The DNA-binding properties of the major regulatory protein alpha 4 of herpes simplex viruses.

The transition from the expression of alpha, the first set of five herpes simplex virus genes expressed after infection, to beta and gamma genes, expressed later in infection, requires the participation of infected cell protein 4 (alpha 4), the major viral regulatory protein. The alpha 4 protein is present in complexes formed by proteins extracted from infected cells and viral DNA fragments derived from promoter domains. This report shows that the alpha 4 protein forms specific complexes with DNA fragments derived from 5' transcribed noncoding domains of late (gamma 2) genes whose expression requires viral DNA synthesis as well as functional alpha 4 protein. Some of the DNA fragments to which alpha 4 binds do not contain homologs of the previously reported DNA binding site consensus sequence, suggesting that alpha 4 may recognize and interact with more than one type of DNA binding site. The alpha 4 proteins can bind to DNA directly. A posttranslationally modified form of the alpha 4 protein designated alpha 4c differs from the alpha 4a and alpha 4b forms with respect to its affinity for DNA fragments differing in the nucleotide sequences of the binding sites.

Rapid genetic selection of inhibitor-resistant protease mutants: clinically relevant and novel mutants of the HIV protease.

Site-specific proteolysis is an important regulatory mechanism in many basic cellular processes as well as playing critical roles in the life cycle of viruses and other pathogenic organisms. For the human immunodeficiency virus (HIV) the encoded protease is required for the replication of the virus and has been the target of novel antiviral therapeutics. However, the emergence of inhibitor-resistant viral strains has become an increasingly significant clinical problem. Using a bacteriophage-based genetic selection, a bank of inhibitor-resistant mutants in this protease has been isolated that includes mutations that correlate with resistant clinical isolates. The rapid selection of such mutations has implications for the prediction of relevant mutations and may be applicable to other viral systems.

The mouse homologue of the human transcription factor C1 (host cell factor). Conservation of forms and function.

The assembly of the herpes simplex virus (HSV) alpha/IE gene enhancer complex is determined by the interactions of the Oct-1 POU domain protein, the viral alphaTIF (alpha-trans-induction factor, VP16, ICP25, VMW65), and the C1 factor (host cell factor, HCF). A unique transcription factor, C1 consists of a family of polypeptides derived from a common precursor by site-specific proteolytic processing. To analyze the role of this factor in the determination of HSV lytic-latent infection, cDNAs and genomic DNAs encoding the mouse homologue have been isolated. This factor is nearly identical to the human protein, contains multiple consensus proteolytic processing sites, and functions efficiently in the assembly of a specific HSV enhancer complex. Interestingly, the differential expression of the C1 factors in both human and mouse tissues may be important for the determination of HSV tissue tropism in these two organisms.

Autocatalytic proteolysis of the transcription factor-coactivator C1 (HCF): a potential role for proteolytic regulation of coactivator function.

Site-specific proteolysis is an important biological mechanism for the regulation of cellular processes such as gene expression, cell signaling, development, and apoptosis. In transcriptional regulation, specific proteolysis regulates the localization and activity of many regulatory factors. The C1 factor (HCF), a cellular transcription factor and coactivator, undergoes site-specific proteolytic processing at a series of unusual amino acid reiterations to generate a family of amino- and carboxyl-terminal polypeptides that remain tightly associated. Expression and purification of bacterially expressed domains of the C1 factor identifies an autocatalytic activity that is responsible for the specific cleavage of the reiterations. In addition, coexpression of the autocatalytic domain with a heterologous protein containing a target cleavage site demonstrates that the C1 protease may also function in trans.

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.

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.

Purification of the cellular C1 factor required for the stable recognition of the Oct-1 homeodomain by the herpes simplex virus alpha-trans-induction factor (VP16).

The assembly of specific multiprotein complexes on the herpes simplex virus alpha/IE (immediate early) enhancer elements requires the interactions of the Oct-1 POU homeodomain, the viral alpha TIF (alpha-trans-induction factor) (VP16), and at least one additional cellular factor, the C1 factor. The C1 factor interacts directly with alpha TIF, likely forming an intermediate protein complex that recognizes the Oct-1 homeodomain-DNA complex. The biochemical purification of the mammalian C1 factor suggests that it is composed of multiple subunits of related, but heterogeneous, polypeptides. The interaction of a subset of these polypeptides with alpha TIF is stimulated by post-translational modifications of the C1 proteins, suggesting that this factor may be a critical target for the regulation of the herpes simplex virus alpha/IE transcription.

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.

Host cell proteins bind to the cis-acting site required for virion-mediated induction of herpes simplex virus 1 alpha genes.

