Analysis of the thymidine kinase genes from acyclovir-resistant mutants of varicella-zoster virus isolated from patients with AIDS.

Patients with AIDS often experience recurrent infections with varicella-zoster virus (VZV) requiring repeated or prolonged treatment with acyclovir (ACV), which may lead to the development of ACV resistance. The ACV resistance of isolates recovered from such patients is associated with diminished VZV thymidine kinase (TK) function. We determined the nucleotide sequences of the TK genes of 12 ACV-resistant VZV strains purified from nine patients with AIDS. Five VZV strains contained nucleotide deletions in their TK genes, introducing a premature termination codon which is expected to result in the production of a truncated protein. No detectable full-length TK protein could be immunoprecipitated from extracts of cells infected with these virus strains. These TK-deficient strains were cross resistant to the TK-dependent antiviral agents ACV, 9-(4-hydroxy-3-hydroxymethylbutyl-yl)guanine (penciclovir), and 1-beta-D-arabinofuranosyl-E-5-(2-bromovinyl) uracil (BVaraU). The remaining seven strains each contained a nucleotide change that resulted in an amino acid substitution in the TK protein. These substitutions occurred throughout the TK protein, namely, in the ATP-binding site, the nucleoside-binding site, between the two binding sites, and at the carboxy terminus of the protein. We determined the effects of these mutations on the stability of TK protein expression in virus-infected cells and on the sensitivity of mutants to the TK-dependent antiviral agents ACV, BVaraU, and penciclovir.     

Directed evolution of thymidine kinase for AZT phosphorylation using DNA family shuffling

The thymidine kinase (TK) genes from herpes simplex virus (HSV) types 1 and 2 were recombined in vitro with a technique called DNA family shuffling. A high-throughput robotic screen identified chimeras with an enhanced ability to phosphorylate zidovudine (AZT). Improved clones were combined, reshuffled, and screened on increasingly lower concentrations of AZT. After four rounds of shuffling and screening, two clones were isolated that sensitize Escherichia coli to 32-fold less AZT compared with HSV-1 TK and 16,000-fold less than HSV-2 TK. Both clones are hybrids derived from several crossover events between the two parental genes and carry several additional amino acid substitutions not found in either parent, including active site mutations. Kinetic measurements show that the chimeric enzymes had acquired reduced K(M) for AZT as well as decreased specificity for thymidine. In agreement with the kinetic data, molecular modeling suggests that the active sites of both evolved enzymes better accommodate the azido group of AZT at the expense of thymidine. Despite the overall similarity of the two chimeric enzymes, each contains key contributions from different parents in positions influencing substrate affinity. Such mutants could be useful for anti-HIV gene therapy, and similar directed-evolution approaches could improve other enzyme-prodrug combinations.     

Conservative mutations of glutamine-125 in herpes simplex virus type 1 thymidine kinase result in a ganciclovir kinase with minimal deoxypyrimidine kinase activities

The herpes simplex virus type 1 thymidine kinase (HSV-1 TK) is the major anti-herpes virus pharmacological target, and it is being utilized in combination with the prodrug ganciclovir as a toxin gene therapeutic for cancer. One active-site amino acid, glutamine-125 (Gln-125), has been shown to form hydrogen bonds with bound thymidine, thymidylate, and ganciclovir in multiple X-ray crystal structures. To examine the role of Gln-125 in HSV-1 TK activity, three site-specific mutations of this residue to an aspartic acid, an asparagine, or a glutamic acid were introduced. These three mutants and wild-type HSV-1 TK were expressed in E. coli and partially purified and their enzymatic properties compared. In comparison to the Gln-125 HSV-1 TK, thymidylate kinase activity of all three mutants was decreased by over 90%. For thymidine kinase activity relative to Gln-125 enzyme, the K(m) of thymidine increased from 0.9 microM for the parent Gln-125 enzyme to 3 microM for the Glu-125 mutant, to 6000 microM for the Asp-125 mutant, and to 20 microM for the Asn-125 mutant. In contrast, the K(m) of ganciclovir decreased from 69 microM for the parent Gln-125 enzyme to 50 microM for the Asn-125 mutant and increased to 473 microM for the Glu-125 mutant. The Asp-125 enzyme was able to poorly phosphorylate ganciclovir, but with nonlinear kinetics. Molecular simulations of the wild-type and mutant HSV-1 TK active sites predict that the observed activities are due to loss of hydrogen bonding between thymidine and the mutant amino acids, while the potential for hydrogen bonding remains intact for ganciclovir binding. When expressed in two mammalian cell lines, the Glu-125 mutant led to GCV-mediated killing of one cell line, while the Asn-125 mutant was equally as effective as wild-type HSV-1 TK in metabolizing GCV and causing cell death in both cell lines.     

