Translational compensation of a frameshift mutation affecting herpes simplex virus thymidine kinase is sufficient to permit reactivation from latency

Herpes simplex virus thymidine kinase is important for reactivation of virus from its latent state and is a target for the antiviral drug acyclovir. Most acyclovir-resistant isolates have mutations in the thymidine kinase gene; however, how these mutations confer clinically relevant resistance is unclear. Reactivation from explanted mouse ganglia was previously observed with a patient-derived drug-resistant isolate carrying a single guanine insertion within a run of guanines in the thymidine kinase gene. Despite this mutation, low levels of active enzyme were synthesized following an unusual ribosomal frameshift. Here we report that a virus, generated from a pretherapy isolate from the same patient, engineered to lack thymidine kinase activity, was competent for reactivation. This suggested that the clinical isolate contains alleles of other genes that permit reactivation in the absence of thymidine kinase. Therefore, to establish whether thymidine kinase synthesized via a ribosomal frameshift was sufficient for reactivation under conditions where reactivation requires this enzyme, we introduced the mutation into the well-characterized strain KOS. This mutant virus reactivated from latency, albeit less efficiently than KOS. Plaque autoradiography revealed three phenotypes of reactivating viruses: uniformly low thymidine kinase activity, mixed high and low activity, and uniformly high activity. We generated a recombinant thymidine kinase-null virus from a reactivating virus expressing uniformly low activity. This virus did not reactivate, confirming that mutations in other genes that would influence reactivation had not arisen. Therefore, in strains that require thymidine kinase for reactivation from latency, low levels of enzyme synthesized via a ribosomal frameshift can suffice.     

Quantitative polymerase chain reaction analysis of herpes simplex virus DNA in ganglia of mice infected with replication-incompetent mutants.

To study the roles of viral genes in the establishment and maintenance of herpes simplex virus (HSV) latency, we have developed a polymerase chain reaction assay that is both quantitative and sensitive. Using this assay, we analyzed the levels of viral DNA in trigeminal ganglia of mice inoculated corneally with HSV mutants that are defective for virus replication at one or more sites in mice and for reactivation upon ganglionic explant. Ganglia from mice infected with thymidine kinase-negative mutants, which replicate at the site of inoculation and establish latency but do not replicate acutely in ganglia or reactivate upon explant, contained a range of levels of HSV DNA that overlapped with the range found in ganglia latently infected with wild-type virus. On average, these mutant-infected ganglia contained one copy of HSV DNA per 100 cell equivalents (ca. 10(4) molecules), which was 50-fold less than the average for wild-type virus. Ganglia from mice infected with a ribonucleotide reductase deletion mutant, which is defective for acute replication and reactivation upon ganglionic explant, also contained on average one copy of HSV DNA per 100 cell equivalents. We also detected substantial numbers of HSV DNA molecules (up to ca. 10(3] in ganglia of mice infected with an ICP4 deletion mutant and other replication-negative mutants that are severely impaired for viral DNA replication and gene expression. These results raise the possibility that such mutants can establish latency, which could have important implications for mechanisms of latency and for vaccine and antiviral drug development.     

Database Searching for Thymidine and Thymidylate Kinase Inhibitors Using Three-dimensional Structure-based Methods

Structure-based drug design methods were used to search for novel inhibitors of herpes simplex virus type 1 (HSV-1) thymidine kinase and Mycobacterium tuberculosis thymidylate kinase. The method involved the use of crystal structure complexes to guide database searching for potential inhibitors. A number of weak inhibitors of HSV-2 were identified, one of which was found to inhibit HSV-1 TK and HSV-1 TK-deficient viral strains. Each compound tested against M. tuberculosis thymidylate kinase was found to have some activity. The best of these compounds was only 4.6-fold less potent than 3′-azido-3′-deoxythymidine-5′-monophosphate (AZTMP). This study demonstrates the utility of structure-based drug design methods in the search for novel enzyme inhibitors.     

Use of [125I]deoxycytidine to detect herpes simplex virus-specific thymidine kinase in tissues of latently infected guinea pigs

The footpad skin and the lumbosacral dorsal root ganglia were removed from inbred guinea pigs at different times after subcutaneous infection with herpes simplex virus type 2 (HSV-2) in both hind footpads. These tissues, shown by our previous study to harbor latent HSV, were dispersed into single cells. The presence of virus-specific thymidine kinase (TK) in these cells was assayed by the uptake and phosphorylation of [125I]deoxycytidine in culture. [125I]deoxycytidine was shown to be a specific substrate for the HSV-coded TK. The method could detect herpes TK activity in a culture of 10(6) cells with less than 0.1% of the cells being virally infected. The enzyme was readily detected in footpad cells of acutely (24 h) but not of latently (14 days to 1 year) infected guinea pigs. No herpes TK was found either in the sensory ganglionic cells of guinea pigs during the early and late phases of latent infection. It is concluded that HSV-2, while residing in the footpads and the lumbosacral ganglia of the guinea pig during latent infection, does not express any viral TK function.     

