Cloning, sequence analysis, and expression of the major capsid protein of the iridovirus frog virus 3.

The nucleotide sequence of the gene encoding in the major capsid protein (MCP) of frog virus 3 (FV3) has been determined and compared to other iridovirus capsid genes. Nucleotide sequence and S1 nuclease analysis showed that the FV3 MCP gene encoded a transcript of 1452 nucleotides containing a 12 nucleotide AU-rich 5' nontranslated region (NTR) and a 50-nucleotide 3' NTR whose terminus was predicted to fold into a hairpin of moderate stability. An open reading frame initiating from the the 5'-most AUG codon encoded a protein of 463 amino acids with a predicted molecular weight of 49,860. Expression of the putative FV3 MCP gene in vitro confirmed that it encoded the major capsid protein of FV3 and supported the suggestion that translation initiated at AUG-1. Pairwise amino acid alignments detected a high degree of sequence identity between the FV3 MCP and other iridoviruses. These results indicate that iridoviruses possess an evolutionarily related major capsid protein and provide information useful not only in studies of viral gene expression, but also in characterizing newly isolated iridoviruses.

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.

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.

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.

Herpesviral deoxythymidine kinases contain a site analogous to the phosphoryl-binding arginine-rich region of porcine adenylate kinase; comparison of secondary structure predictions and conservation.

Twelve herpesviral deoxythymidine kinases were examined for regions of sequence similarity by multiple alignment. Six highly conserved sites were observed. Site 1 corresponded to a glycine-rich loop that forms part of the ATP-binding pocket in porcine adenylate kinase (PAK), and site 5 corresponded to a region in PAK, located on one lobe of the cleft, that contains arginine residues that bind substrate phosphoryl groups. Site 3, consisting of the motif -DRH-, is thought to be involved in thymine/deoxythymidine recognition; site 4, which is nearby, probably participates in this function as well. The functions of sites 2 and 6 have not been identified. Secondary structure predictions were made by the Garnier method and averaged for each position in the multiple alignment. The structure predicted for all six sites was typically a short flexible region (turn or coil) at or adjacent to the site, flanked by rigid structures (helix or sheet) on either side.

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.

Interaction of nucleolar phosphoprotein B23 with nucleic acids.

The interaction of eukaryotic nucleolar phosphoprotein B23 with nucleic acids was examined by gel retardation and filter binding assays, by fluorescence techniques, and by circular dichroism. All studies utilized protein prepared under native conditions by a newly developed purification procedure. Electrophoretic gel mobility shift assays with phage M13 DNA suggested that protein B23 is a single-stranded nucleic acid binding protein. This was confirmed in competition binding assays with native or heat-denatured linearized plasmid pUC18 DNA where the protein showed a marked preference for the denatured form. In other competition assays, there was no apparent preference for single-stranded synthetic ribo- versus deoxyribonucleotides. Equilibrium binding with poly(riboethenoadenylic acid) indicated cooperative ligand binding with a protein binding site size of 11 nucleotides and an apparent binding constant (K omega) of 5 x 10(7) M-1 which includes an intrinsic binding constant (K) of 6.3 x 10(4) M-1 and a cooperativity factor (omega) of 800. In circular dichroism (CD) studies, protein B23, when combined with the single-stranded synthetic nucleic acids poly(rA) and poly(rC), effected a decrease in ellipticity and a shift of the positive peak at 260-270 nm toward higher wavelengths, indicating helix destabilizing activity. No CD changes were seen with double-stranded poly(dA.dT). The change in ellipticity of poly(rA) was sigmoidal upon addition of protein, confirming the cooperative behavior seen with fluorescence methods. These studies indicate that protein B23 binds cooperatively with high affinity for single-stranded nucleic acids and exhibits RNA helix destabilizing activity. These features may be related to its role in ribosome assembly.

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.

Sequence analyses of herpesviral enzymes suggest an ancient origin for human sexual behavior.