猪伪狂犬病毒鲁A株TK基因的序列测定及其结构功能与进化分析

对猪伪狂犬病毒鲁A株(PRV LA株)TK基因进行了克隆和序列测定,并分析比较了该序列与PRV NIA-3株、Ea株、SH以及HSV-1和VZV的同源性,结果表明:在全长1048bp的DNA序列中,包括着一个963bp的开放阅读框(ORF),可编码320个氯基酸组成的多肽;在整个TK基因的ORF内,PRV LA株与PRV NIA-3株、PRV Ea株、PRV SH株、HSV-1、VZV的TK基因比较,核苷酸的同源性分别为98.9%、99.5%、99.3%、36.4%、39.1%,氨基酸的同源性分别为98.4%、99.7%、98.7%、36.6%、37.2%.PRV LA株TK具有疱疹病毒胸苷激酶催化结构域的保守氨基酸共有序列和亚结构域特征序列.将PRV-LA TK、人类和小鼠的胸苷酸激酶、人类脱氧胞苷激酶、人类腺苷酸激酶的对应于这两个亚结构域的氨基酸用DNA Star分析的进化树表明,疱疹病毒的TK与人类和小鼠的胸苷酸激酶的亲缘关系比与人类脱氧胞嘧啶激酶的亲缘关系更近,因此疱疹病毒的TK基因在进化上可能起源于宿主细胞的胸苷酸激酶基因.     

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

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.     

Robustness of hen lysozyme monitored by random mutations

We investigated the robustness of hen lysozyme by using random mutant libraries. Six random mutant libraries containing 1, 1.5, 2, 3, 5 and 14 amino acid mutations per hen lysozyme were systematically constructed by varying the concentrations of Mg(2+) and Mn(2+) on polymerase chain reaction. The mutated genes from the six libraries were cloned to a yeast expression vector and a total of 4000 clones were screened on the basis of lysis activity and ELISA employing monoclonal antibody that recognized only lysozyme with native conformation. About 80% of the clones with an average of two amino acid mutations retained active structure. Almost all clones with an average of five mutations lost active structure. On the other hand, 80% of the clones with an average of two amino acid mutations retained both gross conformation and active structure and 24% of the clones with an average of 14 amino acid mutations retained gross conformation. These results show that gross conformation is robust against mutations and so is active structure to a lesser extent.     

Characterization of pyrimidine deoxyribonucleoside kinase (thymidine kinase) and thymidylate kinase as a multifunctional enzyme in cells transformed by herpes simplex virus type 1 and in cells infected with mutant strains of herpes simplex virus.

Pyrimidine deoxyribonucleoside kinase (thymidine kinase [TK]) was purified from two herpes simplex virus type 1 (HVS-1)-transformed TK-deficient mouse (LMTK-) cell lines and from LMTK- cells infected with HSV-1 mutant viruses coding for variant TK enzymes. These preparations exhibited normal or variant virus-induced thymidylate kinase activities correlating with their relative TK activities. Neither virus-induced activity was detected in LMTK- cells infected with an HSV-1 TK-deficient mutant. These results suggest that HSV-1 thymidylate kinase activity and TK activity are mediated by the same protein.     

Increased thermostability of microbial transglutaminase by combination of several hot spots evolved by random and saturation mutagenesis

The thermostability of microbial transglutaminase (MTG) of Streptomyces mobaraensis was further improved by saturation mutagenesis and DNA-shuffling. High-throughput screening was used to identify clones with increased thermostability at 55°C. Saturation mutagenesis was performed at seven "hot spots", previously evolved by random mutagenesis. Mutations at four positions (2, 23, 269, and 294) led to higher thermostability. The variants with single amino acid exchanges comprising the highest thermostabilities were combined by DNA-shuffling. A library of 1,500 clones was screened and variants showing the highest ratio of activities after incubation for 30 min at 55°C relative to a control at 37°C were selected. 116 mutants of this library showed an increased thermostability and 2 clones per deep well plate were sequenced (35 clones). 13 clones showed only the desired sites without additional point mutations and eight variants were purified and characterized. The most thermostable mutant (triple mutant S23V-Y24N-K294L) exhibited a 12-fold higher half-life at 60°C and a 10-fold higher half-life at 50°C compared to the unmodified recombinant wild-type enzyme. From the characterization of different triple mutants differing only in one amino acid residue, it can be concluded that position 294 is especially important for thermostabilization. The simultaneous exchange of amino acids at sites 23, 24, 269 and 289 resulted in a MTG-variant with nearly twofold higher specific activity and a temperature optimum of 55°C. A triple mutant with amino acid substitutions at sites 2, 289 and 294 exhibits a temperature optimum of 60°C, which is 10°C higher than that of the wild-type enzyme.     