The herpes simplex virus 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. In productively infected cells, the alpha genes are expressed first, and a virion protein, the alpha-trans-inducing factor (alpha-TIF), acts in trans to enhance their expression. Induction of the alpha genes by alpha-TIF requires the presence of a trans-induction cis-acting site (alpha-TIC), and one to three homologs of the alpha-TIC sequence are contained in the regulatory domains of all alpha genes. We report that small DNA fragments from regulatory domains of alpha 0, alpha 4, and alpha 27 genes containing alpha-TIC homologs formed complexes with host but not viral proteins. DNase protection studies indicated that the major host protein complex alpha-H1 detected in DNA gel retardation assays bound asymmetrically across the alpha-TIC site. All DNA fragments containing alpha-TIC homologs, but not those lacking the homolog, competed for the binding of this complex. The location of the binding site of the other host proteins is not yet known. Simian virus 40 DNA fragments containing a homolog of the alpha-TIC sequence also competed with herpes simplex virus DNA fragments carrying authentic alpha-TIC homologs for the alpha-H1 protein complex.

Separation of sequences defining basal expression from those conferring alpha gene recognition within the regulatory domains of herpes simplex virus 1 alpha genes.

The genes of herpes simplex virus 1 form three major groups--alpha, beta, and gamma--whose expression is coordinately regulated and sequentially ordered in a cascade fashion. To determine how the infected cell differentiates between these gene groups, alpha-regulated chimeric genes were constructed in earlier studies by fusing the structural sequences of the thymidine kinase (TK) gene, a beta gene, to the 5' noncoding sequences of alpha genes. These studies showed that (i) one or more structural components of the virion act in trans to increase alpha gene expression and (ii) the 5' noncoding sequences of alpha genes contain cis-acting domains that promote gene expression and confer alpha-gene regulation. These two domains could be moved independently, but the regulatory domain required a promoter for its function. We report here the properties of three sequences containing features common to the regulatory regions of all alpha genes. Sequence 1, containing (G + C)-rich inverted repeats, increased the basal level of TK expression when fused 5' to either the alpha gene 4 promoter or the truncated beta TK promoter. The effect was to some extent orientation dependent. Moreover, sequence 1 restored beta regulation to the truncated beta TK promoter but did not confer alpha-specific regulation on any of the chimeric genes tested. Sequences 2 (49 base pairs) and 3 (29 base pairs), containing an (A + T)-rich homolog from alpha gene 27 and alpha gene 0, respectively, restored alpha-specific regulation to the alpha promoter gene but only sequence 2 conferred alpha regulation on the truncated beta promoter gene. Our results indicate that (i) in natural beta TK the promoter and regulatory domains overlap, (ii) sequence 1 determines basal level of expression and substitutes for a promoter component that is essential for beta but not alpha regulation, and (iii) conversion of a gene with a promoter into an alpha gene requires two elements. Sequence 2 may contain both whereas sequence 3 contains only one.

The novel coactivator C1 (HCF) coordinates multiprotein enhancer formation and mediates transcription activation by GABP.

Transcription of the herpes simplex virus 1 (HSV-1) immediate early (IE) genes is determined by multiprotein enhancer complexes. The core enhancer assembly requires the interactions of the POU-homeodomain protein Oct-1, the viral transactivator alphaTIF and the cellular factor C1 (HCF). In this context, the C1 factor interacts with each protein to assemble the stable enhancer complex. In addition, the IE enhancer cores contain adjacent binding sites for other cellular transcription factors such as Sp1 and GA-binding protein (GABP). In this study, a direct interaction of the C1 factor with GABP is demonstrated, defining the C1 factor as the critical coordinator of the enhancer complex assembly. In addition, mutations that reduce the GABP transactivation potential also impair the C1-GABP interaction, indicating that the C1 factor functions as a novel coactivator of GABP-mediated transcription. The interaction and coordinated assembly of the enhancer proteins by the C1 factor may be critical for the regulation of the HSV lytic-latent cycle.

A genetic screen for the isolation and characterization of site-specific proteases.

Site-specific proteolysis is an important regulatory mechanism in basic cellular and viral processes. Using the protease of the HIV as a model, a genetic system has been developed for the isolation and characterization of site-specific proteases. The system utilizes the well defined bacteriophage lambda regulatory circuit where the viral repressor, cI, is specifically cleaved to initiate the lysogenic-to-lytic switch. The model system is rapid, highly specific, and demonstrates the ability to isolate and characterize enzymes of limited expression or activity. In addition, the system has a significant potential for the selection of clinically relevant mutant enzymes and in the development of anti-protease therapeutics.

Interactions of the Oct-1 POU subdomains with specific DNA sequences and with the HSV alpha-trans-activator protein.

