Quantification and analysis of thymidine kinase expression from acyclovir-resistant G-string insertion and deletion mutants in herpes simplex virus-infected cells

To be clinically relevant, drug-resistant mutants must both evade drug action and retain pathogenicity. Many acyclovir-resistant herpes simplex virus mutants from clinical isolates have one or two base insertions (G8 and G9) or one base deletion (G6) in a homopolymeric run of seven guanines (G string) in the gene encoding thymidine kinase (TK). Nevertheless, G8 and G9 mutants express detectable TK activity and can reactivate from latency in mice, a pathogenicity marker. On the basis of studies using cell-free systems, ribosomal frameshifting can explain this ability to express TK. To investigate frameshifting in infected cells, we constructed viruses that express epitope-tagged versions of wild-type and mutant TKs. We measured TK activity by plaque autoradiography and expression of frameshifted and unframeshifted TK polypeptides using a very sensitive immunoprecipitation-Western blotting method. The G6 mutant expressed ～0.01% of wild-type levels of TK polypeptide. For the G9 mutant, consistent with previous results, much TK expression could be ascribed to reversion. For the G8 mutant, from these assays and pulse-labeling studies, we determined the ratio of synthesis of frameshifted to unframeshifted polypeptides to be 1:100. The effects of stop codons before or after the G string argue that frameshifting can initiate within the first six guanines. However, frameshifting efficiency was altered by stop codons downstream of the string in the 0 frame. The G8 mutant expressed only 0.1% of the wild-type level of full-length TK, considerably lower than estimated previously. Thus, remarkably low levels of TK are sufficient for reactivation from latency in mice.     

High-frequency phenotypic reversion and pathogenicity of an acyclovir-resistant herpes simplex virus mutant

A double-guanine-insertion mutation within a run of guanines in the herpes simplex virus gene encoding thymidine kinase (TK) was previously found in an acyclovir-resistant clinical isolate. This mutation was engineered into strain KOS, and stocks were generated from single plaques. Plaque autoradiography revealed that most plaques in such stocks exhibited low levels of TK activity, while approximately 3% of plaques exhibited high levels of TK activity, indicating a remarkably high frequency of phenotypic reversion. This virus was able to reactivate from latency in mouse ganglia; a fraction of the reactivating virus expressed a high level of TK activity due to an additional G insertion, suggesting that the observed genetic instability contributed to pathogenicity.     

The viral thymidine kinase gene as a tool for the study of mutagenesis in Trypanosoma brucei.

We have tested the use of thymidine kinase as a negative selection system for Trypanosoma brucei. To this end we have targeted a construct containing a Herpes simplex virus thymidine kinase (TK) gene into the ribosomal DNA array of procyclic T. brucei. This resulted in TK activity 30-50-fold above background and in susceptibility to the nucleoside analogues ganciclovir, ethyl-deoxyuridine and 1-[2-deoxy,2-fluoro-8-D-arabinofuranosyl]-5-iodouracil, all of which have no effect on wild-type trypanosomes. TK+ trypanosomes, however, reverted to a ganciclovir resistant phenotype at a rate of 10(-6) per cell-generation. A similar reversion rate was observed using the Varicella-zoster virus TK gene. Loss of TK activity was not due to detectable DNA rearrangements or a decrease in TK mRNA. Sequence analysis of the revertant genes demonstrated, however, the occurrence of point mutations and frameshifts. One revertant line had a mutation in the thymidine binding site leading to the substitution of a conserved arginine by a glycine. Other mutations included single base insertion, single base deletion and the introduction of a premature termination codon by point mutation.     

Substrate binding is a prerequisite for stabilisation of mouse thymidine kinase in proliferating fibroblasts

