Leonard J. Bello

Regulation of thymidine kinase synthesis in human cells.

Abstract The regulation of thymidine (TdR) kinase synthesis in synchronized cultures of KB cells was studied. The enzyme was found to undergo periodic increases and decreases in activity coinciding approximately with the beginning and end of DNA synthesis. The stability of the enzyme in vivo during these periods of increasing and decreasing activity did not differ significantly suggesting that the alterations in enzyme activity reflect periodic changes in the rate of enzyme synthesis rather than changes in the rate of enzyme degradation. The start of TdR kinase synthesis was insensitive to 10 −3 M hydroxyurea although DNA synthesis was inhibited more than 98% by this concentration of the drug. Termination of TdR kinase synthesis, however, was sensitive to hydroxyurea and in the presence of the drug cells continued synthesis of the enzyme for a prolonged period of time. The relationship between DNA synthesis and termination of TdR kinase synthesis was examined further by treating synchronized cells with hydroxyurea after various fractions of DNA had been replicated. The results obtained demonstrate unequivocally that termination of TdR kinase synthesis requires replication of more than 59% of the cellular DNA. Additional results suggest that, in fact, replication of the total complement of DNA is required. A detailed study of the time at which TdR kinase synthesis is terminated indicates that enzyme synthesis ends at about the time cells begin mitosis rather than at the time DNA synthesis is complete. A study of the effect of actinomycin D on thymidine kinase synthesis revealed that termination is caused by a post-transcriptional block in enzyme synthesis which, in turn, is dependent upon RNA synthesis either during the G 2 period of the cell cycle or late in the DNA synthesis period.

The human herpesvirus-8 ORF 57 gene and its properties

Human herpesvirus-8 (HHV-8) is a gamma(2) lymphotropic herpesvirus associated with Kaposis sarcoma, a major neoplasm of AIDS patients, and with other AIDS-related neoplasms. The HHV-8 ORF 57 gene is conserved throughout the herpesvirus family and has a herpes simplex virus type 1 homologue, IE63 (also termed ICP27), which is an essential regulatory protein and acts at both transcriptional and post-transcriptional levels. We show that, contrary to the published HHV-8 sequence, which predicts a protein of 275 amino acids, the ORF 57 gene is spliced, contains a single intron and encodes a protein of 455 amino acids. For several gammaherpesviruses examined, the upstream coding exon is 16-17 amino acids in length and is rich in methionine residues. When ORF 57 was fused to the gene for enhanced green fluorescent protein (EGFP), the fusion protein exhibited a punctate nuclear distribution that co-localized with the cellular splicing factor SC-35. Unlike the IE63-EGFP fusion protein, ORF 57-EGFP did not shuttle from the nucleus to the cytoplasm in the presence of actinomycin D. However, ORF 57-EGFP was capable of shuttling from a transfected monkey nucleus to a recipient mouse nucleus in an interspecies heterokaryon assay. These data indicate that HHV-8 ORF 57 and IE63 possess certain common properties.

Latency and reactivation of a thymidine kinase-negative bovine herpesvirus 1 deletion mutant

SummaryA bovine herpesvirus 1 (BHV 1) mutant variant with a deletion in the thymidine kinase (TK) gene was assessed for its ability to establish latency and be reactivated in cattle. After treatment with dexamethasone, reactivated TK− BHV 1 was isolated from one of four cattle that received virus by intravenous inoculation only, and from four of four cattle that received virus by intranasal, intravaginal, and intravenous inoculation. Results prove that TK− BHV 1 will establish latency and can be reactivated in the natural host.

Fusion to C3d Enhances the Immunogenicity of the E2 Glycoprotein of Type 2 Bovine Viral Diarrhea Virus

ABSTRACT The use of DNA and protein subunit vaccines in animals provides an opportunity to introduce vaccines that are arguably the safest that can be developed. For that reason, considerable effort is under way to devise methods of enhancing the immunogenicity of such vaccines. Seven years ago it was shown that fusing complement fragment C3d to hen egg lysozyme (HEL) enhanced the immunogenicity of HEL 10,000-fold. Based on this observation, we decided to evaluate the effect of C3d on the immunogenicity of the E2 protein of bovine viral diarrhea virus (BVDV). E2 is the major target of neutralizing antibody during BVDV infection. To test the effect of C3d on E2 immunogenicity, expression cassettes encoding a secreted form of E2 alone (E2s) or E2 fused to three copies of murine C3d (E2s-C3d) were constructed. The proteins were purified from the supernatants of transfected cells and used to immunize mice. The immune response was monitored by an enzyme-linked immunosorbent assay (ELISA) for E2s-specific antibody and by a virus neutralization test. The ELISA results indicated that the E2s-C3d protein is 10,000-fold more immunogenic than the E2s protein alone. The maximum primary immune response was elicited with <0.1 μg of E2s-C3d protein without an adjuvant. In addition, we have shown for the first time that high levels of anti-E2s and neutralizing antibodies can be elicited when this same low concentration of E2s-C3d is used to both prime and boost the immune response. We conclude that the E2s-C3d fusion protein has significant potential as a subunit vaccine against BVDV infection.

