Promoter-regulatory Domains Research Articles

Identification of three genes nonessential for growth in cell culture near the right terminus of the unique sequences of long component of herpes simplex virus 1

We report the construction of herpes simplex virus 1 recombinants from which all or part of the coding sequences of four open reading frames have been deleted. In recombinants R7101 and R7108, the BamHI D′ fragment containing the coding sequences of the dUTPase gene and the promoter-regulatory domain of a late gene was either replaced with a fragment containing a thymidine kinase gene or deleted, respectively. The recombinant R7107 lacks, in addition to BamHI D′, 202 of a total of 244 predicted amino acids from the 3′ end of the adjacent open reading frame U L51. The deletion in recombinant R7105 encompasses two genes, i.e., the entire open reading frame of U L47 and the amino terminus of U L46. These genes map next to that specifying the α trans-inducing factor. R7105, R7101, and R7108 do not exhibit demonstrable defects in viral replication. The recombinant R7107 forms minute plaques and replicates best in multiplying cells in subconfluent cultures. The results indicate that the multiplying cells supply at least some of the functions expressed by the protein encoded in U L51.

Functional relevance of specific interactions between herpes simplex virus type 1 ICP4 and sequences from the promoter-regulatory domain of the viral thymidine kinase gene

The herpes simplex virus (HSV) type 1 immediate-early regulatory protein ICP4 is required for induced expression of HSV early and late genes, yet the mechanism by which this occurs is not known. We examined the promoter and flanking sequences of the HSV early gene that encodes thymidine kinase for the ability to interact specifically with ICP4 in gel retardation assays. Protein-DNA complexes containing ICP4 were observed with several distinct regions flanking the tk promoter. cis-Acting elements that interact with cellular transcription factors were apparently not required for these interactions to form. Purified ICP4 formed protein-DNA complexes with fragments from these regions, and Southwestern (DNA-protein blot) analysis indicated that the interaction between ICP4 and these sequences can be direct. None of the tk sequences that interact with ICP4 contains a consensus binding site for ICP4 (S. W. Faber and K. W. Wilcox, Nucleic Acids Res. 14:6067-6083, 1986), reflecting the ability of ICP4 to interact with more than one DNA sequence. A mutated ICP4 protein expressed from the viral genome that retains the ability to bind to a consensus binding site but does not bind specifically to the identified sites flanking the tk promoter results in induced transcription of the tk gene. These data support hypotheses for ICP4-mediated transactivation of the tk promoter in Vero cells that do not require the intrinsic ability of ICP4 to bind specifically in or near the promoter of the tk gene.

The herpes simplex virus type 1 alpha protein ICP27 can act as a trans-repressor or a trans-activator in combination with ICP4 and ICP0.

The herpes simplex virus type 1 (HSV-1) alpha proteins ICP4, ICP0, and ICP27 are trans-acting proteins which affect HSV-1 gene expression. To investigate potential interactions between these alpha products and to determine the specificity of action of the alpha proteins in combination with each other compared with their activities individually, we performed a series of transient-expression assays. In these assays we used plasmids containing the alpha genes encoding ICP4, ICP0, and ICP27 either singly or in combination as effectors and HSV-1 genes of different kinetic classes and heterologous genes as targets. The HSV-1 targets consisted of promoter-regulatory domains from alpha (ICP0 and ICP27), beta (thymidine kinase and alkaline exonuclease), beta-gamma (glycoprotein D, glycoprotein B, and VP5), and gamma (glycoprotein C) genes, each fused to the chloramphenicol acetyltransferase (CAT) gene. The heterologous target genes consisted of the simian virus 40 early promoter with enhancer and the Rous sarcoma virus long terminal repeat promoter and enhancer each fused to the CAT gene. Target promoter activity was measured by the assay of CAT activity in extracts of transfected cells and by Northern (RNA) blot hybridization of CAT mRNA. The results of these experiments showed that ICP4 activated only HSV-1 target genes, whereas ICP0 activated all of the targets and ICP27 had little effect on any of the targets. ICP4 and ICP0 had a synergistic effect when inducing HSV-1 targets, but they did not have this effect on the heterologous targets pSV2-CAT or pRSV-CAT. In fact, lower levels of CAT activity and CAT mRNA were found in the presence of both effectors than with ICP0 alone. Most interestingly, although the effector plasmid containing the ICP27 gene had little effect on its own, two different and marked effects depending on the target were observed when ICP27 was combined with ICP4 or ICP0 or both. A trans-repression of the induction seen with ICP4 and ICP0 was found when ICP27 was present in the transfections with pSV2-CAT, pRSV-CAT, pICP0-CAT, pICP27-CAT, pTK-CAT, pgD-CAT, pgB-CAT, and pgC-CAT. This resulted in CAT activity levels which were similar to or lower than the basal level of expression of the target genes in the absence of effector plasmids. This trans-repression occurred over a wide range of concentrations of input ICP27 plasmid. In contrast to this repressive effect of ICP27, a trans-activation was seen when ICP4, ICP0, and ICP27 plasmids were combined in transfections with pAE-CAT and pVP5-CAT as targets. This trans-activation also occurred over a 10-fold range of input ICP27 plasmid. These results suggest that ICP27 can facilitate both down