Isolation of drug resistant mutants of varicella-zoster virus: cross resistance of acyclovir resistant mutants with phosphonoacetic acid and bromodeoxyuridine

Mutants of Varicella-Zoster Virus (VZV) which are resistant to phosphonoacetic acid (PAA), bromodeoxyuridine (BuDR), and acyclovir (ACV) were obtained by serial passages of VZV with increasing concentrations of these drugs. A PAA-resistant mutant and a BuDR-resistant mutant were found also to be resistant to ACV. Five of 8 ACV-resistant mutants acquired resistance to PAA, but none acquired resistance to BuDR. The BuDR-resistant mutant did not induce viral thymidine kinase (TK) activity, but all the ACV-resistant mutants selected in ACV showed viral TK activity which was suppressed with anti-VZV serum and had almost the same electrophoretic mobility as that of the parent strain on polyacrylamide gel electrophoresis in non-denaturing conditions. However, in competitive TK assay with ACV, 2 of 8 ACV-resistant mutants showed no change of phosphorylation of radioactive thymidine, while the other 6 showed decreased phosphorylation of radioactive thymidine. It was suggested that TK induced by the former 2 ACV-resistant mutants had lost affinity to ACV, and so the mutants could grow in the presence of ACV. Thus of the 8 ACV-resistant mutants selected in ACV, 2 were sensitive to PAA with altered TK activity, 5 were resistant to PAA with unaltered TK activity, and 1 was sensitive to PAA with unaltered TK activity, and may have altered DNA polymerase activity to ACV, retaining sensitivity to PAA. These results suggest that resistance of VZV to ACV results from alterations in the virus-specified TK or DNA polymerase, as demonstrated in HSV resistant to ACV.     

Elevated frequency of microsatellite mutations in TK6 human lymphoblast clones selected for mutations at the thymidine kinase locus.

A major question in carcinogenesis is, How can a normal cell accumulate multiple mutations in different genes on different chromosomes, when the mutation rate of each gene is in the range of 10(-8) to 10(-5) per cell division? We hypothesize that many mutations may not be isolated events but rather are accompanied by concomitant mutations elsewhere in the genome. To test this hypothesis, 331 independent clones selected for new mutations at the thymidine kinase (TK) locus on chromosome 17q, and 243 nonselected control clones were examined for mutations in 12 random microsatellite loci dispersed throughout the genome. A total of 24 second-site mutations were identified in the TK mutant clones, compared with 3 in the control clones not selected for mutations at TK. The mutations include small deletions, insertions, and loss of heterozygosity. These results provide evidence that a global trans-acting mutagenic process exists in human cells. The activation of this process could be responsible for causing multiple essential mutations in tumor cells.     

Evolution of the herpes thymidine kinase: identification and comparison of the equine herpesvirus 1 thymidine kinase gene reveals similarity to a cell-encoded thymidylate kinase.

We have identified the equine herpesvirus 1 (EHV-1) thymidine kinase gene (TK) by DNA-mediated transformation and by DNA sequencing. Alignment of the amino acid sequence of the EHV-1 TK with the TKs from 3 other herpesviruses revealed regions of homology, some of which correspond to the previously identified substrate binding sites, while others have as yet, no assigned function. In particular, the strict conservation of an aspartate within the proposed nucleoside binding site suggests a role in ATP binding for this residue. Comparison of 5 herpes TKs with the thymidylate kinase of yeast revealed significant similarity which was strongest in those regions important to catalytic activity of the herpes TKs, and, therefore we propose that the herpes TK may be derived from a cellular thymidylate kinase. The implications for the evolution of enzyme activities within a pathway of nucleotide metabolism are discussed.     

Sequence analysis of temperature-sensitive mutations in the Saccharomyces cerevisiae gene CDC28.

Eleven independently isolated temperature-sensitive mutations in the cell division cycle gene CDC28 were mapped with respect to the DNA sequence of the wild-type gene and then sequenced to determine the precise nature of each mutation. The set yielded six different point mutations, each of which predicts a single amino acid substitution in the CDC28 product. The positions of the mutations did not correlate in any obvious way with observable biological characteristics of the mutant alleles. When the positions of substitutions were collated with a predicted secondary structural analysis of the CDC28 protein kinase, they were found to correlate strongly with probable regions of structural transition.     