DNA dependent RNA polymerase of KB cells I. Isolation of the enzymes and transcription of viral DNA, mammalian DNA and chromatin

Abstract 1. Two forms (nucleolar and nucleoplasmic) of RNA polymerase (ribonucleoside triphosphate: RNA nucleotidyl transferase, EC 2.7.7.6) have been isolated from KB cells, a neoplastic human cell line. The transcriptional properties of these enzymes have been studied using a variety of natural templates. 2. The properties of RNA polymerase obtained from adenovirus-infected cells are similar to those of polymerase from uninfected cells. 3. Adenovirus DNA is a relatively ineffective template for both forms of KB RNA polymerase. Optimal salt and cation conditions for transcription of adenovirus DNA differ from the conditions which are optimal for transcription of mammalian DNA. 4. Both nucleolar and nucleoplasmic forms of the enzyme transcribe chromatin. Optimal salt conditions for the transcription of chromatin differ from the conditions which are optimal for the transcription of DNA.

Studies on gene activity in synchronized cultures of mammalian cells

Abstract Cultures of KB cells synchronized by excess thymidine treatment were used to study the synthesis of DNA-like RNA (D-RNA) and the enzymes lactic dehydrogenase and fumarase during the mammalian cell cycle. Hybridization competition experiments were employed to determine whether the populations of D-RNA molecules synthesized during two different portions of the mammalian cell cycle were completely different. No difference could be demonstrated suggesting that at least some of the same species of D-RNA were synthesized during both time periods examined. An investigation of the pattern of synthesis of the enzymes lactic dehydrogenase and fumarase in synchronized cells indicated that both enzymes were synthesized continuously. The possibility that some mammalian genes function continuously during interphase is discussed.

Relationship of bovine herpesvirus 1 immediate-early, early, and late gene expression to host cellular gene transcription

Bovine herpesvirus 1 (BHV-1) gene expression was examined by RNA blot hybridization using clones representing immediate-early, early, and late genes. An immediate-early protein gene probe hybridized with two transcripts, 3.4 and 5.8 kb, expressed by infected cells in the presence of cycloheximide (CH). During infection of cells without metabolic inhibitors these transcripts were detected as early as 2 hr postinfection (p.i.) and accumulated to 8 hr p.i. The early gene probe, thymidine kinase, hybridized with a 4.3-kb RNA that was detected in the presence of phosphonoacetic acid (PAA), but not in the presence of CH. The late gene probe, glycoprotein III, (gIII) hybridized with a 1.6-kb transcript that was not expressed by infected cells treated with CH and only in very reduced amounts by infected cells treated with PAA. The gIII RNA was not detected until 4 hr p.i. in total cell RNA. Transcripts for the bovine actin and beta-galactosyltransferase genes did not decrease in BHV-1-infected cells until 6 hr p.i., coincident with the increase of BHV-1 DNA and RNA synthesis. Consequently, shutoff of host cell transcription by BHV-1 may be different than what has been described for herpes simplex virus.

Stabilization of heterogeneous nuclear RNA by intercalating drugs

The effect of the intercalating drugs proflavine, ethidium and daunomycin on the rate of degradation of 14C-labeled heterogeneous nuclear RNA (HnRNA) in KB cells was studied. All three drugs decreased the rate of degradation of 14C-HnRNA to acid soluble products. The most striking effect was produced by proflavine which promptly and completely stabilized 14C-HnRNA against degradation. Ethidium also produced complete stabilization after a delay of 30 to 60 min. Daunomycin decreased the rate of 14C-HnRNA degradation but did not alter the fraction of 14C-HnRNA which was ultimately degraded. The results are consistent with the view that base-paired sequences are present in HnRNA in vivo and play a role in the processing of HnRNA.

Synthesis of DNA-like RNA in synchronized cultures of mammalian cells

Abstract 1. Cultures of KB cells, synchronized by excess thymidine treatment were used to study the synthesis of DNA-like RNA (D-RNA) and total RNA during the various periods of the mammalian cell cycle. The rate of incorporation of [8- 14 C]hypoxanthine into RNA was used as a measure of the rate of total RNA synthesis. D-RNA was assayed by the hybridization of 3 H-labeled RNA with DNA. 2. The results obtained indicate that D-RNA is a relatively constant fraction of the RNA synthesized at all times in the cell cycle. On the basis of this result, together with the observation that total RNA synthesis continues throughout interphase, it is concluded that D-RNA is synthesized during all 3 periods of interphase, G 1 , S and G 2 .

Replication of repeated DNA in human cells

Abstract The replication pattern of the repeated sequence families of human DNA has been studied by means of DNA reassociation curves. Early- and late-replicating DNA fractions were obtained from synchronized cultures of KB cells by labeling cells with bromodeoxyuridine (BUdR) early or late in the DNA synthesis period and isolating the BUdR-containing DNA by CsCl density-gradient centrifugation. Highly repeated and moderately repeated sequence classes labeled with 14 C-deoxycytidine either early or late in the DNA synthesis period were also prepared. The effect of the isolated early- or late-replicating BUdR-DNA on the rate of reassociation of the 14 C-labeled repeated sequences was then tested. Increasing concentrations of early- or late-replicating BUdR-DNA were added to a constant amount of either 14 C-labeled early- or late-replicating repeated sequences, and the fraction of label in double-stranded DNA was determined. Analysis of the DNA reassociation curves so obtained indicates that some repeated sequence families are replicated throughout the DNA synthesis period whereas others are replicated primarily in the second half. This is true for both the highly-repeated and moderately-repeated sequence classes.