Emergence of resistance to acyclovir and penciclovir in varicella-zoster virus and genetic analysis of acyclovir-resistant variants

Varicella zoster virus (VZV) is usually associated with mild to moderate illness in immunocompetent patients. However, older age and immune deficiency are the most important risk factors linked with virus reactivation and severe complications. Treatment of VZV infections is based on nucleoside analogues, such as acyclovir (ACV) and its valyl prodrug valacyclovir, penciclovir (PCV) as its prodrug famciclovir, and bromovinyldeoxyuridine (BVDU; brivudin) in some areas. The use of the pyrophosphate analogue foscarnet (PFA) is restricted to ACV-resistant (ACV r ) VZV infections. Since antiviral drug resistance is an emerging problem, we attempt to describe the contributions of specific mutations in the viral thymidine kinase (TK) gene identified following selection with ACV, BVDU and its derivative BVaraU (sorivudine), and the bicyclic pyrimidine nucleoside analogues (BCNAs), a new class of potent and specific anti-VZV agents. The string of 6 Cs at nucleotides 493 to 498 of the VZV TK gene appeared to function as a hot spot for nucleotide insertions or deletions. Novel amino acid substitutions (G24R and T86A) in VZV TK were also linked to drug resistance. Six mutations were identified in the "palm domain" of VZV DNA polymerase in viruses selected for resistance to PFA, PCV, and the 2-phophonylmethoxyethyl (PME) purine derivatives. The investigation of the contributions of specific mutations in VZV TK or DNA polymerase to antiviral drug resistance and their impacts on the structures of the viral proteins indicated specific patterns of cross-resistance and highlighted important differences, not only between distinct classes of antivirals, but also between ACV and PCV.

Section: Discussionmentioning

confidence: 99%

In Vitro -Selected Drug-Resistant Varicella-Zoster Virus Mutants in the Thymidine Kinase and DNA Polymerase Genes Yield Novel Phenotype-Genotype Associations and Highlight Differences between Antiherpesvirus Drugs

Andrei

Topalis

Fiten

et al. 2012

“…While a recent analysis of the Acv r Kawaguchi strain of varicella-zoster virus (VZV) revealed TK-deficient variants similar to the classical Acv r HSV mutants, with deletions which result in frameshifts and premature termination (19), the VZV TK gene lacks a G 7 element, and the location of the single C 6 tract is not conserved relative to its HSV-1 and HSV-2 homologs. Most ACV-resistant VZV mutants reportedly lack frameshift mutations and are more likely to express full-length TK than Acv r HSV mutants (40).…”

Section: Discussionmentioning

confidence: 99%

Characterization of Herpes Simplex Viruses Selected in Culture for Resistance to Penciclovir or Acyclovir

Sarisky¹,

Quail²,

Clark³

et al. 2001

Penciclovir (PCV), an antiherpesvirus agent in the same class as acyclovir (ACV), is phosphorylated in herpes simplex virus (HSV)-infected cells by the viral thymidine kinase (TK).Resistance to ACV has been mapped to mutations within either the TK or the DNA polymerase gene. An identical activation pathway, the similarity in mode of action, and the invariant cross-resistance of TK-negative mutants argue that the mechanisms of resistance to PCV and ACV are likely to be analogous. A total of 48 HSV type 1 (HSV-1) and HSV-2 isolates were selected after passage in the presence of increasing concentrations of PCV or ACV in MRC-5 cells. Phenotypic analysis suggested these isolates were deficient in TK activity. Moreover, sequencing of the TK genes from ACV-selected mutants identified two homopolymeric G-C nucleotide stretches as putative hot spots, thereby confirming previous reports examining Acv r clinical isolates. Surprisingly, mutations identified in PCV-selected mutants were generally not in these regions but distributed throughout the TK gene and at similar frequencies of occurrence within A-T or G-C nucleotides, regardless of virus type. Furthermore, HSV-1 isolates selected in the presence of ACV commonly included frameshift mutations, while PCV-selected HSV-1 mutants contained mostly nonconservative amino acid changes. Data from this panel of laboratory isolates show that Pcv r mutants share cross-resistance and only limited sequence similarity with HSV mutants identified following ACV selection. Subtle differences between PCV and ACV in the interaction with viral TK or polymerase may account for the different spectra of genotypes observed for the two sets of mutants.The introduction of penciclovir [PCV;9-(4-hydroxy-3-hydroxymethylbut-1-yl)guanine] and its prodrug, famciclovir, (FCV), resulted in the use of antivirals alternative to acyclovir (ACV) for treatment of herpes simplex virus (HSV) infections. Biochemical studies have indicated that PCV, like ACV, is phosphorylated by the viral thymidine kinase (TK) to a monophosphate and subsequently converted by cellular enzymes to a triphosphate, which inhibits the HSV DNA polymerase (Pol) (44). Although PCV and ACV have identical activation pathways and similar modes of action (14, 44), and the frequencies with which resistance in HSV arises to PCV and ACV in cell culture are identical (36), the affinities and therefore the fine molecular interactions of PCV, ACV, and their triphosphates with TK and Pol differ (14). The last point raises the possibility that drug-resistant mutants selected by these antiviral agents may differ.Resistance to acyclovir typically arises by a single mutation in either the TK or Pol gene (11,23,29). The viral TK, unlike DNA polymerase, is not essential for virus replication in cell culture (13), although in vivo analyses implicate it in HSV virulence, pathogenicity, and reactivation from latency (9,15,20,41). Mutations in HSV TK are the most common causes of clinical resistance to ACV (7,34), and the majority of mutants completely lac...

