duration. Since this episode, the patient has been treated successfully with ACV.

Effects of Antiviral Usage on Transmission Dynamics of Herpes Simplex Virus Type 1 and on Antiviral Resistance: Predictions of Mathematical Models

“…At present, it is unclear whether resistance can be acquired when a symptomatic episode of sensitive HSV-1 in an immunocompetent person is treated with a nucleoside analogue. There are four reports in which treatment of an immunocompetent host may have resulted in the acquisition of resistance in HSV-1 or HSV-2 (22,33,38,58,59). In none of these is it certain whether treatment itself was responsible for the appearance of resistant virus.…”

“…H-10, p. 317, 1998). Because of the self-limiting nature of HSV reactivations in these patients, resistance has minor clinical consequences and resistant virus may occur only transiently (22). In severely immunocompromised people, however, resistance is more common.…”

Antiviral therapy: current concepts and practices

“…Both of the last two mechanisms result in lack of recognition of acyclovir as a substrate for the enzyme. Among patient isolates, thymidine kinase-deficient mutants are most common, but viruses expressing the other mechanisms have also been found (49,76,123,399,426,490).…”

“…They may also be unreliable in that they can select for virus strains that grow well in cell culture but are not necessarily representative of the virus population present in the patient. It is increasingly appreciated that viruses isolated from patients are not clonal derivatives homogeneous in their properties but instead are heterogeneous populations of viruses with differing drug susceptibilities and other properties, including the ability to grow in different cell lines (123,149). As a result, new methods of measuring resistance are being developed for the research laboratory, including use of the polymerase chain reaction for detection of resistance genes, cell lines which allow growth of a broader spectrum of viral strains, and improved methods of nucleic acid hybridization (226,264,266).…”

Herpes Simplex Virus Type 1 Entry Is Inhibited by the Cobalt Chelate Complex CTC-96

“…Not surprisingly, drug-resistant strains are appearing with increasing frequency (13,14,17,28,29,54,70,85). Resistance arises from mutations in the TK gene (18,27) or mutations in the gene encoding DNA polymerase (13,14,47,70,85). Therefore, new drugs need to be developed that target other aspects of the virus life cycle in order to find more effective treatments against the existing drug-resistant strains as well as all the known herpesviruses.…”