The trans-activation of herpes simplex virus gene expression: comparison of two factors and their cis sites

In herpes simplex virus-infected cells, gene expression is tightly regulated. In this review, we compare the properties of two trans-activating factors which regulate the expression of viral genes. The first, α trans-minducing factor (αTIF) is a structural component which induces the 5 α genes, the first set of genes transcribed after infection. αTIF requires for induction a cis-acting site present in promoter-regulatory domains of all α genes. The cis site binds 2 host proteins, αH1 and αH2-αH3. These host proteins have a maximum bound molecular weight of 110 000 and 64 000, respectively. DNase 1 protection assays indicate that αH1 protects the entire cis site, whereas αH2-αH3 binds the 3′ domain of the cis site. The methylation interference assays indicate that the contact points of αH1 and αH2-H3 are at the 5′ and 3′ termini of the cis site, respectively. Both proteins can bind to the cis site concurrently. αTIF does not bind directly to DNA but was shown to be present in DNA-protein complexes. The binding of αTIF to these DNA-protein complexes requires the participation of αH1. In contrast to αTIF, the product of the α4 gene, a protein 163 000 in apparent molecular weight binds to DNA directly and regulates genes both positively and negatively. The data indicate that α4 protein can bind to at least 2 binding sites differing in nucleotide sequence and which can be present in promoters, across the transcription initiation sites, and in 5′ transcribed non-coding sequences. The regulatory functions of the α4 protein may reflect both the nature and location of the binding site. The biological implications of the viral trans-acting proteins are discussed.

Differentiation and DNA contact points of host proteins binding at the cis site for virion-mediated induction of alpha genes of herpes simplex virus 1.

Transcriptional trans-activation of the five herpes simplex virus 1 alpha genes by the alpha trans-inducing factor requires a cis-acting site (alpha TIC; with the consensus 5'-GyATGnTAATGArATTCyTTGnGGG-3') located in the promoter-regulatory domains of the alpha genes. In DNA band shift assays with nuclear extracts from either mock-infected or infected cells, the DNA fragments containing an alpha TIC sequence from the alpha 0, alpha 4, and alpha 27 genes formed several cellular protein-DNA complexes designated alpha H1, alpha H2, and alpha H3. The host proteins that formed the alpha H2 and alpha H3 complexes were differentiated from those that formed the alpha H1 complex but not from each other by chromatography and specificity of the DNA-binding sites. The alpha H1 proteins protected the alpha TIC sequence of all three genes from DNase I digestion. Methylation of the purines in the sequence 5'-GyATGnTAAT-3' located at the 5' terminus of the alpha TIC sites precluded the binding of alpha H1. The binding site of the alpha H2-alpha H3 proteins in the alpha 27 gene alpha TIC overlapped, in part, with the alpha H1-binding site. The binding of these proteins was precluded by methylation of the purine residues in the sequence 5'-GCCACGTG-3' located at the 3' terminus of the DNase I footprint. The maximum apparent molecular weight of alpha H1 was 110,000, whereas that of alpha H2-alpha H3 was 64,000. A protein designated alpha H2', resembling alpha H2-alpha H3 with respect to molecular weight and chromatographic properties but differing in sequence specificity, bound to a site adjacent to the alpha H1 site in the fragment carrying an alpha TIC sequence of the alpha 4 gene. alpha H1 and alpha H2-alpha H3 or alpha H2' bound concurrently, notwithstanding the apparent overlap in the DNase I footprints.