Thymidine kinase (TK) expression in mammalian cells is strictly growth regulated, with high levels of the enzyme present in proliferating cells and low levels in resting cells. We have shown that mouse TK expressed from a constitutive promoter is still subject to this regulation. The drastic decline in TK enzyme levels in resting cells is largely due to a pronounced reduction in the half-life of the protein. Deletion of the 30 C-terminal amino acid residues from TK abrogates growth regulation, rendering the enzyme very stable. Moreover, the substrate thymidine was sufficient to stabilise the labile TK protein in quiescent cells. Here, we report that the ability of TK to bind substrates is essential for both growth-dependent regulation and stabilisation by the substrate. By mutation or elimination of the binding sites for either of the two substrates, ATP and thymidine, we expressed TK proteins lacking enzymatic activity which abolished growth-regulated expression in both cases. Mutant TK proteins impaired in substrate binding were subject to rapid degradation in exponentially growing cells and thymidine was no longer sufficient to inhibit this rapid decay. A C-terminal truncation known to stabilise the TK wild-type protein in resting cells did not affect the rapid turnover of enzymatically inactive TK proteins. Proteasome inhibitors also failed to stabilise these substrate-binding mutants. By cross-linking experiments, we show that TK proteins with mutated substrate-binding sites exist only as monomers, whereas active TK enzyme forms dimers and tetramers. Our data indicate that, In addition to the C terminus intact substrate-binding sites are required for growth-dependent regulation of TK protein stability.     

Engineering of a single conserved amino acid residue of herpes simplex virus type 1 thymidine kinase allows a predominant shift from pyrimidine to purine nucleoside phosphorylation

Studies of herpes simplex virus type 1 (HSV-1) thymidine (dThd) kinase (TK) crystal structures show that purine and pyrimidine bases occupy distinct positions in the active site but approximately the same geometric plane. The presence of a bulky side chain, such as tyrosine at position 167, would not be sterically favorable for pyrimidine or pyrimidine nucleoside analogue binding, whereas purine nucleoside analogues would be less affected because they are located further away from the phenylalanine side chain. Site-directed mutagenesis of the conserved Ala-167 and Ala-168 residues in HSV-1 TK resulted in a wide variety of differential affinities and catalytic activities in the presence of the natural substrate dThd and the purine nucleoside analogue drug ganciclovir (GCV), depending on the nature of the amino acid mutation. A168H- and A167F-mutated HSV-1 TK enzymes turned out to have a virtually complete knock-out of dThd kinase activity (at least approximately 4-5 orders of magnitude lower) presumably due to a steric clash between the mutated amino acid and the dThd ring. In contrast, a full preservation of the GCV (and other purine nucleoside analogues) kinase activity was achieved for A168H TK. The enzyme mutants also markedly lost their binding capacity for dThd and showed a substantially diminished feedback inhibition by thymidine 5'-triphosphate. The side chain size at position 168 seems to play a less important role regarding GCV or dThd selectivity than at position 167. Instead, the nitrogen-containing side chains from A168H and A168K seem necessary for efficient ligand discrimination. This explains why A168H-mutated HSV-1 TK fully preserves its GCV kinase activity (Vmax/Km 4-fold higher than wild-type HSV-1 TK), although still showing a severely compromised dThd kinase activity (Vmax/Km 3-4 orders of magnitude lower than wild-type HSV-1 TK).     

The A167Y mutation converts the herpes simplex virus type 1 thymidine kinase into a guanosine analogue kinase

The thymidine (dThd) kinase (TK) encoded by herpes simplex virus type 1 (HSV-1) is not only endowed with dThd kinase, but also with thymidylate (dTMP) kinase and 2'-deoxycytidine (dCyd) kinase (dCK) activity. HSV-1 TK also recognizes a variety of antiherpetic guanine nucleoside analogues such as acyclovir (ACV), ganciclovir (GCV), lobucavir (LBV), penciclovir (PCV), and others (i.e., A5021). Site-directed mutagenesis of the highly conserved Ala-167 to Tyr in HSV-1 TK completely abolished TK, dTMP-K, and dCK activity, but maintained ACV-, GCV-, LBV-, PCV-, and A5021-phosphorylating capacity. A variety of 5-substituted pyrimidine nucleoside substrates, but also a number of selective HSV-1 TK inhibitors structurally related to thymine lost significant binding affinity for the mutant enzyme and did not markedly compete with GCV phosphorylation by the mutant enzyme. These findings could be explained by computer-assisted modeling data that revealed steric hindrance of the pyrimidine ring in the HSV-1 TK active site by the large 4-hydroxybenzyl ring of 167-Tyr, while the positioning of the purine ring of guanine-based HIV-1 TK substrates in the active site was kept virtually unaltered. Surprisingly, the efficiency of conversion the antiherpetic 2'-deoxyguanosine analogues ACV, GCV, LBV, PCV, and A5021 to their phosphorylated forms by the A167Y mutant HSV-1 TK was far more pronounced than for the wild-type enzyme. Therefore, the single A167Y mutation converts the wild-type HSV-1 TK from a predominantly pyrimidine nucleos(t)ide kinase into a virtually exclusive purine (guanine) nucleoside analogue kinase.     