Thymidine kinase from sea urchin oocytes

Thymidine kinase was isolated and purified 1600-fold from sea urchin (Strongylocentrotus intermedius) oocytes. The molecular mass of the enzyme is 66 kDa, pI is 5.2. The enzyme activity needs Mg2+, ATP and the corresponding phosphate acceptor. The pH optimum of the enzyme activity is at 9.0-9.5. The isolated enzyme does not exhibit any strict substrate specificity and can phosphorylate thymidine, deoxycytidine and some other pyrimidine nucleosides and their derivatives, the phosphorylation rate being maximal for thymidine, ATP or dATP. The TMP formed via the enzymatic reaction does not influence the thymidine kinase activity.     

Thymidine kinase with altered substrate specificity of acyclovir resistant varicella-zoster virus

The acyclovir resistant mutant of varicella-zoster virus ACV-R (A 8) induced the same level of thymidine kinase activity in infected cells as the parent Kawaguchi strain. However, it induced less deoxycytidine kinase activity and did not induce phosphorylating activity for the nucleotide analogue, 9-(2 hydroxy-ethoxymethyl)-guanine-(acyclovir). Another acyclovir resistant mutant, ACV-R (A 4), which is cross-resistant to phosphonoacetate and is thought to be a viral DNA polymerase mutant, induced the same level of phosphorylating activities for thymidine, deoxycytidine and acyclovir as the parent strain. The altered substrate specificity of thymidine kinase induced by ACV-R (A 8) is concluded to confer resistance to acyclovir on ACV-R (A 8).     

Association of thymidylate kinase activity with pyrimidine deoxyribonucleoside kinase induced by herpes simplex virus.

Thymidine kinase derived from LMTK+ does not exhibit thymidylate kinase activity. However, protein isolated by affinity column chromatography from thymidine kinase-deficient mouse cells (LMTK-) infected by herpes simplex virus type 1 shows thymidylate kinase activity in addition to thymidine kinase and deoxycytidine kinase activities. The virus-induced multifunctional enzyme has a molecular weight of 85,000, whereas the molecular weight of thymidylate kinase from uninfected LMTK- mouse cells is 71,000. The virus-induced enzyme has a Km for thymidine of 0.8 micromolar, and for thymidylate of 25 micromolar, and for thymidylate of 25 micromolar; the ratio of Vmax for thymidylate kinase to thymidine kinase is 1.7. When subjected to isoelectric focusing, thymidylate kinase activity is not separated from thymidine kinase activity, and even though four peaks of activity are observed they have a constant ratio of thymidylate kinase to thymidine kinase activity. The isoelectric points (pI) of these four peaks are 4.8, 5.8, 6.2, and 6.6, respectively. Thymidylate kinase, derived from uninfected cells when subjected to isoelectric focusing, separates into a major component with an isoelectric point at pH 8.2 and a minor component at pH 7.7. Although thymidine and thymidylate kinase activities derived from the virus-infected cells cannot be separated either by affinity column chromatography, glycerol density gradient centrifugation, or isoelectric focusing, there is a differential rate of inactivation when the enzyme is subjected to incubation at 37 degrees, with thymidylate kinase activity being more labile than thymidine kinase activity.     

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.     

THYMIDINE TRIPHOSPHATE SYNTHESIS IN TETRAHYMENA : I. Studies on Thymidine Kinase

The amount of thymidine-H³ converted to thymidine-H³ monophosphate in 30 min formed the basis for assays of thymidine kinase in cell extracts from Tetrahymena pyriformis. The optimal concentration of adenosine triphosphate is lower than that required by other cell types. Thymidine triphosphate does not exercise any feedback control of the enzyme. Other deoxyprimidine nucleotides were tested, but these also failed to exhibit any feedback inhibition. At suboptimal adenosine triphosphate levels, thymidine triphosphate and other deoxypyrimidine nucleotides stimulate the reaction, suggesting that these nucleotides may act either directly or indirectly as phosphate donors in the crude enzyme preparations. This possibility was affirmed when thymidine triphosphate and deoxycytidine triphosphate were shown to be capable of limited phosphorylation of thymidine. Comparison of enzymatic activities in logarithmically growing culture and stationary phase culture, in which nuclear DNA synthesis has virtually ceased, reveals no change in enzymatic activity. The results suggest that thymidine kinase is a constitutive enzyme in Tetrahymena.     

