Biomedical subjects

R D Everett

Publications and source records attributed to R D Everett.

At least 55 records · Page 3Linked to original sources

PubMed1991

Purification of the DNA binding domain of herpes simplex virus type 1 immediate-early protein Vmw175 as a homodimer and extensive mutagenesis of its DNA recognition site.

The herpes simplex virus type 1 (HSV-1) Immediate-Early (IE) polypeptide Vmw175 is essential for the activation of transcription from viral early and late promoters. Vmw175 also reduces the activity of its own (IE-3) promoter in transfection assays. Both transactivation and repression mediated by Vmw175 require the integrity of a conserved domain of the polypeptide which has been shown to bind to specific DNA sequences. We have investigated the DNA sequence requirements for Vmw175 binding using a randomly mutated target. The results indicate that the binding site covers a region of 13 nucleotides divided into proximal and distal parts which are consistent with the consensus ATCGTNNNNNYSG. We have also expressed several different constructs encompassing the DNA binding domain of Vmw175 in bacteria, and obtained preparations of greater than 90% purity. The DNA binding domain is a dimer in solution, and binds DNA with a specificity similar to that of the intact protein, although the smallest DNA binding competent protein has a slightly reduced specificity.

Base Sequence↗

PubMed1991

Nucleotides within both proximal and distal parts of the consensus sequence are important for specific DNA recognition by the herpes simplex virus regulatory protein ICP4.

The herpes simplex virus type 1 regulatory protein ICP4 is a sequence specific DNA binding protein which associates with a number of different sites, some of which include the consensus ATCGTCnnnnYCGRC. In order to investigate the involvement in DNA binding of conserved bases within the consensus, we have synthesised a family of mutant oligonucleotides and tested their ability to form a complex with ICP4. We have also compared the binding specificities of bacterially expressed fragments of ICP4 which include the DNA binding domain. Mutation of most (but not all) bases in the proximal part of the consensus greatly reduced binding by ICP4, as did a mutation affecting the distal part. Most (but not all) G residues identified in methylation interference assays were required for efficient binding. While a bacterially expressed ICP4 peptide encompassing amino acid residues 252-523 bound to DNA with a specificity similar to that of the whole protein, a shorter protein (residues 275-523) had a slightly relaxed DNA binding specificity.

Base Sequence↗

PubMed1991

The Vmw175 binding site in the IE-1 promoter has no apparent role in the expression of Vmw110 during herpes simplex virus type 1 infection.

The immediate-early (IE) genes of herpes simplex virus type 1 (HSV-1) are the first to be expressed during infection in tissue culture. Since they are transcribed at abnormally high levels in the absence of IE protein synthesis they appear to be subject to repression during normal infection. One of the major HSV-1 regulatory proteins, Vmw175 (the product of IE gene 3), is required for normal IE gene regulation since mutations which inactivate it lead to abnormally high levels of IE gene expression. The mechanism of repression of the IE-3 promoter requires both the ability of Vmw175 to bind to DNA and the presence of a Vmw175 recognition DNA binding sequence at the cap site of the IE-3 promoter. A similar Vmw175 DNA binding sequence has been defined within the IE-1 promoter. This paper describes the construction of a variant of HSV-1 with a mutation within the IE-1 Vmw175 DNA binding site. Although the mutation destroyed the ability of Vmw175 to bind to the site, and greatly reduced the ability of Vmw175 to repress the IE-1 promoter in transfection assays, the mutation had no effect on the levels of Vmw110 expression during normal HSV-1 infection.

Animals↗

PubMed1991

Construction and characterization of herpes simplex type 1 viruses without introns in immediate early gene 1.

Herpes simplex virus type 1 (HSV-1) encodes at least 70 distinct genes in a DNA genome sequence of about 150 kb. In contrast to most cellular genes and those of several other DNA viruses, the overwhelming majority of HSV-1 transcripts are not spliced. One exception is immediate early (IE) gene 1, which contains two introns in the Vmw110 coding region. This study investigated the possibility that IE-1 intron sequences have a role during HSV-1 infection. IE-1 genes lacking the first, second or both introns were constructed by site-directed deletion mutagenesis and recombined into the viral genome. Viruses lacking the IE-1 introns were essentially indistinguishable from the parent virus in terms of growth, particle to p.f.u. ratio or viral polypeptide expression in a variety of cell types. The lack of introns did not affect the time-course or efficiency of expression of Vmw110 either during normal infection or in cycloheximide reversal experiments. In contrast, in transfection assays, the loss of both intron sequences resulted in the elimination of the ability of a plasmid-encoded IE-1 to activate gene expression. These results imply that in certain situations the introns in IE-gene 1 may contribute to the efficient expression of Vmw110 but such an effect is not readily apparent using a recombinant virus in the tissue culture systems tested. In the course of this work a mutant of Vmw110 was fortuitously isolated which had lost the majority of an extremely acidic section of the polypeptide; this mutation appeared to have little effect on Vmw110 function.