Establishment of latency in mice by herpes simplex virus 1 recombinants that carry insertions affecting regulation of the thymidine kinase gene.

Herpes simplex virus 1 recombinants carrying alpha-, beta-, and late gamma (gamma 2)-regulated thymidine kinase (TK) genes were tested for the ability to establish latency in BALB/c mice inoculated by the eye route. The significant findings were as follows. Representatives of alpha- and gamma 2-regulated TK recombinants all established and maintained latent infections, but the efficiency was somewhat lower than that of wild-type virus. Of the three alpha TK recombinants tested, one (R316) spontaneously deleted portions of the inserted sequences which conferred alpha regulation to the TK gene. The viruses carrying these deletions expressed considerably lower TK activity than did wild-type virus, i.e., 2 to 40% of the levels expressed by the wild-type virus carrying the beta TK gene. However, the ability of these viruses to establish latency was not related to the efficiency of expression of the TK gene. These results indicate the following: (i) conversion of the TK gene into an alpha or gamma 2 gene did not preclude the establishment of latent infections; (ii) there was no correlation between the levels of TK activity expressed in cell culture and the ability to establish latency; and (iii) rearrangement of the genome by insertions or deletions which interrupt gene domains did not automatically result in an inability to establish latent infections.     

Abnormal formation of polyhedra resulting from a single mutation in the polyhedrin gene of Autographa californica multicapsid nucleopolyhedrovirus

A spontaneous mutant that produces a single abnormally large cubic polyhedron per infected cell was isolated from a polyhedra-positive recombinant Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV). Both wild-type and mutant virus produce two forms of virus particles, budded virions and occluded virions. However, occluded virions are not found within the polyhedra of cells infected with mutant virus, as with the wild-type virus. These large cubic polyhedra do not have the typical lattice-like structure normally seen in wild-type polyhedra and are noninfectious. Spodoptera frugiperda 9 (SF9) cells which were infected with this virus had low infectivity to larvae. No significant alterations were found in the viral genome by restriction enzyme analysis, and no mutations were found in the 25K gene. A single point mutation resulting in an amino acid change of Gly25 to Asp was identified in the polyhedrin gene. A transfer vector containing the entire polyhedrin gene including the point mutation was constructed and used to cotransfect Sf9 cells with a polyhedron-negative recombinant virus. Large cubic polyhedra were once again observed, confirming that the Gly25 to Asp mutation is responsible for the formation of abnormal polyhedra.     

Characterization of three essential residues in the conserved ATP-binding region of Epstein-Barr virus thymidine kinase

The thymidine kinase encoded by Epstein-Barr virus (EBV TK) is an important target for antiviral therapy and the treatment of EBV-associated malignancies. Through computer-assisted alignment with other human herpesviral TK proteins, EBV TK was shown to contain a conserved ATP-binding motif as for the other TK enzymes. To investigate functional roles of three highly conserved residues (G294, K297, T298) within this region, site-directed mutagenesis was employed to generate various mutants. The TK enzyme activity and ATP-binding ability of these mutant TK enzymes were determined and compared with EBV wild-type TK (wtTK). Mutant G294V lost its ATP-binding ability and was inactive in enzyme activity assay. As the enzyme activity of G294A was reduced to 20% of that of wtTK, the K(m) for ATP binding of G294A was 48.7 microM as compared with 30.0 microM of EBV wtTK. These results suggested that G294 participates in ATP binding and contributes to maintenance of structure. EBV TK mutants K297E, K297Q, and K297R lost their ATP-binding ability and enzyme activity. However, K297R was shown to have a preference for usage of GTP (K(m): 43.0 microM) instead of ATP (K(m): 87.6 microM) as the phosphate donor. This implies that, in addition to nucleotide binding, K297 was involved in the selection of phosphate donor. While EBV TK mutant T298S retained approximately 80% of wtTK enzyme activity, T298A lost its enzyme activity, suggesting that a hydroxyl group at this position is important for the enzyme activity. Interestingly, T298A retained its ATP-binding ability, suggesting a role of T298 in the catalytic process but not in the coordination of ATP. This study demonstrated that amino acid residues G294, K297, and T298 in the ATP-binding motif of EBV TK enzyme are essential for the enzymatic activity but are involved in different aspects of its action.     

