Drug resistance of herpes simplex virus type 1

Abstract

Several drug-resistant strains of herpes simplex virus type 1 (HSV1) isolated in vivo or from tissue culture, have exhibited a mutated thymidine kinase (TK). Moreover, various site-directed-mutagenesis experiments conducted on HSV1 TK allowed the assignment of specific amino acid residues to specific functional properties. From this, a range of hypotheses was generated related to substrate binding of TK at the molecular level. A site-directed-mutagenesis study on Q125 was performed to clarify the contribution of this residue to the binding of thymidine or aciclovir beyond the hydrogen-bonding pattern observed in the crystal structure. While Q125L is only able to phosphorylate thymidine, Q125N accepts thymidine and aciclovir as substrates. Q125E shows no phosphorylation activity. Several mutations identified previously as relevant in drug resistance were studied in an attempt to further understand their role in these processes. Four amino acid positions are described (T63, A168, R176 and C336) that confer drug resistance when mutated ; however, the molecular mechanisms are considerably different in each case. Analysis of the crystal structures and the molecular modeling presented in this paper suggest that T63 is essential for the binding of Mg 2ϩ and thus the catalytic activity of the enzyme, while A168 limits steric accessibility and if mutated to a bulkier residue will exclude binding of larger substrate analogues. R176 appears to be essential for electrostatic balance within the active site, and C336, which is located at the surface of TK and directed toward the ATP-binding site, disrupts the three-dimensional structure of the whole active site by shifting the LID-domain. The present work contributes to a detailed understanding of nucleoside binding to TK, thereby facilitating the rational design of substrates for HSV1 TK and of drug-specific TK for gene therapy.Keywords : herpes simplex virus ; thymidine kinase ; mutation; aciclovir ; resistance.Thymidine kinase (TK) is a key enzyme in the pyrimidine-herpesviral TK isoenzymes is the molecular basis of selective salvage pathway catalyzing the transfer of γ-phosphate from antiviral therapy. Furthermore, HSV1 TK was used recently in ATP to thymidine, to synthesize thymidine monophosphate combination with aciclovir or gangciclovir as suicide enzyme in (dTMP). Herpes viruses encode their own thymidine kinases gene therapy of cancer [2Ϫ4] and AIDS [5]. Therefore, further which differ considerably from the human host cell isoenzyme. understanding of the molecular mechanism of HSV1 TK and its In particular, viral TKs are able to phosphorylate a broad drug-resistant mutants could lead to rational design of antiviral spectrum of pyrimidine and purine substrate analogues including drugs and to customized drug-specific TK for gene therapy. those carrying an acyclic sugar moiety such as the antiviral There are four classes of single mutations of the HSV gedrugs aciclovir and ganciclovir [1]. Compounds such as nome that confer drug resistance. Three of these affect the th...

Crystal Structure of Varicella Zoster Virus Thymidine Kinase

“…3b). This is in marked contrast to HSV1-tk where the base is sandwiched by an aliphatic and aromatic residue, Met-128 and Tyr-172, respectively, with Ile-100 also making some contacts (19,26,36). These differences in interactions with the pyrimidine ring result in the base binding in a significantly different position to that seen in the HSV1-tk structure.…”

“…These changes in combination with the differing stacking of the pyrimidine ring between two aromatic phenylalanine residues in VZV-tk compared with methionine/ tyrosine side chains in HSV1-tk result in a significant reorientation of the BVDU in the binding site. Interestingly, despite these differences in the modes of interaction the k cat /K m , parameters for BVDU as a substrate are similar for both VZV and HSV1-tks (15,19,22).…”

“…The efficacy of BVDU is similar for both HSV1 and VZV. Both tks have K i values of 0.1 M and similar k cat /K m values (15,19,22). The most potent compounds against VZV are found within the BVDU-related halogen-vinyl araU series.…”

“…However, the K i values determined for the nucleosides against VZV-tk in the same study show only a 20-fold difference, indicating that a relatively minor component of this selectivity is attributable to the tk activation step. ACV has been the main drug of choice against VZV and HSV infections (16), although its antiviral activity is much greater against HSV (types 1 and 2) than it is against VZV (4, 17), a result of the differing affinities for the thymidine kinases where the K i for ACV against VZV-tk is 830 M (18) compared with 200 M for HSV1-tk (19,20). Even more marked is the difference in potency for GCV, which binds with a similar affinity to VZV-tk as ACV but more tightly to HSV1-tk with a K i of 48 M (7,21).…”

Reduced Rates of Gene Loss, Gene Silencing, and Gene Mutation in Dnmt1-Deficient Embryonic Stem Cells

“…The Mlh1 control primers used were 5Ј-AGG AGC TGA TGC TGA GGC-3Ј (OL 117/sense) and 5Ј-TTT CAT CTT GTC ACC CGA TG-3Ј (OL 118/anti-sense) (4). Screening of the TKNeo gene for mutations was performed by amplification of a 946-bp region spanning all previously described hot spots for TK mutation (2,9,21,42,57). The primers used for primary PCR amplification were 5Ј-CGA CCA GGC TGC GCG TTC TCG-3Ј (TK-MC1) and 5Ј-CCA GGA TAA AGA CGT GCA TGG AAC GG-3Ј (TKMC-2).…”