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.

Binding of the virion protein mediating alpha gene induction in herpes simplex virus 1-infected cells to its cis site requires cellular proteins.

In herpes simplex virus 1-infected cells, the transcription of alpha genes, the first set of genes to be expressed, is induced by a virion component, the alpha-trans-induction factor, and requires a cis site. Homologs of the cis site are present in the promoter-regulatory domains of all alpha genes and bind two cellular proteins designated as alpha H1 and alpha H2-alpha H3. We report that alpha-trans-induction factor, synthesized in vitro or present in nuclear extracts of infected cells, forms complexes with viral DNA fragments containing its cis-acting site only in the presence of cellular proteins and only under conditions that also enable the binding of the alpha H1 protein to the DNA. The induction of alpha genes by alpha-trans-induction factor appears, therefore, to be mediated by the interaction of the viral protein with cellular proteins at its cis-acting site.

DNA-binding site of major regulatory protein alpha 4 specifically associated with promoter-regulatory domains of alpha genes of herpes simplex virus type 1.

Herpes simplex virus type 1 genes form at least five groups (alpha, beta 1, beta 2, gamma 1, and gamma 2) whose expression is coordinately regulated and sequentially ordered in a cascade fashion. Previous studies have shown that functional alpha 4 gene product is essential for the transition from alpha to beta protein synthesis and have suggested that alpha 4 gene expression is autoregulatory. We have previously reported that labeled DNA fragments containing promoter-regulatory domains of three alpha (alpha 0, alpha 4, and alpha 27) and a gamma 2 gene form stable complexes with proteins from lysates of infected cells as detected by a gel electrophoresis binding assay and that monoclonal antibody to alpha 4 protein reduced the electrophoretic mobility of the complex of labeled DNA and protein from infected cells. In this study we identified one monoclonal antibody, H950, from a panel of monoclonal antibodies to the alpha 4 protein that blocks the formation of specific infected cell complexes with labeled DNA fragments containing promoter and regulatory domains of alpha genes. We also report the nucleotide sequence of the alpha 0 promoter domain protected from exonuclease III digestion by alpha 4 protein in the absence of H950 monoclonal antibody but not in its presence. In addition, we identified a 59-base-pair sequence from the regulatory domain of the alpha 4 gene that binds alpha 4 protein. Deletion clones of this fragment localize sequence elements required for formation of the alpha 4 protein-DNA complex. Furthermore, deletion of the in vitro binding site of the SP1 transcription factor from the 59-base-pair fragment did not affect the formation of the alpha 4 protein-DNA complex.

Alpha 4, the major regulatory protein of herpes simplex virus type 1, is stably and specifically associated with promoter-regulatory domains of alpha genes and of selected other viral genes.

Herpes simplex virus type 1 genes form at least five groups (alpha, beta 1, beta 2, gamma 1, and gamma 2) whose expression is coordinately regulated and sequentially ordered in a cascade fashion. Previous studies have shown that functional alpha 4 gene product is essential for the transition from alpha to beta protein synthesis and have suggested that alpha 4 gene expression is autoregulatory. However, the mechanism by which alpha 4 regulates gene expression remained unknown. We report that labeled DNA fragments containing promoter-regulatory domains of three alpha (alpha 0, alpha 4, and alpha 27) and a gamma 2 gene form stable complexes with proteins from infected-cell lysates as detected by a gel electrophoresis binding assay. The protein(s) exhibits sequence specificity since autologous DNA fragments but not heterologous DNA fragments, synthetic polydeoxynucleotide chains, or salmon sperm DNA competitively displace the DNA probe from the complexes. Murine monoclonal antibody to alpha 4 protein added before or after DNA-protein complex formation further retarded the electrophoretic mobility of the complexes whereas monoclonal antibody to alpha 0, alpha 27, or to a viral glycoprotein had no effect. Complexes consisting of the promoter-regulatory domain of the beta-class thymidine kinase gene and infected cell proteins were low in abundance and could be detected only in the presence of antibody to alpha 4 protein. The alpha 4 protein, therefore, forms stable complexes with promoter-regulatory domains of alpha genes and of selected other herpes simplex virus type 1 genes either alone or in combination with other proteins.