Comparison of the amino acid sequences of the deoxythymidine kinases of herpes simplex (HSV) and of marmoset herpes viruses (MHV) suggests a divergence time of 8 to 10 million years ago for HSV-1 and -2. Like MHV, HSV-1 and -2 cause local infections in their natural hosts, and direct contact between two individuals during the brief period of infectivity is needed for transmission. Because B virus, a nearer relative of HSV, depends on both oral and genital routes of transmission, we postulate that ancestral HSV (aHSV) was similar, and that for HSV-1 and -2 to diverge, genital and oral sites had to become microbiologically somewhat isolated from each other, while oral--oral and genital--genital contact had to be facilitated to maintain both aHSV strains. We propose that acquisition of continual sexual attractiveness by the ancestral human female and the adoption of close face-to-face mating, two hallmarks of human sexual behavior, provided the conditions for the divergence.

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.

Isolation and differentiation of herpes simplex virus and Trichomonas vaginalis in cell culture.

During the period January 1982 to January 1985, 2,234 specimens were cultured for isolation of herpes simplex virus (HSV). HSV was isolated from 23% of these, Trichomonas vaginalis was isolated from 1.6%, and 75.3% were negative. In 0.2% of these, HSV and T. vaginalis were isolated from the same specimen. Cytopathic effects produced by HSV were identified by their sensitivity to arabinosylthymine, whereas those produced by T. vaginalis were identified by their lack of sensitivity to arabinosylthymine and by observation of motility. Cytopathic effects produced by T. vaginalis were reproduced by trophozoites from axenic cultures of T. vaginalis as well as by lysates of T. vaginalis added to serum-free BHK cells.

Herpes simplex virus type 1 and neuronal cells--a special cell-virus interaction.

Rat brain glioma cells were semipermissive for herpes simplex virus (HSV) replication, because the growth of HSV was multiplicity-dependent in these cells. By using this property, we successfully isolated 'survivor' glioma cells following HSV infection at low multiplicity and without using any special treatment (such as UV irradiation) either of the cells or of the virus. Under the same conditions there were no survivor BHK or 3T3 cells, which suggests the uniqueness of the glioma cell-HSV interaction. The survivor cells ceased to produce infectious virus after two subcultures, but were highly resistant to superinfection for at least 20 subcultures. Parental cells were significantly more permissive for homologous virus growth than survivor cells. Interferon was apparently not induced in the survivor cells, because they were as susceptible as the parental cells to infection with vesicular stomatitis virus. The survivor cells produced HSV-specific antigens and contained HSV-specific DNA.

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.

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.

Alterations in the recognition of nucleoside analogues as substrates by the deoxythymidine kinase of a 5-methoxymethyldeoxyuridine-resistant mutant of herpes simplex virus type 1.

Inhibition constants (Kis) were used as an estimate of the ability of various nucleoside analogues to be recognized as substrates by the deoxythymidine kinases (dTKs) of a 5-methoxymethyldeoxyuridine-resistant (MMdUr) mutant of herpes simplex virus type 1 (HSV-1) and its parent wild-type (wt). It was found that the Kis for the 5-position analogues MMdU, [E]-5-(2-bromovinyl)deoxyuridine, bromodeoxyuridine and iododeoxyuridine were increased approximately three-to fivefold, suggesting that they were poorer substrates for the MMdUr dTK than for the wt dTK. With the 2' analogues arabinosylthymine and 2' fluoro 5-methylarabinosyluracil, however, the Kis were increased to a much greater extent, 80- and 240-fold, respectively. These findings suggest that the resistance of the mutant MMdUr to these analogues may be due to a mutation(s) in the viral dTK that directly affects binding at the 2' recognition site and indirectly at the 5, while still allowing substantial activity with the natural substrate deoxythymidine.

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).

Thymine salvage, mitochondria, and the evolution of the herpesviruses.

The salvage of thymine is an apparently ubiquitous feature of free-living lifeforms as well as of mitochondria, chloroplasts and most of the large DNA viruses. Assumptions and data are described which explain the evolution of thymine salvage in prokaryotes, animal cells, and large DNA viruses, in terms of deoxythymidine kinase and its relationship to mitochondria. Specifically, it is suggested that regulation of deoxythymidine kinase (by end-product inhibition) has evolved as a means of assuring a constant supply of thymine compounds for the mitochondria and that the degree to which this regulation is present in the deoxythymidine kinases of the various herpesviruses correlates with the degree of dependence of their replicative cycle on the continued health of the mitochondria of their host cells.