Characterization of herpes simplex virus type 1 thymidine kinase mutants selected under a single round of high-dose brivudin

A broad variety of herpes simplex virus type 1 clones was selected under a single round of high-dose selection with brivudin. Mutations in the thymidine kinase (TK) genes consisted of 42% frameshift mutations within homopolymer repeats of G's and C's and single nucleotide substitutions (58%) that produced stop codons (Q261 and R281) or a new codon at the site of the substitution (A168T, R51W, G59W, G206R, R220H, Y239S, and T287 M). The A168T change, associated with an altered TK phenotype, proved to be the most commonly selected substitution. For the different mutants, a correlation between phenotype, genotype, and in vivo neurovirulence was observed.     

Kinetic properties of mutant human thymidine kinase 2 suggest a mechanism for mitochondrial DNA depletion myopathy

Thymidine kinase 2 (TK2) is a mitochondrial (mt) pyrimidine deoxynucleoside salvage enzyme involved in mtDNA precursor synthesis. The full-length human TK2 cDNA was cloned and sequenced. A discrepancy at amino acid 37 within the mt leader sequence in the DNA compared with the determined peptide sequence was found. Two mutations in the human TK2 gene, His-121 to Asn and Ile-212 to Asn, were recently described in patients with severe mtDNA depletion myopathy (Saada, A., Shaag, A., Mandel, H., Nevo, Y., Eriksson, S., and Elpeleg, O. (2001) Nat. Genet. 29, 342-344). The same mutations in TK2 were introduced, and the mutant enzymes, prepared in recombinant form, were shown to have similar subunit structure to wild type TK2. The I212N mutant showed less than 1% activity as compared with wild type TK2 with all deoxynucleosides. The H121N mutant enzyme had normal K(m) values for thymidine (dThd) and deoxycytidine (dCyd), 6 and 11 microm, respectively, but 2- and 3-fold lower V(max) values as compared with wild type TK2 and markedly increased K(m) values for ATP, leading to decreased enzyme efficiency. Competition experiments revealed that dCyd and dThd interacted differently with the H121N mutant as compared with the wild type enzyme. The consequences of the two point mutations of TK2 and the role of TK2 in mt disorders are discussed.     

Antiviral Activity of 1-β-D-Arabinofuranosyl-E-5-(2-Bromovinyl)Uracil against Thymidine Kinase Negative Strains of Varicella-Zoster Virus

Mechanism of antiviral activity of 1-β-d -arabinofuranosyl-E-5-(2-bromovinyl)uracil (BV-araU) against the YSR strain of varicella-zoster virus (VZV), which is a mutant derived from the wild YS strain and is completely deficient in viral thymidine kinase (TK), was searched in comparison with antiviral activity of other thymidine analogues, guanosine analogue and thymidylate synthase (TS) inhibitor in human embryo lung fibroblast cells. Thymidine analogues, such as BV-araU, 5-iododeoxyuridine (IUDR), 1-β-d -arabinofuranosylthymine (araT), and guanosine analogue, such as 9-(2-hydroxyethoxymethyl)guanine (ACV), showed higher antiviral activity to the YS strain than to the YSR strain. Though, BV-araU also had the antiviral activity of a microgram level against the YSR strain. In contrast to these results, TS inhibitor, 5-fluorodeoxyuridine (FUDR), had higher antiviral activity to the YSR strain than to the YS strain. Highly synergistic antiviral activities of FUDR to the YS strain and the YSR strain were observed in combination with IUDR, araT, or ACV. However, weakly synergistic or additive inhibition to the YSR strain was shown in combination of BV-araU and FUDR, in spite of highly synergistic effect of this combination to the YS strain. The viral and cellular TS activity was partially inhibited by BV-araU monophosphate, but not by BV-araU. These results indicate that BV-araU is converted into BV-araU monophosphate by cellular TK, and the inhibition of TS activity by BV-araU monophosphate in the YSR strain-infected cells results in the suppression of viral replication.     