Trans-activation by the herpes simplex virus (HSV) protein, alpha TIF (VP16), is dependent on an inducible enhancer sequence that contains a homolog of the octamer element. An ordered series of multiprotein complexes can be assembled on this enhancer, requiring the interactions of Oct-1, alpha TIF, and two additional cellular factors (C1 and C2). Oct-1 binds to the octamer homolog, whereas alpha TIF, also a sequence-specific DNA-binding protein, recognizes sequences within the HSV enhancer core. The partially purified C1 factor interacts directly with alpha TIF in the absence of DNA and is required to form a stabile Oct-1/alpha TIF/C1 factor complex. The POU domain of Oct-1 is a bipartite sequence recognition structure, as both the POU-specific box and the POU homeo box contribute directly to the recognition of the octamer element. Surprisingly, the POU homeo box alone is sufficient to direct the cooperative binding of alpha TIF and to assemble the Oct-1/alpha TIF/C1 factor complex.

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.

The octamer-binding proteins form multi-protein--DNA complexes with the HSV alpha TIF regulatory protein.

The herpes simplex virus transactivator, alpha TIF, stimulates transcription of the alpha/immediate early genes via a cis-acting site containing an octamer element and a conserved flanking sequence. The alpha TIF protein, produced in a baculovirus expression system, nucleates the formation of at least two DNA--protein complexes on this regulatory element. Both of these complexes contain the ubiquitous Oct-1 protein, whose POU domain alone is sufficient to allow assembly of the alpha TIF-dependent complexes. A second member of the POU domain family, the lymphoid specific Oct-2 protein, can also be assembled into similar complexes at high concentrations of alpha TIF protein. These complexes contain at least two cellular proteins in addition to Oct-1. One of these proteins is present in both insect and HeLa cells and probably recognizes sequences in the cis element. The second cellular protein, only present in HeLa cells, probably binds by protein-protein interactions.

Recognition of the surface of a homeo domain protein.

Homeo domain proteins exhibit distinct biological functions with specificities that cannot be predicted by their sequence specificities for binding DNA. Recognition of the surface of the Oct-1 POU homeo domain provides a general model for the contribution of selective protein-protein interactions to the functional specificity of the homeo domain family of factors. The assembly of Oct-1 into a multiprotein complex on the herpes simplex virus alpha/IE enhancer is specified by the interactions of its homeo domain with ancillary factors. This complex (C1 complex) is composed of the viral alpha TIF protein (VP16), Oct-1, and one additional cellular component, the C1 factor. Variants of the Oct-1 POU homeo domain were generated by site-directed mutagenesis, which altered the residues predicted to form the exposed surface of the domain-DNA complex. Proteins with single amino acid substitutions on the surface of either helix 1 or 2 of the Oct-1 POU homeo domain had decreased abilities to form the C1 complex. The behavior of these mutants in a cooperative DNA-binding assay with alpha TIF suggested that the Oct-1 POU homeo domain is principally recognized by alpha TIF in the C1 complex. The preferential recognition of Oct-1 over the closely related Oct-2 protein is critically influenced by a single residue on the surface of helix 1 because the introduction of this residue into the Oct-2 POU homeo domain significantly enhanced its ability to form a C1 complex.

Nuclear localization of the C1 factor (host cell factor) in sensory neurons correlates with reactivation of herpes simplex virus from latency.

After a primary infection, herpes simplex virus is maintained in a latent state in neurons of sensory ganglia until complex stimuli reactivate viral lytic replication. Although the mechanisms governing reactivation from the latent state remain unknown, the regulated expression of the viral immediate early genes represents a critical point in this process. These genes are controlled by transcription enhancer complexes whose assembly requires and is coordinated by the cellular C1 factor (host cell factor). In contrast to other tissues, the C1 factor is not detected in the nuclei of sensory neurons. Experimental conditions that induce the reactivation of herpes simplex virus in mouse model systems result in rapid nuclear localization of the protein, indicating that the C1 factor is sequestered in these cells until reactivation signals induce a redistribution of the protein. The regulated localization suggests that C1 is a critical switch determinant of the viral lytic-latent cycle.

The cellular C1 factor of the herpes simplex virus enhancer complex is a family of polypeptides.

The alpha/immediate early genes of herpes simplex virus are regulated by the specific assembly of a multiprotein enhancer complex containing the Oct-1 POU domain protein, the viral alpha-transinduction factor alpha TIF, (VP16, ICP25), and the C1 cellular factor. The C1 factor from mammalian cells is a heterogeneous but related set of polypeptides that interact directly with the alpha-transinduction factor to form a heteromeric protein complex. The isolation of cDNAs encoding the polypeptides of the C1 factor suggests that these proteins are proteolytic products of a novel precursor. The sequence of the amino termini of these polypeptide products indicate that the proteins are generated by site-specific cleavages within a reiterated 20-amino acid sequence. Although the C1 factor appears to be ubiquitously expressed, it is localized to subnuclear structures in specific cell types.