Analysis of the Relationship between Cellular Thymidine Kinase Activity and Virulence of Thymidine Kinase-Negative Herpes Simplex Virus Types 1 and 2

The virulence of thymidine kinase-negative herpes simplex virus type 1 (HSV-1; VRTK^? strain) and type 2 (HSV-2; UWTK^? strain) was studied in comparison with that of their parental strains (VR-3 and UW-268, respectively) in an encephalitis model of adult (4-week-old) and newborn (3-day-old) mice. Viral thymidine kinase (TK) activity was essential for the maximum expression of virulence of HSV-1, because the 50% lethal dose (LD₅₀) of VRTK^? was 60 times higher than that of VR-3 in the brains of newborn mice expressing high levels of cellular TK activity. However, the UWTK^? strain showed the same virulence as the parental strain in newborn mice, despite the lack virulence in adults, suggesting that replication of the UWTK^? strain was completely supported by cellular TK activity. This difference in the role of viral and cellular TKs for virus growth between HSV-1 and HSV-2 was confirmed with the one-step growth of virus strains in L-M and L-M(TK^?) cells.     

Evidence for CDK-dependent and CDK-independent functions of the murine gammaherpesvirus 68 v-cyclin

Gamma-2 herpesviruses encode homologues of mammalian D-type cyclins (v-cyclins), which likely function to manipulate the cell cycle, thereby providing a cellular environment conducive to virus replication and/or reactivation from latency. We have previously shown that the v-cyclin of murine gammaherpesvirus 68 is an oncogene that binds and activates cellular cyclin-dependent kinases (CDKs) and is required for efficient reactivation from latency. To determine the contribution of v-cyclin-mediated cell cycle regulation to the viral life cycle, recombinant viruses in which specific point mutations (E133V or K104E) were introduced into the v-cyclin open reading frame were generated, resulting in the disruption of CDK binding and activation. While in vitro growth of these mutant viruses was unaffected, lytic replication in the lungs following low-dose intranasal inoculation was attenuated for both mutants deficient in CDK binding as well as virus in which the entire v-cyclin open reading frame was disrupted by the insertion of a translation termination codon. This replication defect was not apparent in spleens of mice following intraperitoneal inoculation, suggesting a cell type- and/or route-specific dependence on v-cyclin-CDK interactions during the acute phase of virus infection. Notably, although a v-cyclin-null virus was highly attenuated for reactivation from latency, the E133V v-cyclin CDK-binding mutant exhibited only a modest defect in virus reactivation from splenocytes, and neither the E133V nor K104E v-cyclin mutants were compromised in reactivation from peritoneal exudate cells. Taken together, these data suggest that lytic replication and reactivation in vivo are differentially regulated by CDK-dependent and CDK-independent functions of v-cyclin, respectively.     

Origin of Thymidine Kinase in Adenovirus-Infected Human Cell Lines

Human adenovirus type 5 enhances the thymidine kinase activity of KB cells but does not induce the enzyme in kinase-deficient HeLa (BU25) cells. Vaccinia induces thymidine kinase activity in both KB and HeLa (BU25) cells. Human adenovirus types 2, 4, 7, and 12 also fail to induce the enzyme in HeLa (BU25) cells. Vaccinia replicates equally well in the presence or absence of HATG (hypoxanthine-aminopterin-thymidine-glycine) in KB and HeLa (BU25) cells. Adenovirus type 5 replicates in KB and in HeLa (BU25) cells in the absence of HATG, and adenovirus type 5 replicates in kinase-positive KB cells in the presence of HATG. However, replication of adenovirus type 5 is grossly inhibited in HeLa (BU25) cells in the presence of HATG. These results suggest that human adenoviruses do not code for a new virus-specific thymidine kinase.     