Regulation of herpes simplex virus 1 genes: α gene sequence requirements for transient induction of indicator genes regulated by β or late ( γ2) 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 ( γ 2) genes were regulated as bonafide γ 2 genes when resident in the herpes simplex virus 1 genome but could not be differentiated from β 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 β-TK and the chimeric γ 2-TK gene transfected into TK − baby hamster kidney (BHKtk −) were induced by α4 and α0 but not by the other α genes. Specifically: (i) Both TK genes were induced by cotransfection with DNA fragments carrying an intact α4 or an intact α0 gene, but not by fragments carrying only the promoter-regulatory domain or the structural sequences of the α4 gene or intact α22, α27, and α47 genes. (ii) An α4 gene carrying a 2700-bp deletion in its 3′ coding sequence also induced both genes, although less efficiently. (iii) RNA homologous to the α4 gene recovered from the cytoplasm of cells transfected with either the intact or truncated α4 gene mapped to the bonafide site of transcription initiation of the a4 gene. RNA homologous to the chimeric TK gene extracted from the cytoplasm of cells transfected with both γ 2-TK and the α4 gene was transcribed from the bonafide γ 2 gene capping site fused to the TK gene. These results indicate that the α4 gene and the α0 gene are each capable of inducing the expression of both β and γ 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 α4 protein, and reinforce the conclusion that γ 2 genes resident in the environment of the host cell cannot be used to identify the authentic determinants of γ 2 gene regulation by currently available tests.

The terminal a sequence of the herpes simplex virus genome contains the promoter of a gene located in the repeat sequences of the L component.

The herpes simplex virus DNA genome consists of two covalently linked components, L and S. The unique sequences of the L component are flanked by 9-kilobase-pair inverted repeat sequences ab and b'a', whereas those of the S component are flanked by 6.5-kilobase-pair inverted repeat sequences c'a' and ca. We report that the 500-base-pair a sequence contains the promoter-regulatory domain and the transcription initiation site of a diploid gene, the coding sequences of which are located in the b sequences of the inverted repeats of the L component. The chimeric gene constructed by fusion of the a sequence to the coding sequences of the thymidine kinase gene and recombined into the viral genome was regulated as a gamma 1 gene. The size of the protein predicted from its sequence is 358 amino acids; it was designated as infected cell protein (ICP) 34.5. Thus, the inverted repeats flanking the unique sequences of the L component contain two genes specifying ICP0 and ICP34.5, respectively. Moreover, in addition to the cis-acting sites for the inversion of L and S components relative to each other, for cleavage of unit length DNA molecules from head-to-tail concatemers, and for packaging of the DNA into capsids, the a sequence also contains the promoter-regulatory domain and transcription initiation sites of a gene.

Nucleotide sequence and predicted amino acid sequence of a protein encoded in a small herpes simplex virus DNA fragment capable of trans-inducing alpha genes.

The five alpha genes of herpes simplex virus 1 are the first set of genes to be expressed after infection. Previous studies have shown that alpha genes resident in eukaryotic cells are induced by infection with herpes simplex virus 1 or 2 but not by other herpesviruses and indicate that the alpha trans-inducing factor was a structural component of the virion. This factor induces genes linked to a bona fide promoter and containing at the 5' end a small sequence derived from the promoter-regulatory domains of alpha genes. We report the sequence of a small DNA fragment shown previously to be capable of expressing the alpha trans-inducing factor in transient expression systems. The only gene encoded in its entirety in this fragment is predicted to specify a 479 amino acid protein with a Mr of 53,053. The precise termini of the 1.74-kilobase mRNA specifying this protein were determined in our 5' and 3' S1 nuclease protection studies.

