Biomedical subjects

J Hubenthal-Voss

Publications and source records attributed to J Hubenthal-Voss.

5 recordsLinked to original sources

PubMed1988

Mapping of functional and antigenic domains of the alpha 4 protein of herpes simplex virus 1.

Monoclonal antibodies to alpha 4, the major regulatory protein of herpes simplex virus 1, have been shown to differ in their effects on the binding of the protein to its DNA-binding site in the promoter-regulatory domain of an alpha gene. To map the epitopes, we expressed truncated genes in transient expression systems. All 10 monoclonal antibodies tested reacted with the N-terminal 288-amino-acid polypeptide. To map the epitopes more precisely, 29 15-mer oligopeptides, overlapping by five amino acids at each end, were synthesized and reacted with the monoclonal antibodies. The nine reactive monoclonal antibodies were mapped to seven sites. Of the two monoclonal antibodies which blocked the binding of alpha 4 to DNA, one (H950) reacted with oligopeptide no. 3 near the N terminal of the protein, whereas the second (H942) reacted with oligopeptide no. 23 near the C terminus of the 288-amino-acid polypeptide. In further tests, oligopeptide no. 19 was found to compete with two host proteins, designated as alpha H1 and alpha H2-alpha H3, for binding to DNA as well as to retard DNA in a band shift assay, whereas oligopeptides no. 26, 27, and 28 enhanced the binding of alpha 4 to DNA. Moreover, oligopeptide no. 27 was also found to retard DNA in a band shift assay. Polypeptide no. 19 competed with alpha 4 for binding to DNA, whereas no. 27 neither enhanced nor competed with the binding of the host polypeptide alpha H1 to its binding site in the promoter-regulatory domain of an alpha gene, but did enhance the binding of the alpha H2-alpha H3 protein to its binding site. In contrast to these results, the truncated alpha 4 polypeptide, 825 amino acids long, bound to the viral DNA, whereas a shorter, 519-amino-acid-long, truncated polypeptide did not. The 825-amino-acid polypeptide was previously shown to induce in transient expression of a late (gamma 2) viral gene.

Amino Acid Sequence↗

PubMed1987

The herpes simplex virus origins of DNA synthesis in the S component are each contained in a transcribed open reading frame.

In the herpes simplex virus 1 genome, the origins of viral DNA synthesis are located in the unique sequences of the L component (Oril) and in the reiterated sequences of the S component (OriS) located between the 5' terminus of the alpha 4 gene and the 5' terminus of either the alpha 22 (left terminus of the S component) or the alpha 47 (right terminus of the S component) gene. Studies prompted by the finding that only one, but not both, OriS sequence is dispensable for growth in cell culture indicate that each OriS sequence is contained in an open reading frame designated as OriSORF. The transcription of OriSORF is initiated approximately 860 nucleotides upstream from that of the alpha 4 gene and 162 nucleotides downstream, but on the opposite strand from the transcription initiation site of the alpha 22 or alpha 47 genes within the inverted repeat c sequence. The OriSORF transcript is 3' coterminal with the mRNA of the alpha 4 gene, polyadenylated but not spliced, transported into the cytoplasm, and capable of directing the synthesis of a 330-amino-acid protein with a translated molecular weight of approximately 34,000. Transcription is cycloheximide but not phosphonoacetate sensitive and is therefore regulated as either a beta or a gamma 1 gene. The implications of the transcription of OriS and of possible functions of the product of OriSORF are discussed.

Animals↗

PubMed1986

Regulation of herpes simplex virus 1 genes: alpha gene sequence requirements for transient induction of indicator genes regulated by beta or late (gamma 2) promoters.

This laboratory reported earlier that chimeric genes consisting of the structural sequences of the thymidine kinase (TK) gene fused to the promoter-regulatory domains of late (gamma 2) genes were regulated as bonafide gamma 2 genes when resident in the herpes simplex virus 1 genome but could not be differentiated from beta genes when introduced by transfection and stably integrated into the environment of the host genome (S. Silver and B. Roizman, Mol. Cell. Biol. 5, 518-528, 1985). We report here that beta-TK and the chimeric gamma 2-TK gene transfected into TK- baby hamster kidney (BHKtk-) were induced by alpha 4 and alpha 0 but not by the other alpha genes. Specifically: Both TK genes were induced by cotransfection with DNA fragments carrying an intact alpha 4 or an intact alpha 0 gene, but not by fragments carrying only the promoter-regulatory domain or the structural sequences of the alpha 4 gene or intact alpha 22, alpha 27, and alpha 47 genes. An alpha 4 gene carrying a 2700-bp deletion in its 3' coding sequence also induced both genes, although less efficiently. RNA homologous to the alpha 4 gene recovered from the cytoplasm of cells transfected with either the intact or truncated alpha 4 gene mapped to the bonafide site of transcription initiation of the alpha 4 gene. RNA homologous to the chimeric TK gene extracted from the cytoplasm of cells transfected with both gamma 2-TK and the alpha 4 gene was transcribed from the bonafide gamma 2 gene capping site fused to the TK gene. These results indicate that the alpha 4 gene and the alpha 0 gene are each capable of inducing the expression of both beta and gamma 2 genes resident in the environment of the cellular genome, that the active site responsible for induction is located near the N terminus of the alpha 4 protein, and reinforce the conclusion that gamma 2 genes resident in the environment of the host cell cannot be used to identify the authentic determinants of gamma 2 gene regulation by currently available tests.