Thymidine kinase, thymidylate synthase, and endothelial cell growth under hyperoxia

To determine the respective role of thymidine kinase and thymidylate synthase activities in the hyperoxia-induced decrease in DNA synthesis and their relationship with cell replication, we measured these two enzyme activities in primary cultures of porcine aortic endothelial cells under different O2 concentrations for various durations. In confluent cells, exposure to 95% O2 for 5 days reduced thymidine kinase activity to 15% of control values; thymidylate synthase activity was unaffected. In preconfluent cells exposed to 95% O2 for 2 days, similar results were obtained, together with evidence for arrest in cell proliferation. Thymidylate synthase activity could therefore not be related to decreased cell proliferation under hyperoxia. [3H]thymidine incorporation into DNA, thymidine kinase activity, and cell proliferation were all similarly affected under exposure to graded O2 concentration for 2 days. Thymidine kinase appears to be a key enzyme in the modulation of DNA synthesis from thymidine and in its replication in endothelial cells.     

Immunity in the female genital tract after intravaginal vaccination of mice with an attenuated strain of herpes simplex virus type 2.

Herpes simplex virus type 2 is a common human venereal pathogen which causes lethal neurological illness after intravaginal inoculation into BALB/cJ mice. To investigate whether an attenuated, nonlethal strain of this virus would confer immunity after inoculation of mice, we constructed a strain containing a partial deletion of the thymidine kinase gene, which is necessary for viral replication and spread in sensory ganglia. Unlike its wild-type counterpart, this deletion-containing strain of herpes simplex virus type 2 caused mild clinical disease and was not lethal when studied in an age-dependent murine model of intravaginal infection. Furthermore, after intravaginal infection, the deletion-containing strain could not be isolated from sensory ganglia at a time when wild-type virus was abundant. Of greater significance, intravaginal inoculation with the deletion-containing strain rendered mice completely resistant to rechallenge with a 10-fold 50% lethal dose of wild-type virus. These results suggest that a strain of herpes simplex virus type 2 containing a deletion of the thymidine kinase gene will be useful in studying the cellular basis of mucosal immunity in the genital tract.     

Substrate Specificities of Mitochondrial Thymidine Kinase and Cytosolic Deoxycytidine Kinase Against 5-Aryl Substituted Pyrimidine-2′-deoxyribose Analogues

Abstract

Some 5-aryl-2′-deoxyuridine and -deoxycytidine analogues, many with known antiviral activity, were evaluated as substrates for pure deoxycytidine kinase (dCK) and pure mitochondrial thymidine kinase (TK2). Some of the deoxyuridine compounds were also tested with pure cytosolic thymidine kinase (TK1). TK2 showed the highest activity with this type of analogues.     

Thymidine kinase not required for antiviral activity of acyclovir against mouse cytomegalovirus.

Previous studies of herpesvirus infections have indicated that a virus-specified thymidine kinase is required for the initial phosphorylation of acyclovir [acycloguanosine or 9-(2-hydroxyethoxymethyl)guanine] in the formation of acycloguanosine triphosphate. The latter compound accumulates in infected cells and competitively inhibits the viral DNA polymerase. We found that mouse cytomegalovirus, which does not express a thymidine kinase, was sensitive to the antiviral effects of acyclovir at a 50% inhibitory dose of approximately 0.23 microM. Acyclovir was equally effective against mouse cytomegalovirus in normal 3T3 cells and in 3T3 cells deficient in cellular thymidine kinase. Furthermore, the activity of acyclovir could not be reversed by excess thymidine, which easily reversed the antiviral activity of acyclovir against herpes simplex virus. Using a high-pressure liquid chromatography technique that easily detected acycloguanosine triphosphate in cells infected with herpes simplex virus, we could not detect acycloguanosine triphosphate in mouse cytomegalovirus-infected cells. These experiments demonstrated that the activity of acyclovir against mouse cytomegalovirus is not dependent on a thymidine phosphorylation pathway. Additional experiments are underway to determine whether acycloguanosine triphosphate is produced by another pathway in concentrations sufficient to inhibit mouse cytomegalovirus DNA polymerase.     

Thymidine kinase activity in mouse embryo fibroblast cells infected with murine cytomegalovirus

Thymidine kinase activity was found in whole cell extracts of growing and stationary mouse embryo fibroblast cells after infection with murine cytomegalovirus. Determination of the kinetic constants and heat stability characteristics indicated that the enzyme activity from infected cells was different to that found in uninfected cells in the growth phase. The expression of thymidine kinase activity during virus replication was reflected by the incorporation of (6-³H) thymidine into acid precipitable fractions of infected cell cultures. Preliminary data from kinetic studies showed a reduction in the phosphorylation of thymidine by this enzyme activity in the presence of Acyclovir, a potent inhibitor of herpes virus replication.