Herpes Simplex Virus Isolates from an Immunocompromised Patient who Failed to Respond to Acyclovir Treatment Express Thymidine Kinase with Altered Substrate Specificity

Abstract

Ten sequential post-treatment herpes simplex virus type 1 (HSV-1) isolates were obtained from an immunocompromised patient whose infection, during prolonged treatment, became unresponsive to acyclovir (ACV). Of the ten isolates, eight later isolates were resistant in vitro to ACV and ganciclovir (DHPG), but remained sensitive to 9-[3-D-arabinofuranosyladenine (ara-A) and phosphonoformate (PFA). Biochemical characterization of plaque-purified clones of the resistant isolates revealed an altered thymidine kinase (TK) substrate specificity phenotype. The comparative nucleotide sequence analysis of polymerase chain reaction (PCR)-amplified DNA encoding the TK genes of one sensitive and two resistant clones ,showed a single mutation at nucleotide 527. This change would result in a substitution of arginine by glutamine at residue 176 ofthe polypeptide, a mutation previously observed in a laboratory isolated variant, SC16 Tr7 (Darby et al., 1986).

A Comparative Study of the in vitro and in vivo Antiviral Activities of Acyclovir and Penciclovir

“…They are predominantly TK deficient and would therefore not be susceptible to PCV. Two HSV clinical isolates with ACV-resistant DNA polymerase mutations have been described (Parker et el., 1987;Collins et el., 1989;Sacks et al, 1989), as well as several TK substrate specificity mutants (Hill et et., 1991;Nugier et al, 1991). The laboratory-derived ACV-resistant polymerase mutants and the TK substrate specificity mutant reported here were all found to be cross-resistant to PCV, although one laboratory-generated ACV-resistant polymerase mutant that was sensitive to PCV has been 4 4 minimal reduction in virus recovery from the primary site.…”

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

“…Isolate 11 contained a substitution at nt 527 that resulted in an amino acid substitution at codon 176 within the putative nucleoside-binding site as well as two other changes (codons 42 and 89) associated with gene polymorphism (21,25). The amino acid change (Arg176Glu) has been previously reported in a laboratory-derived HSV-1 mutant (12) and in ACV-resistant HSV-1 isolates (30). A 3G-for-3C substitution was observed in isolate 12 at positions 175 to 179 within the proposed ATPbinding site (42).…”

“…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).…”

Drug resistance of herpes simplex virus type 1

“…The loss in affinity was compared with the wild-type the solved crystal structures [9, 10, 23], is incomplete as the essential Mg 2ϩ ion is absent. Therefore, modeling work has been previously, exhibit the same mutations as those obtained in vitro, namely A168→T and R176→Q [33,34]. performed to account for the lack of structural evidence.…”