Genetic Characterization of Thymidine Kinase from Acyclovir‐Resistant and ‐Susceptible Herpes Simplex Virus Type 1 Isolated from Bone Marrow Transplant Recipients

Abstract

Emergence of acyclovir (Acy)-resistant herpes simplex virus (HSV) is a major concern in bone marrow transplant recipients. Phenotypic and genetic characterization of thymidine kinase (TK) was done for 7 Acy-susceptible and 11 Acy-resistant HSV-1 isolated from 11 patients. In total, 19 amino acid substitutions were detected that were not related to Acy resistance but to TK gene polymorphism, including 5 mutations that have not been previously reported. The Acy-resistant strain from 1 patient presented no TK gene mutation related to resistance. Five patients (45%) had isolates that harbored point mutations leading to amino acid substitutions that could be associated with Acy resistance. Of the 5 substitutions detected, 3 have not been previously reported (codons 51, 83, and 175). A nucleotide insertion or deletion was detected in resistant isolates from 5 patients (45%); these mutations are located in homopolymer repeats at codon 92 (1 subject) and at codon 146 (4 subjects).

Highly Reliable Heterologous System for Evaluating Resistance of Clinical Herpes Simplex Virus Isolates to Nucleoside Analogues

“…Isolate 6b also contained a mutation in the DNA pol gene conferring resistance to foscarnet (42). Isolate 7 contained a deleted A at nt 1065 and three other nonsilent mutations of which two (Pro42Leu and Arg89Gln) have been found in other pretherapy isolates from this study and elsewhere (21,25) and thus presumably are associated with gene polymorphism. Isolates 8 and 9 had deletions of G at positions 779 and 180, respectively.…”

“…Furthermore, Sasadeusz et al have identified mutational hot spots consisting of frameshift mutations within homopolymer nucleotide stretches of G's and C's (41). Recent studies by our group (16) and others (25) have demonstrated that about 50% of the clinical ACV-resistant strains contain an insertion or a deletion of one or two nucleotides in homopolymer runs of G's and C's, whereas the other half presents single-base substitutions in conserved or nonconserved regions of the TK gene.…”

“…Furthermore, Sasadeusz et al have identified mutational hot spots consisting of frameshift mutations within homopolymer nucleotide stretches of G's and C's (41). Recent studies by our group (16) and others (25) have demonstrated that about 50% of the clinical ACV-resistant strains contain an insertion or a deletion of one or two nucleotides in homopolymer runs of G's and C's, whereas the other half presents single-base substitutions in conserved or nonconserved regions of the TK gene.Characterization of the TK gene from ACV-susceptible isolates frequently reveals the presence of nonsilent mutations outside the active sites of the TK gene, reflecting a certain degree of viral polymorphism (21,24,25). At this time, identification of TK mutations conferring resistance to nucleoside analogues is fastidious and requires transfection of the mutated TK gene in a wild-type virus by a rare homologous recombination event followed by selection of the recombinant strain with an antiviral drug.…”

“…Darby et al have proposed a preliminary model for the active center of the HSV type 1 (HSV-1) TK enzyme including three distinct regions: an ATP-binding site (amino acids 51 to 63), a nucleoside-binding site (amino acids 168 to 176), and amino acid 336 (12). Indeed, single-point mutations in one or more of these conserved regions have been found in ACVresistant HSV isolates (16,20,25,30,41,42). Furthermore, Sasadeusz et al have identified mutational hot spots consisting of frameshift mutations within homopolymer nucleotide stretches of G's and C's (41).…”

“…Characterization of the TK gene from ACV-susceptible isolates frequently reveals the presence of nonsilent mutations outside the active sites of the TK gene, reflecting a certain degree of viral polymorphism (21,24,25). At this time, identification of TK mutations conferring resistance to nucleoside analogues is fastidious and requires transfection of the mutated TK gene in a wild-type virus by a rare homologous recombination event followed by selection of the recombinant strain with an antiviral drug.…”