Biomedical subjects

L I Pizer

Publications and source records attributed to L I Pizer.

At least 19 recordsLinked to original sources

PubMed1997

Identification of a promoter-specific transactivation domain in the herpes simplex virus regulatory protein ICP4.

ICP4 is expressed during the immediate-early phase of infection by herpes simplex virus (HSV) and activates transcription of viral genes during subsequent phases of productive infection. Several members of the alpha-herpesvirus family encode regulatory proteins that have extensive homology with ICP4 and exhibit a transactivation domain (TAD) at the N terminus. The portions of ICP4 required for nuclear localization, DNA binding, and dimerization have been defined, but a domain that is specifically required for transactivation has not been identified. We have defined a promoter-specific ICP4 TAD by analysis of the activity of GAL4-ICP4 fusion proteins cotransfected into HeLa cells with a luciferase reporter gene linked to a promoter with five GAL4 binding sites. The transactivation activity of GAL4-ICP4 hybrids is located entirely within the first 139 residues of ICP4 and is significantly less potent than the activity of GAL4-TAD hybrids derived from ICP4 homologs. ICP4 residues 97 to 109 are a critical component of this N-terminal TAD. Transient transfection assays performed with nonfusion forms of ICP4 and luciferase genes linked to the HSV glycoprotein D (gD) or thymidine kinase (tk) promoter revealed that ICP4 residues 97 to 109 are required for induction of the gD promoter but are not required for induction of the tk promoter. Comparative experiments with ICP4 homologs revealed that the pseudorabies virus TAD is a potent activator of the gD promoter and a weak activator of the tk promoter. Complementation assays revealed that loss of ICP4 residues 97 to 109 reduced the yield of virus from infected cells nearly 500-fold compared to wild-type ICP4. We conclude that ICP4 residues 97 to 109 are a core component of a promoter-specific transactivation domain that is required for efficient replication of herpes simplex virus.

Animals↗

PubMed1992

Replication, latent infection, and reactivation in neuronal culture with a herpes simplex virus thymidine kinase-negative mutant.

Herpes simplex virus type 1 (HSV-1) mutant viruses lacking functional viral thymidine kinase activity are reported to be incapable of replication in neurons. To investigate the role of viral thymidine kinase (TK) activity in the HSV-1 infection of the neuron, we studied a thymidine kinase-negative (TK-) mutant virus engineered to eliminate TK function without affecting the other known transcripts encoded in this region of the genome. Studies using the mouse eye model demonstrated that the mutant behaved as is reported for other TK- viruses: DNA of the mutant virus was detected in the ganglia during the latent infection by polymerase chain reaction, but virus did not reactivate after explantation of the ganglia. Utilizing the neuronal cultures, we investigated the ability of the mutant virus to replicate in neurons and the capacity of the mutant virus to establish latency and reactivate. With a low multiplicity of infection (m.o.i.), replication of the TK- mutant virus in sensory neurons in culture was significantly delayed compared to that of the wild-type virus. However, when a high m.o.i. was used, the mutant and the wild-type viruses replicated with similar kinetics. The TK- mutant virus was capable of establishment of latency and reactivation from the latent infection in sensory neurons in culture. These data suggest that HSV-1 thymidine kinase activity facilitates viral replication, but that TK activity is not essential for either replication or reactivation from latent infections in neurons in vitro.

Animals↗

PubMed1992

Activation of second-messenger pathways reactivates latent herpes simplex virus in neuronal cultures.

Herpes simplex virus type 1 (HSV-1) establishes latent infections in neurons of sympathetic and sensory ganglia in humans, and reactivation of latent virus results in recurrent disease. Previously, we reported establishment of latent HSV-1 infections in neuronal cultures derived from rats, monkeys, and humans; reactivation occurs following nerve growth factor (NGF) deprivation. The processes controlling HSV latency are not understood. Using the in vitro neuronal latency system, we have shown that latent HSV-1 reactivated in response to stimulation of at least two second-messenger pathways. Stimulation of cAMP-dependent pathways by several mechanisms or activation of protein kinase C by phorbol myristate acetate (PMA) resulted in reactivation of latent HSV-1. The reactivation kinetics following treatment with activators of protein kinase A and C were accelerated compared with those following NGF deprivation. 2-Aminopurine, which inhibits NGF-stimulated protein kinases and other classes of protein kinases, but does not effect protein kinase A or C, blocked reactivation produced by NGF deprivation or treatment with a cAMP analog, but not reactivation by PMA treatment. These results demonstrate that latent HSV-1 reactivates in neurons in vitro in response to activation of second-messenger pathways.