Polymorphic HLA-DR7 beta 1 chain residues that are involved in T cell allorecognition

The contributions to allorecognition of polymorphic amino acids in the HLA-DR7 beta 1 chain were analyzed by using mutant DR7 beta 1 chains with single amino acid substitutions at position 4, 11, 13, 25, 30, 37, 57, 60, 67, 70, 71, 74, or 78. Transfectants expressing mutant DR7 molecules were used as stimulators for six DR7-alloreactive T cell clones. The majority of the substitutions had profound effects on the ability of the DR7 molecule to stimulate one or more T cell clones. Nine of the 13 substitutions completely abrogated recognition by at least one clone. The finding that each of the substitutions in the beta-strands in the floor of the peptide binding groove affected T cell allorecognition supports the model of allorecognition in which the complex of a self-peptide bound to a class II molecule is recognized by the TCR. Interestingly, the substitution at position 4, which is predicted to be located outside the peptide binding groove, decreased the ability of the DR7 molecule to stimulate some clones. Each of the DR7-alloreactive T cell clones had a unique reactivity pattern in response to the different mutant molecules, indicating that the TCR of each clone recognized the DR7 molecule differently. Surprisingly, many of the mutant DR7 molecules induced proliferation by one or more clones that was greater than 125% of the proliferation induced by the wild-type DR7 molecule. These data indicate that multiple polymorphic residues, predicted in the class II model to be located in both the beta-strands and alpha-helix of the DR7 beta 1 chain, contribute to allorecognition of the DR7 molecule.     

In vitro mutagenesis of a mouse MHC class I gene for the examination of structure-function relationships

Oligonucleotide-directed, site-specific mutagenesis has been employed to elucidate the role of individual amino acids on the expression and function of a MHC class I antigen. Two oligonucleotides were synthesized to introduce single amino acid substitutions in the murine H-2Ld gene. The highly conserved glycosylation site at amino acid position 86 was changed from asparagine to lysine to remove the carbohydrate moiety from the first external domain of the H-2 molecule, and the phenylalanine at position 116 was changed to tyrosine, replacing the Ld residue with the Kb type amino acid analogous to Kb mutants: bm5 and bm16 mutants derived from the Kb antigen have the Ld-type residue at this position. The mutant genes were constructed by annealing the mutagenic oligomers to the single stranded H-2Ld gene, followed by chain elongation reaction. The expected mutations were confirmed by DNA sequence determination. The mutant genes were introduced into mouse L cells by DNA-mediated gene transfer. Both mutant genes expressed the antigens on the cell surface, as detected by antibody binding; these antigens were reactive with the cytotoxic T cells specific for the H-2Ld antigen. Detailed examination with 16 monoclonal anti-H-2Ld antibodies revealed that the binding of some antibodies was significantly reduced in the glycosylation mutant, implying a certain contribution of the carbohydrates to the antigenic activity of some determinants. No detectable changes have been observed in the mutant of the substitution at position 116 by the parameters we tested.     

Gene-enzyme relationships of aromatic acid biosynthesis in Bacillus subtilis

Mutants have been isolated which correspond to every step concerned with the biosynthesis of the aromatic amino acids in Bacillus subtilis. Each mutant has been characterized, and the lesion it bore was analyzed by deoxyribonucleic acid transformation and PBS-1 mediated transduction. The biochemical analysis revealed that each of the mutations appears to have affected a single enzyme, except for two groups of pleiotropic mutations. All aroF mutants (chorismic acid synthetase) lack dehydroquinic acid synthetase (aroB) activity. The gene that specifies aroB is closely linked to the gene coding for the aroF enzyme. Both genes are a part of the aro cluster. Mutants lacking chorismate mutase activity also lack d-arabino-heptulosonic acid-7-phosphate synthetase and shikimate kinase activity, presumably as a result of these three activities forming a multi-enzyme complex. Another mutant, previously undescribed, had been isolated. The affected gene codes for the tyrosine and phenylalanine aminotransferase activity. All of the mutations have been located on the B. subtilis genome except those in the genes specifying shikimate kinase activity and tyrosine-phenylalanine aminotransferase activity.     

Simultaneous enhancement of thermostability and catalytic activity of phospholipase A(1) by evolutionary molecular engineering

The thermal stability and catalytic activity of phospholipase A(1) from Serratia sp. strain MK1 were improved by evolutionary molecular engineering. Two thermostable mutants were isolated after sequential rounds of error-prone PCR performed to introduce random mutations and filter-based screening of the resultant mutant library; we determined that these mutants had six (mutant TA3) and seven (mutant TA13) amino acid substitutions. Different types of substitutions were found in the two mutants, and these substitutions resulted in an increase in nonpolar residues (mutant TA3) or in differences between side chains for polar or charged residues (mutant TA13). The wild-type and mutant enzymes were purified, and the effect of temperature on the stability and catalytic activity of the enzymes was investigated. The melting temperatures of the TA3 and TA13 enzymes were increased by 7 and 11 degrees C, respectively, compared with the melting temperature of the wild-type enzyme. Thus, we found that evolutionary molecular engineering was an effective and efficient approach for increasing thermostability without compromising enzyme activity.