Low-level expression and reversion both contribute to reactivation of herpes simplex virus drug-resistant mutants with mutations on homopolymeric sequences in thymidine kinase

Many acyclovir-resistant herpes simplex virus isolates from patients contain insertions or deletions in homopolymeric sequences in the thymidine kinase (TK) gene (tk). Viruses that have one (G8) or two (G9) base insertions in a run of seven G's (G string) synthesize low levels of active TK (TK-low phenotype), evidently via ribosomal frameshifting. These levels of TK can suffice to permit reactivation from latently infected mouse ganglia, but in a majority of ganglia, especially with the G9 virus, reactivation of virus that has reverted to the TK-positive phenotype predominates. To help address the relative contributions of translational mechanisms and reversion in reactivation, we generated viruses with a base either inserted or deleted just downstream of the G string. Both of these viruses had a TK-low phenotype similar to that of the G8 and G9 viruses but with less reversion. Both of these viruses reactivated from latently infected trigeminal ganglia, albeit inefficiently, and most viruses that reactivated had a uniformly TK-low phenotype. We also generated viruses that have one insertion in a run of six C's or one deletion in a run of five C's. These viruses lack measurable TK activity. However, they reactivated from latently infected ganglia, albeit inefficiently, with the reactivating viruses having reverted to the wild-type TK phenotype. Therefore, for G-string mutants, levels of active TK as low as 0.25% generated by translational mechanisms can suffice for reactivation, but reversion can also contribute. For viruses that lack TK activity due to mutations on other homopolymeric sequences, reactivation can occur via reversion.     

Herpes simplex virus 1 mutant deleted in the alpha 22 gene: growth and gene expression in permissive and restrictive cells and establishment of latency in mice.

R325-beta TK+, a herpes simplex virus 1 mutant carrying a 500-base-pair deletion in the alpha 22 gene and the wild-type (beta) thymidine kinase (TK) gene, was previously shown to grow efficiently in HEp-2 and Vero cell lines. We report that in rodent cell lines exemplified by the Rat-1 line, plating efficiency was reduced and growth was multiplicity dependent. A similar multiplicity dependence for growth and lack of virus spread at low multiplicity was seen in resting, confluent human embryonic lung (HEL) cells. The shutoff of synthesis of beta proteins was delayed and the duration of synthesis of gamma proteins was extended in R325-beta TK+-infected HEL cells relative to cells infected with the wild-type parent, but no significant differences were seen in the total accumulation of viral DNA. To quantify the effect on late (gamma 2) gene expression, a recombinant carrying the deletion in the alpha 22 gene and a gamma 2-TK gene (R325-gamma 2 TK) was constructed and compared with a wild-type virus (R3112) carrying a chimeric gamma 2-TK gene. In Vero cells, the gamma 2-TK gene of R325-gamma 2TK was expressed earlier than and at the same level as the gamma 2-TK gene of R3112. In the confluent resting HEL cells, the expression of the gamma 2-TK gene of the alpha 22- virus was grossly reduced relative to that of the alpha 22+ virus. Electron microscopic studies indicated that the number of intranuclear capsids of R325-beta TK+ virus was reduced relative to that of the parent virus in resting confluent HEL cells, but the number of DNA-containing capsids was higher. Notwithstanding the grossly reduced neurovirulence on intracerebral inoculation in mice, R325-beta TK+ virus was able to establish latency in mice. We conclude that (i) the alpha 22 gene affects late (gamma 2) gene expression, and (ii) a host cell factor complements that function of the alpha 22 gene to a greater extent in HEp-2 and Vero cells than in confluent, resting HEL cells.     

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.     

Replication ofAutographa californica nuclear polyhedrosis virus in a thymidine kinse deficientSpodoptera exigua cell line (SE-UCR-1A)

