Metabolism of deoxycytidine, thymine, and deoxythymidine in the hamster.

The ability of growing and of mature Syrian hamsters to anabolize (to liver DNA) or catabolize (to 14CO2) graded amounts of [2-14C]deoxythymidine (TdR), thymine, or deoxycytidine (CdR) was measured in vivo. Of the three precursors, CdR labeled DNA most efficiently and, as expected, incorporation of all three into DNA was greater in younger animals. The catabolism of [2-14C]CdR to respired 14CO2 was dose dependent and showed no signs whatsoever of saturation, even with the highest dose (greater than 20 mumoles/g liver). In contrast, TdR and thymine were catabolized more slowly and saturation was approached with modest doses. The excretion of CdR in the urine was low and independent of dose, while excretion of TdR and thymine was greater and was dose dependent. Rats tested with an intermediate dose of CdR did not catabolize significant quantities to 14CO2, but did excrete considerably more [C]CdR into the urine than did hamsters. These and other findings suggest that, while the rat and the hamster metabolize thymine (and TdR as well) in a similar fashion, they metabolize CdR quite differently, probably because the hamster has a much higher level of nucleoside aminohydrolase which deaminates CdR and related compounds. Because the human also has a very high level of this enzyme, the hamster appears to be a superior animal model for the study of cytosine-containing compounds intended for human use.

Viral thymidine kinases and their relatives.

Thymidine kinases were described for cellular life long before it was shown that they could also be encoded by viruses, but the viral thymidine kinase genes were the first to be sequenced. These enzymes have been extraordinarily useful to the researcher, serving first to help label DNA, then to get thymidine analogs incorporated into DNA for therapeutic and other purposes and more recently to move genes from one genome to another. Knowledge of the nucleotide and amino acid sequences of these enzymes has allowed some deductions about their possible three-dimensional structure, as well as the location on the polypeptide of various functions; it has also allowed their classification into two main groups: the herpesviral thymidine/eukaryotic deoxycytidine kinases and the poxviral and cellular thymidine kinases; the relationships of the mitochondrial enzyme are still not clear.

Phylogeny of immune recognition: fine specificity of fish immune repertoires to cytochrome C.

Using the structurally defined protein antigen cytochrome C, studies were conducted in an attempt to delineate the fine specificities of channel catfish immune repertoires. We have previously reported that species variants of cytochrome C were cross-stimulatory to peripheral blood leukocytes (PBL) from catfish immunized with the pigeon variant. Molecular database analyses revealed the existence of overlapping epitopes that appear to define the specificity of the immune response to a "family" of closely related antigens. To further explore these observations, studies were conducted to determine the contribution of peptide 81-104 to the immunogenicity of cytochrome C. Current data showed that peptide 81-104 and intact cytochrome C were stimulatory to PBL from fish previously immunized with the native molecule. In contrast, PBL from fish previously primed with the peptide 81-104 responded only to the immunizing peptide as well as to some, but not all, variants of the peptide 81-104. The differences in the stimulatory capacities of the peptide variants appeared to correlate with amino acid substitutions at various positions of the peptide and changes in their predicted secondary structures.

Characterization of a secreted cytoactive factor from Trichomonas vaginalis.

Trichomonas vaginalis, grown in Dulbecco's modified Eagle's medium with or without serum, produced a factor (TVF) which altered the morphology of certain mammalian cells in vitro. TVF had a Mr of approximately 250 kDa by gel filtration, approximately 50 kDa by SDS-PAGE, and was heat (56 degrees C, 30 min) and pH (greater than 6 or less than 8) labile. Co-incubation of TVF with adherent target cells caused a marked rounding and clumping of BHK-21 or CHO-K1 cells, but had no effect on RK-13 or WEHI-3 cells. These morphologic changes were concentration, time, and energy dependent. Reversibility was attained by exogenous serum addition (greater than 10%) or TVF washout. Target cell perturbations were not accompanied by significant changes in growth (as measured by nuclei counts, DNA content, or 3H-thymidine incorporation), in cell leakage (as assessed by lactate dehydrogenase release), or in cell viability (by trypan blue dye exclusion). TVF-induced effects were independent of cyclic AMP and cyclic GMP levels in BHK cells exposed for 5 min-24 hr.

Thymine arabinoside (Ara-T) topical and iontophoretic application for herpes simplex virus type 1 and type 2 skin infections in hairless mice.