Animals↗

PubMed1986

Construction and properties of a cell line constitutively expressing the herpes simplex virus glycoprotein B dependent on functional alpha 4 protein synthesis.

We report the construction of a cell line constitutively expressing the glycoprotein B (gB) of herpes simplex virus (HSV) 1. The cell line was constructed in two steps. In the first, a baby hamster kidney cell line was transfected with the DNA of a plasmid containing the neomycin phosphotransferase gene that confers resistance to the antibiotic G418 and the gene specifying a temperature-sensitive (ts-) alpha 4 protein of HSV-1, the major viral regulatory protein. A clonal cell line, alpha 4/c113, selected for resistance to the antibiotic G418, expressed high levels of alpha 4 protein constitutively. Superinfection of these cells with HSV-2 resulted in twofold induction of the resident HSV-1 alpha 4 gene. In the second step, alpha 4/c113 cells were transfected with the DNA of a plasmid carrying the gB gene and the mouse methotrexate resistance dihydrofolate reductase gene. A clonal cell line, alpha 4/c113/gB, selected for methotrexate resistance expressed gB constitutively. Expression of both gB and alpha 4 continued unabated for at least 32 serial passages. Cells passaged serially in medium containing both methotrexate and G418 after passage 10 contained a higher copy number of the alpha 4 gene and produced larger amounts of both gB and alpha 4 proteins than did cells maintained in medium containing methotrexate alone. Expression of gB was dependent on the presence of functional alpha 4 protein inasmuch as expression of gB ceased on shift up to nonpermissive temperatures, when shifted to permissive temperatures, the cell line reinitiated expression of gB after a delay commensurate with the length of incubation at the nonpermissive temperature, and the cell-resident HSV-1 gB gene was expressed at the nonpermissive temperature in cells infected with a recombinant expressing a ts+ alpha 4 protein and an HSV-2 gB. The properties of the alpha 4/c113 cell line suggest that it may express other viral genes induced by alpha 4 protein constitutively, provided that the product is not toxic to the cells.

Animals↗

PubMed1985

Herpes simplex virus 1 reiterated S component sequences (c1) situated between the a sequence and alpha 4 gene are not essential for virus replication.

The herpes simplex virus 1 genome consists of two components, L and S, each containing unique sequences flanked by inverted repeats. Each of the 6.5-kilobase pair inverted repeats of the S component, designated a'c' and ca, contains an approximately 700-base pair sequence (designated c1) located between the a sequence and the 3' terminus of the alpha 4 gene. Like the a sequence, c1 consists of direct repeats and unique sequences. Its function is not known. To probe for its function, we constructed a plasmid containing a viral thymidine kinase (TK) gene inserted into the c1 sequence. The construct was recombined into the genome of a TK- virus by cotransfection with intact viral DNA and selection for TK+ virus. As predicted from previous studies (Knipe et al., Proc. Natl. Acad. Sci. U.S.A. 75:3896-3900, 1978), the TK gene was found to be present in both copies of the c1 sequence in the R3104 virus. To delete the c1 sequence we constructed a plasmid containing 4 kilobase pairs of pBR322 flanked by an a sequence and by structural sequences of the alpha 4 gene. In this instance the cells were transfected with the construct and R3104 DNA; the progeny of the transfection was plated in the presence of 5-bromo-2'-deoxyuridine, and the selection was for TK- virus (R3158). The pBR322 DNA sequences replaced the c1 at both termini of the S component in R3158 DNA, but a sequence homologous to c1 was present in proximity to the 3' terminus of the alpha 4 gene. The results indicate that the c1 region has no significant role in the replication of the virus in cell culture. The advantage of inserting the pBR322 sequence is that it permits efficient cloning of large herpes simplex virus 1 DNA fragments by simple ligation of digests and transformation of appropriate Escherichia coli strains. The effortless selection of recombinants carrying inserts in both copies of the c1 restates the usefulness of this technique for selection of insertion deletion recombinants and underscores the rapid emergence of sequence identity at both ends of the reiterated regions of the S component as previously reported (Knipe et al., Proc. Natl. Acad. Sci. U.S.A. 75:3896-3900, 1978).

DNA, Viral↗