Gamma 2-Thymidine kinase chimeras are identically transcribed but regulated a gamma 2 genes in herpes simplex virus genomes and as beta genes in cell genomes.

True gamma or gamma 2 genes, unlike alpha, beta, and gamma 1 (beta gamma) genes of herpes simplex virus 1 (HSV-1), stringently require viral DNA synthesis for their expression. We report that gamma 2 genes resident in cells were induced in trans by infection with HSV-1 but that the induction did not require amplification of either the resident gene or the infecting viral genome. Specifically, to test the hypothesis that expression of these genes is amplification dependent, we constructed two sets of gamma 2-thymidine kinase (TK) chimeric genes. The first (pRB3038) consisted of the promoter-regulatory region and a portion of 5'-transcribed noncoding region of the domain of a gamma 2 gene identified by Hall et al. (J. Virol. 43:594-607) in the HSV-1(F) BamHI fragment D' to the 5'-transcribed noncoding and coding regions of the TK gene. The second (pRB3048) contained, in addition, an origin of HSV-1 DNA replication. Cells transfected with either the first or second construct and selected for the TK+ phenotype were then tested for TK induction after superinfection with HSV-1(F) delta 305, containing a deletion in the coding sequences of the TK gene, and viruses containing, in addition, a ts lesion in the alpha 4 regulatory protein (ts502 delta 305) or in the beta 8 major DNA-binding protein (tsHA1 delta 305). The results were as follows: induction by infection with TK- virus of chimeric TK genes with or without an origin of DNA replication was dependent on functional alpha 4 protein but not on viral DNA synthesis; the resident chimeric gene in cells selected for G418 (neomycin) resistance was regulated in the same fashion; the chimeric gene recombined into the viral DNA was regulated as a gamma 2 gene in that its expression in infected cells was dependent on viral DNA synthesis; the gamma 2-chimeric genes resident in the host and in viral genomes were transcribed from the donor BamHI fragment D' containing the promoter-regulatory domain of the gamma 2 gene. The significance of the differential regulation of gamma 2 genes in the environments of host and viral genomes by viral trans-acting factors is discussed.

Gamma 2-Thymidine kinase chimeras are identically transcribed but regulated a gamma 2 genes in herpes simplex virus genomes and as beta genes in cell genomes.

True gamma or gamma 2 genes, unlike alpha, beta, and gamma 1 (beta gamma) genes of herpes simplex virus 1 (HSV-1), stringently require viral DNA synthesis for their expression. We report that gamma 2 genes resident in cells were induced in trans by infection with HSV-1 but that the induction did not require amplification of either the resident gene or the infecting viral genome. Specifically, to test the hypothesis that expression of these genes is amplification dependent, we constructed two sets of gamma 2-thymidine kinase (TK) chimeric genes. The first (pRB3038) consisted of the promoter-regulatory region and a portion of 5'-transcribed noncoding region of the domain of a gamma 2 gene identified by Hall et al. (J. Virol. 43:594-607) in the HSV-1(F) BamHI fragment D' to the 5'-transcribed noncoding and coding regions of the TK gene. The second (pRB3048) contained, in addition, an origin of HSV-1 DNA replication. Cells transfected with either the first or second construct and selected for the TK+ phenotype were then tested for TK induction after superinfection with HSV-1(F) delta 305, containing a deletion in the coding sequences of the TK gene, and viruses containing, in addition, a ts lesion in the alpha 4 regulatory protein (ts502 delta 305) or in the beta 8 major DNA-binding protein (tsHA1 delta 305). The results were as follows: induction by infection with TK- virus of chimeric TK genes with or without an origin of DNA replication was dependent on functional alpha 4 protein but not on viral DNA synthesis; the resident chimeric gene in cells selected for G418 (neomycin) resistance was regulated in the same fashion; the chimeric gene recombined into the viral DNA was regulated as a gamma 2 gene in that its expression in infected cells was dependent on viral DNA synthesis; the gamma 2-chimeric genes resident in the host and in viral genomes were transcribed from the donor BamHI fragment D' containing the promoter-regulatory domain of the gamma 2 gene. The significance of the differential regulation of gamma 2 genes in the environments of host and viral genomes by viral trans-acting factors is discussed.