Animals↗

PubMed1991

Activation of gene expression by human herpesvirus 6 is reporter gene-dependent.

Infection with human herpesvirus 6 (HHV-6) was found to up-regulate expression of human immunodeficiency virus and human T cell leukaemia virus type I (HTLV-I) long terminal repeat sequence (LTR), and herpes simplex virus type 1 (HSV-1) gD chloramphenicol acetyltransferase (CAT) constructs transfected into the T cell line, J. Jhan. Activation by HHV-6 was due to one or more viral proteins produced early in infection and, in the case of the HTLV-I LTR, was synergistic to induction mediated by the HTLV-I tax gene product. Neither the HTLV-I enhancer nor basal promoter elements of the HSV-1 gD gene were essential for activation and no increase in accumulated HTLV-I mRNA was observed due to HHV-6 infection. Induction by HHV-6 was found to be dependent on the reporter construct used, because the CAT gene and, to a lesser extent, the HSV-1 thymidine kinase gene were responsive to HHV-6 infection although no significant activation of growth hormone constructs was observed. Our results bear a strong resemblance to those obtained for the Epstein-Barr virus BMLF1 gene, indicating that the major HHV-6 trans-activator may be a homologue of this gene.

Chloramphenicol O-Acetyltransferase↗

PubMed1990

The major transcriptional regulatory protein of herpes simplex virus type 1 includes a protease resistant DNA binding domain.

Herpes simplex virus type 1 expresses five immediate-early (IE) polypeptides. In the absence of functional Vmw175 (the product of IE gene 3) activation of transcription of later classes of viral genes and repression of IE gene expression does not occur. The recognition of specific DNA sequences by Vmw175 requires, as determined by sensitivity to mutation, a part of the protein highly conserved in the corresponding proteins of related herpes viruses. However, mutations in other parts of the protein can also disrupt specific DNA binding. This paper shows that the DNA binding domain of Vmw175 can be liberated as a functional unit by digestion with proteinase K. Analysis of mutant Vmw175 proteins showed that the proteinase K resistant domain has an amino terminus between amino acid residues 229 and 292, while its carboxy terminus is between residues 495 and 518. Mutations outside this region which affect DNA binding by the intact protein do not eliminate binding of the proteinase K resistant domain. This implies that direct DNA binding by Vmw175 involves a linear subsection of the polypeptide, and that mutations in other parts of the polypeptide which affect DNA binding of the whole protein do so by indirect means.

DNA, Viral↗

PubMed1990

A herpes simplex virus type 1 recombinant with both copies of the Vmw175 coding sequences replaced by the homologous varicella-zoster virus open reading frame.

Varicella-zoster virus (VZV) gene 62 encodes a protein with a predicted Mr of 140,000 (VZV 140K) that shares considerable amino acid homology with the immediate early (IE) regulatory protein Vmw175 of herpes simplex virus type 1 (HSV-1) and is believed to be its functional equivalent. We have tested this hypothesis by insertion of VZV gene 62 (expressed from the HSV-1 IE3 promoter) into both IE3 gene loci in the short region repeats of the HSV-1 genome. The parent virus used for this manipulation was D30EBA, which is a variant of HSV-1 from which the majority of the Vmw175 coding sequences have been deleted. Like other HSV-1 viruses lacking Vmw175 functions, D30EBA is able to grow only in cell lines which express Vmw175 constitutively. The resulting recombinant virus. HSV-140, is able to propagate (but unable to form obvious plaques) on normal cell lines. The properties of HSV-140 were studied by monitoring the time course of polypeptide expression and DNA replication during normal infection. We found that at high multiplicity HSV-140 synthesized apparently normal amounts of many viral polypeptides but that the expression of certain late genes was reduced; this slight defect may be related to less efficient DNA replication by HSV-140. At low multiplicity HSV-140 expressed viral proteins inefficiently. Surprisingly, VZV 140K was produced in large amounts at later times of a normal infection, indicating that the polypeptide fails to autoregulate the IE3 promoter. The results strongly suggest that VZV 140K is able to perform most of the functions of Vmw175 during growth of HSV-1, but that differences in detail lead to less efficient virus growth.

Animals↗

PubMed1990

Inactivation of the shutoff gene (UL41) of herpes simplex virus types 1 and 2.