“…Unfortunately, despite these medications, PHN remains a major problem, and the currently available drugs have a limited effect on prevention of this condition. Thus, there is a need for additional, more effective medications to treat herpes zoster in adults.The development of antiviral resistance remains a concern, particularly when treatment relies on only a small number of approved drugs with common modes of action (13,14,25,26). ACV, valaciclovir (which is metabolized to acyclovir), and famciclovir (the prodrug of penciclovir) are all phosphorylated by the viral thymidine kinase as an initial step for intracellular activation.…”

mentioning

confidence: 99%

Identification of Small Molecule Compounds That Selectively Inhibit Varicella-Zoster Virus Replication

Visalli¹,

Fairhurst²,

Srinivas

et al. 2003

A series of nonnucleoside, N-␣-methylbenzyl-N-arylthiourea analogs were identified which demonstrated selective activity against varicella-zoster virus (VZV) but were inactive against other human herpesviruses, including herpes simplex virus. Representative compounds had potent activity against VZV early-passage clinical isolates and an acyclovir-resistant isolate. Resistant viruses generated against one inhibitor were also resistant to other compounds in the series, suggesting that this group of related small molecules was acting on the same virus-specific target. Sequencing of the VZV ORF54 gene from two independently derived resistant viruses revealed mutations in ORF54 compared to the parental VZV strain Ellen sequence. Recombinant VZV in which the wild-type ORF54 sequence was replaced with the ORF54 gene from either of the resistant viruses became resistant to the series of inhibitor compounds. Treatment of VZV-infected cells with the inhibitor impaired morphogenesis of capsids. Inhibitor-treated cells lacked DNA-containing dense-core capsids in the nucleus, and only incomplete virions were present on the cell surface. These data suggest that the VZV-specific thiourea inhibitor series block virus replication by interfering with the function of the ORF54 protein and/or other proteins that interact with the ORF54 protein.Varicella-zoster virus (VZV) causes a disseminated primary infection, chickenpox, and later may reactivate to cause herpes zoster, commonly known as shingles (3,26). Herpes zoster can result in pain lasting over 1 month from the onset of the rash, termed postherpetic neuralgia (PHN). PHN is often a debilitating condition that is quite difficult to treat (15,32,33).Acyclovir (ACV), valaciclovir, and famciclovir are the primary antivirals used for treating VZV infections in both immunocompetent and immunocompromised individuals (25). Unfortunately, despite these medications, PHN remains a major problem, and the currently available drugs have a limited effect on prevention of this condition. Thus, there is a need for additional, more effective medications to treat herpes zoster in adults.The development of antiviral resistance remains a concern, particularly when treatment relies on only a small number of approved drugs with common modes of action (13,14,25,26). ACV, valaciclovir (which is metabolized to acyclovir), and famciclovir (the prodrug of penciclovir) are all phosphorylated by the viral thymidine kinase as an initial step for intracellular activation. They are subsequently phosphorylated by cellular kinases, and the activated compounds inhibit the viral DNA polymerase. Sorivudine and brivudine are also potent inhibitors of VZV, and although they represent a different class of nucleoside analogues from ACV, they also require phosphorylation by the viral thymidine kinase for activation (18,31). While thymidine kinase-deficient VZV can be treated with foscarnet, a direct inhibitor of the viral DNA polymerase (25), this drug requires intravenous administration and has a marked potential for...