Summary Cultivation of aSpodoptera exigua cloned cell line (SE-UCR-1A) for 8 to 9 mo. in a medium containing increasing amounts of bromodeoxyuridine (BUdr) resulted in the selection of a BUdr-resistant subline unable to grow in TNMFH medium supplemented with HAT (hypoxanthine, 10⁻⁴ M; aminopterin, 10⁻⁷ M; thymidine, 10⁻³ M). Subsequent assay of this subline revealed an absence of thymidine kinase (TK) activity. The specific activity of the wild-type (wt) cells was 878±192 counts per min (cpm)/μg supernatant protein compared to 9 cpm/μg for the BUdr-resistant, HAT-sensitive subline. In addition the wt activity was inhibited >90% by addition of BUdr to the assay, indicating that the activity is predominantly due to TK and not to a nonspecific nucleoside phosphotransferase. The morphology of the TK-deficient (−) cells was indistinguishable from that of wt cells. The doubling time for wt cells in TNMFH was 16 h; however, in TNMFH-HAT it was 36 h. In comparison the TK(−) cells in TNMFH had a doubling time of 61 h. Cultivation of TK(−) cells in nonselective TNMFH for 14 mo. to date has not changed the TK(−) characteristic of the subline. The host-cell TK was not required for development of progeny virus fromAutographa californica NPV inoculum. Although similar numbers of cells were infected (80 to 90%) in the wt and TK(−) lines and extracellular virus was generated at similar rates to similar titers in both media, the initial appearance of virus in the medium of TK(−) cell was delayed 10 to 20 h compared to wt cells. In addition, polyhedra appearance was similarly delayed in TK(−) cells and only 20 to 25% of the cells contained >10 polyhedra per cell compared to 75 to 90% for wt cells. Also, during infection of wt cells, specific activity of TK increased twofold peaking at 20 to 30 h postinfection, yet there was no stimulation of TK activity in infected TK(−) cells.     

Genetically Engineered Vesicular Stomatitis Virus in Gene Therapy: Application for Treatment of Malignant Disease

We report here the generation of recombinant vesicular stomatitis virus (VSV) able to produce the suicide gene product thymidine kinase (TK) or cytokine interleukin 4 (IL-4). In vitro cells infected with the engineered viruses expressed remarkably high levels of biologically active TK or IL-4 and showed no defects in replication compared to the wild-type virus. Recombinant viruses retained their ability to induce potent apoptosis in a variety of cancer cells, while normal cells were evidently more resistant to infection and were completely protected by interferon. Significantly, following direct intratumoral inoculation, VSV expressing either TK or IL-4 exhibited considerably more oncolytic activity against syngeneic breast or melanoma tumors in murine models than did the wild-type virus or control recombinant viruses expressing green fluorescent protein (GFP). Complete regression of a number of tumors was achieved, and increased granulocyte-infiltrating activity with concomitant, antitumor cytotoxic T-cell responses was observed. Aside from discovering greater oncolytic activity following direct intratumoral inoculation, however, we also established that VSV expressing IL-4 or TK, but not GFP, was able to exert enhanced antitumor activity against metastatic disease. Following intravenous administration of the recombinant viruses, immunocompetent BALB/c mice inoculated with mammary adenocarcinoma exhibited prolonged survival against lethal lung metastasis. Our data demonstrate the validity of developing novel types of engineered VSV for recombinant protein production and as a gene therapy vector for the treatment of malignant and other disease.     

Transfer of Thymidine Kinase to Thymidine Kinaseless L Cells by Infection with Ultraviolet-Irradiated Herpes Simplex Virus

L cells lacking thymidine kinase (TK) activity (Ltk(-) cells) have been stably transformed to a TK-positive phenotype by infection with ultraviolet-irradiated herpes simplex virus (HSV-UV). The highest frequency of the Ltk(-) to Ltk(+) transformation observed in these experiments was approximately 10(-3), whereas no measurable transformation was observed (less than 10(-8)) in the absence of HSV-UV infection. Cell lines of HSV-transformed Ltk(+) cell lines contain 7 to 24 times as much TK activity as do the parental Ltk(-) cells, and they have been maintained in culture for a period exceeding 8 months. The kinetics of thermal inactivation of the TK activity derived from an Ltk(+) HSV-transformed cell line and the TK activity from Ltk(-) cells lytically infected with infectious HSV are similar. Both of these TK activities are much more thermolabile than the TK activity present in wild-type L cells. A mutant strain of HSV which does not induce TK activity during lytic infection does not cause the Ltk(-) to Ltk(+) transformation. These data suggest that either an HSV TK gene has been transferred to Ltk(-) cells or that an HSV gene product has caused the expression of a previously repressed cellular enzyme.     

Enzymatic activity of poliovirus RNA polymerases with mutations at the tyrosine residue of the conserved YGDD motif: isolation and characterization of polioviruses containing RNA polymerases with FGDD and MGDD sequences.