Gene UL41 of herpes simplex virus type 1 (HSV-1) and the corresponding gene of HSV-2, which control the virion-mediated early suppression of cellular protein synthesis, have been inactivated by inserting a beta-galactosidase expression cassette into their coding regions. The resulting recombinants grew well in tissue culture, although with the type 2 recombinant viral protein synthesis was slightly delayed. As a result of inactivation of UL41 host protein synthesis was not suppressed in the presence of actinomycin or early in normal infection, although it declined at a late stage. Polyribosomes were not broken down early in infection, cellular DNA synthesis was not inhibited and in the presence of cycloheximide stable alpha (immediate early) mRNA accumulated, in marked contrast to that of the parent HSV-2 strain. Comparison of the proteins of purified virions of HSV-1 and shutoff-defective recombinant virus revealed discrepancies consistent with the presence of the UL41 gene product in the enveloped virion.

Animals↗

PubMed1990

The product of varicella-zoster virus gene 62 autoregulates its own promoter.

Varicella-zoster (VZV) gene 62 encodes a protein with a predicted Mr of 140,000 (140K) which has considerable amino acid identity with the major immediate early (IE) protein Vmw175 (ICP4) of herpes simplex virus type I (HSV-1). Vmw175 is an essential virus polypeptide with a pivotal role in the activation of early and late viral gene expression and also in the repression of IE gene expression. The VZV 140K protein has been shown to function as a strong transcriptional activator in transfection assays and largely complements for the loss of Vmw175 function in HSV-1. We report the results of cotransfection experiments which demonstrate that the 140K protein strongly represses expression from its own promoter, that of gene 62, thus establishing further functional similarity between it and Vmw175. However, whereas Vmw175 can substitute for the 140K protein in repression of the gene 62 promoter, the 140K protein does not repress the HSV-1 IE3 promoter in the reciprocal experiment. The integrity of a domain of Vmw175 (designated region 2), previously shown to be crucial for repression of the HSV-1 IE3 promoter, is also required for repression of the gene 62 promoter. Moreover, a similar requirement for the highly similar region 2 of the 140K protein for repression is demonstrated, suggesting that VZV 140K protein and HSV-1 Vmw175 autoregulate IE gene expression by a related mechanism.

Animals↗

PubMed1990

Transfer of UL41, the gene controlling virion-associated host cell shutoff, between different strains of herpes simplex virus.

Studies with mutant viruses have suggested that the product of gene UL41 of herpes simplex virus type 1 (HSV-1) controls the virion-mediated inhibition of cellular protein synthesis as well as the rate of degradation of viral mRNAs. HSV-1 strain 17+ has a weak host shutoff function, whereas HSV-2 strain G shuts off strongly. A gene of HSV-2(G), judged from its position in the genome to be the probable analogue of gene UL41 of HSV-1, was inserted into the nonessential thymidine kinase gene of HSV-1(17+). The recombinant virus, 17G41, exhibited a strong shutoff function and its immediate early mRNA did not accumulate in the presence of cycloheximide. It resembled HSV-2(G) in these respects and not the parent, confirming the function of the transferred gene. Recombinant virus 17G41 carries the UL41 genes of both strains, 17+ and G, and in this situation the strong shutoff function was dominant. However, after mixed infection with equal multiplicities of 17G41 and HSV-1(17+) the weak shutoff function was dominant. The recombinant, 17G41, was further modified by insertion of a lacZ expression cassette into the coding region of the original gene UL41 (17+). The resulting virus, 17(41-)G41, also had a strong shutoff activity but grew poorly in tissue culture.

Animals↗

PubMed1990

A mutant of herpes simplex virus type 1 immediate early polypeptide Vmw175 binds to the cap site of its own promoter in vitro but fails to autoregulate in vivo.

Vmw175, the product of herpes simplex virus type 1 immediate early (IE) gene 3, is essential for viral replication. It is required for the activation of transcription from both early and late gene promoters and also for the repression of IE gene expression. Vmw175 is able to bind specifically to certain DNA sequences, some of which (including that at the cap site of IE gene 3) contain the consensus sequence ATCGTC. The presence of this sequence at the cap site has been correlated with the ability of Vmw175 to autoregulate its own promoter. This report describes the characterization of five viruses with temperature-sensitive (ts) lesions in Vmw175. Four of these mutants express Vmw175 which is ts in its ability to bind to DNA in vitro and to autoregulate IE-3 gene expression in the infected cell. Although Vmw175 produced by the remaining mutant, ts1225, fails to autoregulate IE-3 expression at the non-permissive temperature (NPT) its DNA-binding properties are indistinguishable from those of the wild-type protein. This suggests that the ability of Vmw175 to bind to the IE-3 cap site (as measured in vitro) is insufficient for autoregulation (in vivo). All five newly characterized ts mutants are partially permissive for early gene transcription at the NPT, although Vmw175 expressed by four of them is unable to bind to the IE-3 cap site sequence at elevated temperatures. This suggests that binding to one class of recognition sequences by Vmw175, as measured in vitro, is not absolutely required for the activation of early gene promoters during virus infection. The lesions in these five ts mutants lie in the carboxy-terminal third of the polypeptide; three of the mutations (those in ts1219, ts1221 and ts1225) were identified by DNA sequence analysis and were found to affect amino acid residues that are conserved in the homologous proteins from varicella-zoster virus and pseudorabies virus.