The antiviral agent thymine arabinoside (9-beta-D-arabinofuranosyl thymine, Ara-T) was applied topically and by anodal (+) iontophoresis in a NaCl solution to Herpes simplex virus type 1 (HSV-1), and Herpes simplex virus type 2 (HSV-2) skin infections in hairless mice. The chemotherapeutic efficacy of Ara-T was evaluated employing the parameters of mean lesion score, number of mice with signs of neurological involvement (paralysis), mortality and mean survival time. Anodal (+) iontophoresis of 0.4% Ara-T in a 0.1 M NaCl solution significantly suppressed HSV-1 skin infection, but the infection was not altered by topical application of 3% Ara-T. HSV-2 infection was refractory to both topical and iontophoretic application of Ara-T. This is the first report of the in vivo efficacy of Ara-T for HSV-1 skin infections.

Equine herpes virus 1 and pseudorabies virus resistance to 2'-fluoropyrimidine analogs and to bromovinyldeoxyuridine: implications for dTMP kinase activity.

The 2'-fluoropyrimidine nucleoside analogs 1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-5-iodocytosine (FIAC). 1(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-5-methyluracil (FMAU), and 1(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-5-iodouracil (FIAU) showed higher in vitro activity against herpes simplex virus type 1 (HSV-1), than equine herpesvirus 1 (EHV-1) or pseudorabies virus (PRV). Comparison of the 50% plaque inhibitory doses for HSV-1 and its mutant MMdUr-20 in cell cultures with inhibition constants (Ki's) for the viral deoxythymidine kinases (dTKs) suggests that in the infected cell FMAU is phosphorylated by host enzymes. As compared to HSV-1, EHV-1 and PRV were more resistant to E-5-(2-bromovinyl-2'-deoxyuridine (BVdU) and to the 2'-fluoropyrimidine analogs, as are HSV-2 and the HSV-1 mutants MMdUr-20 and S1. Because the dTKs of the latter lack deoxythymidylate kinase (dTMPK) activity, there appears to be a correlation between resistance to these analogs and BVdU on the one hand, and lack of dTMPK activity on the other. We predict that EHV-1 and PRV dTKs will be shown to lack significant dTMPK activity.

Herpesvirus-induced availability of host DNA to exogenous endonuclease.

Deoxyribonuclease I penetrates herpesvirus-infected L cells and degrades host DNA without interfering with viral multiplication. The DNA of uninfected control monolayers of L cells, disrupted by scraping, is similarly attacked. In contrast, undisturbed L cells are not affected. This suggests that altered cell membrane permeability allows the nuclease to enter the cell, thereby permitting access of exogenous deoxyribonuclease to cellular DNA.

Studies on interactions of dTK-HSV mutants with neurons in vitro.

Thymidine kinase negative (dTK-) mutants of herpes simplex virus type 1 (HSV-1) multiplied well in rat brain glioma cells. A proportion (less than 1%) of glioma cells survived the infection with HSV and were designated "survivor" glioma cells. Survivor cells of dTK- mutant virus infection ceased to produce infectious virus after two passages and were highly resistant to both HSV-1 and HSV-2 but not to vesicular stomatitis virus (VSV). Flow cytometric studies indicated morphological differences between parental and survivor glioma cells, and HSV-1 specific antigens as well as DNA were detected in the survivor glioma cells, but only in early passages. Sensitivity to superinfection with HSV appears to correlate to loss of HSV-specific viral DNA in the survivor glioma cells. Survivor glioma cells after several subcultures lost their ability to resist superinfecting HSV, reverted morphologically to the appearance of parental glioma cells and also lost significant amount of HSV-1 specific DNA. These transient survivor glioma cells became persistently infected-virus producer cells upon HSV infection.

Locating a nucleotide-binding site in the thymidine kinase of vaccinia virus and of herpes simplex virus by scoring triply aligned protein sequences.

Computer techniques were used to locate related segments of amino acid sequences in the thymidine kinases of vaccinia virus and of herpes simplex virus type 1 and in porcine adenylate kinase. As determined by a procedure that evaluates triply aligned sequences, the probability that the similarities among the segments described here arose by chance was no greater than 0.001. Because the sequence in porcine adenylate kinase is a nucleotide phosphate-binding site it is concluded that the segments in the vaccinia virus and herpes simplex virus thymidine kinases perform similar functions. The segments are residues 16-23 in porcine adenylate kinase, 11-19 in vaccinia virus thymidine kinase, and 56-64 in herpes simplex virus thymidine kinase.

HSV1-specific thymidylate kinase activity in infected cells.