The poliovirus RNA-dependent RNA polymerase (3Dpol) shares a region of homology with all RNA polymerases, centered around the amino acid motif YGDD, which has been postulated to be involved in the catalytic activity of the enzyme. Using oligonucleotide site-directed mutagenesis, we substituted the tyrosine at this motif of the poliovirus RNA-dependent RNA polymerase with cysteine, histidine, isoleucine, methionine, phenylalanine, or serine. The enzymes were expressed in Escherichia coli, and in vitro enzyme activity was tested. The phenylalanine and methionine substitutions resulted in enzymes with activity equal to that of the wild-type enzyme. The cysteine substitution resulted in an enzyme with approximately 50% of the wild-type activity, while the serine substitution resulted in an enzyme with approximately 10% of the wild-type activity; the isoleucine and histidine substitutions resulted in background levels of enzyme activity. To assess the effects of the mutants in viral replication, the mutant polymerase genes were subcloned into the infectious cDNA clone of poliovirus. Transfection of poliovirus cDNA containing the phenylalanine mutation in 3Dpol gave rise to virus in all of the transfection trials, while cDNA containing the methionine mutation resulted in virus in only 3 of 40 transfections. Transfection of cDNAs containing the other substitutions at the tyrosine residue did not result in infectious virus. The recovered viruses demonstrated kinetics of replication similar to those of the wild-type virus, as measured by [3H]uridine incorporation at either 37 or 39 degrees C. RNA sequence analysis of the 3Dpol gene of both viruses demonstrated that the tyrosine-to-phenylalanine or tyrosine-to-methionine mutation was still present. No other differences in the 3Dpol gene between the wild-type and phenylalanine-containing virus were found. The virus containing the methionine mutation also contained two other nucleotide changes from the wild-type 3Dpol sequence; one resulted in a glutamic acid-to-aspartic acid change at amino acid 108 of the polymerase, and the other resulted in a C-to-T base change at nucleotide 6724, which did not result in an amino acid change. To confirm that the second amino acid mutation found in the 3Dpol gene of the methionine-substituted virus allowed for replication ability, a mutation corresponding to the glutamic acid-to-aspartic acid change was made in the polymerase containing the methionine substitution, and this double-mutant polymerase was expressed in E. coli. The double-mutant enzyme was as active as the wild-type enzyme under in vitro assay conditions.(ABSTRACT TRUNCATED AT 400 WORDS)     

Inhibition of viral gene expression by human ribonuclease P.

External guide sequences (EGSs) are small RNA molecules which consist of a sequence complementary to a target mRNA and render the target RNA susceptible to degradation by ribonuclease P (RNase P). EGSs were designed to target the mRNA encoding thymidine kinase (TK) of herpes simplex virus 1 for degradation. These EGSs were shown to be able to direct human RNase P to cleave the TK mRNA sequence efficiently in vitro. A reduction of about 80% in the expression level of both TK mRNA and protein was observed in human cells that steadily expressed an EGS, but not in cells that either did not express the EGS or produced a "disabled" EGS which carried a single nucleotide mutation that precluded RNase P recognition. Thus, EGSs may represent novel gene-targeting agents for inhibition of gene expression and antiviral activity.     

Mutation of herpesvirus thymidine kinase to generate ganciclovir-specific kinases for use in cancer gene therapies

Understanding the functional and mechanistic properties of the multi-substrate herpes simplex virus type-1 thymidine kinase (HSV-1 TK) remains critical to defining its role as a major pharmacological target in herpesvirus and gene therapies for cancer. An inherent limitation of the activity of HSV-TK is the >70-fold difference in the K(m)s for phosphorylation of thymidine over the pro-drug ganciclovir (GCV). To engineer an HSV-1 TK isoform that is specific for GCV as the preferred substrate, 16 site-specific mutants were generated. The mutations were concentrated at conserved residues involved in nucleoside base binding, Gln125 and near sites 3 and 4 involved in catalysis and substrate binding. The substrate preferences of each mutant enzyme were compared with wild-type HSV-1 TK. One mutant, termed Q7530 TK, had a lower K(m) for GCV than thymidine. Expression of the Q7530 TK in tumor cells indicated comparable metabolism to and improved sensitivity to GCV over wild-type HSV-1 TK, with minimal thymidine phosphorylation activity. A molecular modeling simulation of the different HSV-1 TK active-sites was done for GCV and thymidine binding. It was concluded that mutations at Gln125 and near site 4, especially at Ala168, were responsible for loss of deoxypyrimidine substrate binding.