Amino Acid Sequence↗

PubMed1990

Control of expression of the varicella-zoster virus major immediate early gene.

The cis-acting DNA sequences and trans-acting proteins that control the expression of the major immediate early (IE) gene of varicella-zoster virus (VZV) were investigated. The location of the IE mRNA 5' terminus was determined by primer extension and S1 nuclease analyses and the functional activities of DNA sequences upstream of this site were analysed by a transfection assay. The VZV IE promoter exhibited low activity in BHK and HeLa cells, but was transactivated by the herpes simplex virus type 1 (HSV-1) virion protein Vmw65. DNA sequences between positions -131 and +57 were responsible for promoter activity, whereas sequences between -410 and -131 mediated the response to Vmw65. Two short elements in the -410 to -131 region formed protein-DNA complexes with HeLa cell nuclear proteins and formed a ternary complex when Vmw65 was added. One of the elements, ATGTAAATGAAAT, possessed a strong similarity to the HSV-1 TAATGARAT. The VZV homologue of Vmw65, encoded by open reading frame (ORF) 10, failed to trans-activate expression from HSV-1 or VZV IE promoters and did not form a ternary complex with functional TAATGARAT elements and HeLa cell proteins. Therefore, stimulation of VZV IE transcription by Vmw65 can occur by a mechanism similar to that employed by HSV-1, but VZV ORF 10 does not function as a trans-activator of IE gene expression.

Animals↗

PubMed1990

Comparative DNA sequence analysis of the host shutoff genes of different strains of herpes simplex virus: type 2 strain HG52 encodes a truncated UL41 product.

Herpes simplex virus (HSV) particles contain a factor which can shut off host protein synthesis during the earliest stages of infection. The efficiency of shutoff varies between different strains of virus, type 2 strains being generally more active than type 1 strains. However, HSV-2 strain HG52 is deficient in host cell shutoff. We have investigated the basis of the different shutoff phenotypes of a strong shutoff strain (HSV-2 strain G), a weak shutoff virus (HSV-1 strain 17 syn+) and HG52 by comparative DNA sequence analysis of gene UL41, which encodes the virion-associated host shutoff factor. The results show that the UL41 genes of strains G and 17 are 86% homologous and that the lack of shutoff by HG52 is likely to be explained by a frameshift mutation within its UL41 coding sequence.

Amino Acid Sequence↗

PubMed1990

A prominent serine-rich region in Vmw175, the major transcriptional regulator protein of herpes simplex virus type 1, is not essential for virus growth in tissue culture.

Herpes simplex virus type 1 (HSV-1) encodes five immediate early (IE) genes of which at least three are involved in the transcriptional regulation of later classes of viral genes. Perhaps the most important of these regulatory proteins is Vmw175, a nuclear phosphoprotein of 1298 predicted amino acid residues. In the absence of functional Vmw175 the virus fails to activate early or late genes or to repress IE gene expression. All viruses of the sub-family alphaherpes-virinae encode polypeptides that are closely related to Vmw175. Mutational studies have shown that regions of homology within this family of gene regulators are generally of functional importance. One of the most striking conserved stretches of amino acid sequence is a run of serine residues followed by a highly acidic region in the amino-terminal fifth of the polypeptide. We have constructed an HSV-1 virus which lacks this serine-rich run within Vmw175. Surprisingly, the virus was viable in tissue culture cells and expressed apparently normal amounts of viral polypeptides. In plaque assays it was very slightly temperature-sensitive and, depending on the state of the host cells, could generate plaques with a syncytial morphology. The mutant protein was able to bind to DNA in a manner indistinguishable from that of the wild-type polypeptide. We conclude that despite its conservation in all of the alphaherpes-virinae so far sequenced, the serine-rich homology is not important for virus growth in tissue culture.

Amino Acid Sequence↗

PubMed1989

Construction and characterization of herpes simplex virus type 1 mutants with defined lesions in immediate early gene 1.