Several 5-methoxymethyldeoxyuridine (MMdU)-resistant mutants of herpes simplex virus type 1 (HSV1) were classified by measuring their sensitivities to the deoxythymidine kinase (dTK)-dependent antiviral drugs 9-(2-hydroxyethoxymethyl)-guanine (acyclovir, ACV), 1-beta-D-arabinofuranosylthymine (araT), and E-(2)-5-bromovinyldeoxyuridine (BVdU) and to the dTK-independent antiviral drug phosphonoacetate (PAA). Compared to wild-type (WT) virus, all five of the dTK- mutants were highly resistant (greater than or equal to 500-fold) to BVdU and MMdU, moderately resistant to ACV (50- to 100-fold) and araT (10- to 20-fold), but not resistant to PAA. The dTK of the mutant MMdUr-20 (dTK+) appeared to phosphorylate dTMP less well than that of the WT virus, while its affinity for deoxythymidine was not altered. Two other drug-resistant HSV mutants-S1 (isolated against ACV) and B3 (isolated against BVdU)--also showed reduced phosphorylation of dTMP. This suggests that alterations in both dTK and thymidylate kinase activities may determine sensitivity to antiviral drugs.

Alterations in substrate specificity and physicochemical properties of deoxythymidine kinase of a drug-resistant herpes simplex virus type 1 mutant.

The deoxythymidine kinase (dTK) activity of a 5-methoxymethyldeoxyuridine-resistant mutant (MMdU(r)-20) of herpes simplex virus type 1 was compared with that of the parental wild-type (WT) virus. The dTK activity induced by the mutant was consistently less than that induced by the WT virus, was inhibited by antibody specific for herpes simplex virus dTK, and was more thermostable than the WT dTK. Further, it was inhibited to a lesser degree than the WT dTK by the nucleoside analogs MMdU and arabinosylthymine (araT), which suggests that one of the effects of the mutation was a selective alteration in substrate recognition by the dTK. The loss of ability to inhibit the mutant dTK by E-(2)-5-bromovinyldeoxyuridine was not as great as that seen with araT and MMdU. This agrees well with our previous observation that the MMdU(r)-20 mutant of herpes simplex virus is only partially resistant to this analog, as compared with araT and MMdU (V. Veerisetty and G. A. Gentry, Virology 114:576-579, 1981). [2-(14)C]araT was used to explore further the resistance to araT. Extracts of cells infected with the mutant, although producing a small amount of [(14)C]araTMP, were unable to produce [(14)C]araTTP, in contrast to extracts of cells infected with the WT virus. Both extracts, however, produced [(14)C]dTTP from [(14)C]deoxyribosylthymine. Finally, the ability of the extracts to phosphorylate [(14)C]dTMP was examined. It was found that this activity was greatly reduced relative to dTK activity in the case of the mutant. These findings suggest that a mutation in the dTK polypeptide has affected recognition not only of nucleoside substrates but of the nucleotide substrate dTMP as well, which agrees with the suggestion of Chen et al. that both activities are located on the same polypeptide (M. S. Chen and W. H. Prusoff, J. Biol. Chem. 253:1325-1327, 1978; M. S. Chen, J. Walker, and W. H. Prusoff, J. Biol. Chem. 254:10747-10753, 1979; M. S. Chen, W. P. Summers, J. Walker, W. C. Summers, and W. H. Prusoff, J. Virol. 30:942-945, 1979).

Mycoplasmal deoxyribonuclease activity in virus-infected L-cell cultures.

Cell-free extracts of Mycoplasma hominis and medium from 72-hr broth cultures had deoxyribonuclease activity like that of deoxyribonuclease I. Mg(++) stimulated activity, and the pH optimum was between 8.0 and 9.0. Double-stranded or heatdenatured deoxyribonucleic acid (DNA) served as a substrate, and oligonucleotides were produced. Cell-free extracts of L cells infected with M. hominis or M. hominis plus equine abortion virus (equine herpes virus, EAV) had greatly increased activity over that of extracts of L cells or of L cells infected with EAV alone. In the absence of M. hominis, however, extracts had little activity, most of which was in virus-infected cell cultures. Activity was found in the culture medium only in those systems in which M. hominis was present. It is concluded that M. hominis can contribute significant deoxyribonuclease activity to virus-infected as well as virusfree cell cultures. Perhaps the most interesting question arising concerns the ability of EAV, a DNA virus, to replicate successfully despite the presence of deoxyribonuclease activity at the site of replication (the nucleus).

The Epstein-Barr virus genome encodes deoxythymidine kinase activity in a nested internal open reading frame.

The Epstein-Barr virus (EBV) BXLF1 fragment open reading frame (LORF), though to encode deoxythymidine kinase (dTK) activity, and a shorter frame (SORF), starting at an internal in-frame AUG, were isolated by polymerase chain reaction from a plasmid containing the EcoR1 fragment of EBV strain FF-41. These were transfected into dTK-Escherichia coli, producing multiple SORF- or LORF-containing colonies, which expressed dTK. The 243 NH2-terminal residues of the LORF-encoded polypeptide thus are not essential for dTK activity. SORF, with 1,092 bp, is predicted to encode a 36- to 37-kD polypeptide, in the size range of other herpesviral dTKs.