Transcription from the early and late classes of the herpes simplex virus type 1 (HSV-1) promoters requires prior immediate early (IE) gene expression. Although the product of IE gene 1, Vmw110, is not absolutely essential for virus growth in tissue culture, transfection experiments have demonstrated that Vmw110 can activate gene expression both by itself and in a synergistic manner with the product of IE gene 3, Vmw175. This paper describes the construction of 10 mutant HSV-1 viruses with deletion and insertion mutations in Vmw110. The mutant viruses were then studied in single-step growth curve experiments, by assaying for plaques in a variety of cell types and by analysis of viral polypeptide synthesis during productive infection at high and low multiplicities. The results show that mutations in Vmw110 reduce the efficiency of plaque formation by HSV-1; the extent of this reduction depends on cell type and the position of the mutation in the polypeptide. In particular, a potential zinc finger domain is crucial for Vmw110 function. The patterns and amounts of viral polypeptide synthesis during high multiplicity infections with mutant and wild-type viruses were similar in all cell types. At low multiplicity, mutations in Vmw110 reduced viral gene expression in the least permissive cell type. The data suggest that the role of Vmw110 during virus infection in tissue culture is at a very early stage of low multiplicity infections; its inactivity leads to the failure to express viral genes so that the virus does not enter the lytic cycle.

Chromosome Deletion↗

PubMed1989

ICP4-binding sites in the promoter and coding regions of the herpes simplex virus gD gene contribute to activation of in vitro transcription by ICP4.

The herpes simplex virus immediate-early gene product ICP4 activates the transcription of viral early and late genes. We characterized the DNA sequence elements of the early glycoprotein D (gD) gene that play a role in the response to ICP4 in vitro. Using gel mobility shift assays and DNase I footprinting, we identified three ICP4-binding sites, two 5' to the mRNA start site and a third within the coding region. Site II, which gave a footprint between nucleotides -75 and -111 relative to the RNA start site, was previously identified by Faber and Wilcox and contained the reported consensus ICP4-binding site. Site III, which was located between nucleotides +122 and +163, was very similar to the site II sequence, including a core consensus binding sequence, TCGTC. The site I sequence (nucleotides -308 to -282), however, did not share significant homology with either site II or site III. In vitro transcription experiments from mutant constructs of the gD promoter indicated that all three ICP4-binding sites contribute to the stimulation of transcription by ICP4. DNase I footprinting of the gD promoter with uninfected nuclear extracts of HeLa cells showed protection of two very G-rich sequences between nucleotides -33 and -75. We propose that optimal transcription of the gD gene depends on the interaction of ICP4 with multiple binding sites across the gene and cellular factors that recognize specific sequence elements in the promoter.

Animals↗

PubMed1989

Herpes simplex virus type 1 immediate-early protein Vmw110 reactivates latent herpes simplex virus type 2 in an in vitro latency system.

Reactivation of latent herpes simplex virus type 2 (HSV-2) by the immediate-early protein Vmw110 was studied by using an in vitro latency system. Adenovirus recombinants that express Vmw110 reactivated latent HSV-2. An HSV-1 mutant possessing a deletion in a carboxy-terminal region of Vmw110 reactivated latent HSV-2, whereas mutant FXE, which has a deletion in the second exon, did not. Therefore, Vmw110 alone is required to reactivate latent HSV-2 in vitro, and the region of Vmw110 defined by the deletion in FXE is important for this process.

Adenoviridae↗

PubMed1988

The regions of the herpes simplex virus type 1 immediate early protein Vmw175 required for site specific DNA binding closely correspond to those involved in transcriptional regulation.

The immediate-early (IE) protein Vmw175 (ICP4) of HSV-1 is required for the transcription of later classes of viral genes and the repression of IE gene expression. We have previously constructed a panel of plasmid-borne insertion and deletion mutants of the gene encoding Vmw175 and assayed their ability to regulate transcription in transient transfection assays. By this approach we have mapped the regions of the Vmw175 amino acid sequence that are required for transcriptional activation and repression of herpes virus promoters. This paper describes the use of nuclear extracts, made from cells transfected with these mutant plasmids, in gel retardation DNA binding assays in order to define the regions of Vmw175 involved in binding to a specific Vmw175 DNA binding site. The results show that amino acid residues 275-495 (a region which is highly conserved between Vmw175 and the varicella-zoster virus "IE" 140K protein) include structures which are critically required for specific DNA binding, transactivation and repression. This raises the interesting paradox that although the specific DNA sequence recognized by Vmw175 is not commonly found in its target promoters, the protein domain required for recognition of this sequence is required for promoter activation.

Chromosome Deletion↗