Cell Line↗

PubMed1992

The ICP4 binding sites in the herpes simplex virus type 1 glycoprotein D (gD) promoter are not essential for efficient gD transcription during virus infection.

Activation of the early and late genes of herpes simplex virus type 1 during infection in tissue culture requires functional immediate-early regulatory protein ICP4. ICP4 is a specific DNA-binding protein which recognizes a variety of DNA sequences, many of which contain the consensus ATCGTC. In general, mutations which impair the ability of ICP4 to bind to DNA also eliminate its ability to activate viral early and late promoters both in transfection assays and in the infected cell. However, the role of ICP4 binding sites in the viral genome is unclear; many early and late promoters do not contain consensus binding sites in their vicinity. The glycoprotein D (gD) gene contains two well-characterized ICP4 binding sites upstream of its promoter and a third downstream of the transcription start site. Multimerization of one of these sites has been shown to increase the response of the gD promoter to ICP4 in transfection assays, while their removal reduces stimulation of the gD promoter by ICP4 in vitro. To assess the role of these binding sites during virus infection, we have constructed a recombinant viral genome which has mutations affecting all three. Comparison of the amounts of gD RNA synthesized by the recombinant and wild-type viruses indicated that the mutations had little or no effect on the activity of the gD promoter. Therefore, either the sites have no essential role in gD promoter regulation in the presence of all of the herpes simplex virus type 1 IE polypeptides during a normal infection or they can be functionally substituted by other ICP4 binding sites elsewhere in the genome.

Animals↗

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

Detection of the latency-associated transcript in neuronal cultures during the latent infection with herpes simplex virus type 1.

The transcriptional studies reported in this paper indicate that the latency-associated transcript (LAT) is present in neuronal cultures during the latent infection with herpes simplex virus type 1 (HSV-1). During the latent infection glycoprotein D (gD) mRNA, a mRNA characteristic of the productive infection, is not detected. However, following reactivation by nerve growth factor (NGF) deprivation, gD mRNA is detected in the neuronal cultures. Thus, the restricted viral gene expression in the in vitro neuronal model indicates that the latent infection in culture is analogous to that observed in vivo.

Cells, Cultured↗

PubMed1991

An antigen encoded by the latency-associated transcript in neuronal cell cultures latently infected with herpes simplex virus type 1.

During latent infection of neurons with herpes simplex virus type 1, viral transcription is restricted to the latency-associated transcripts (LATs). These RNAs contain open reading frames, but detection of a protein encoded by the LATs has not been reported. We used immunocytochemical techniques to demonstrate that an antiserum directed against a bacterially expressed fusion protein containing part of a LAT-encoded polypeptide recognized an antigen present in primary neurons latently infected in vitro. This antigen (called LAA, for latency-associated antigen) was not detected in mock-infected neurons, in productively infected Vero cells, or in neurons latently infected with a mutant virus carrying a deletion in the LAT gene. By Western immunoblot analysis, we demonstrated the presence of a protein with an apparent molecular mass of 80 kDa recognized by the anti-LAA antiserum in latently infected neurons.

Animals↗

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

Replication of herpes simplex virus in blood monocytes and placental macrophages from human neonates.

Increased permissiveness of macrophages for herpes simplex virus (HSV) replication may be a mechanism for the dissemination and severity of neonatal herpetic infection. We have assessed the replication of HSV in neonatal blood monocytes and placental macrophages using several criteria for viral permissiveness. Assay of production of infectious progeny virus indicated that cord blood monocytes, like adult monocytes, were nonpermissive for HSV (about 1% of cells producing virus). In vitro culture of cord blood monocytes resulted in increased replication of HSV, but no greater extent than virus production in cultured adult cells. HSV infection of fetal placental macrophages was weak but present (4.4% of cells). Assay of production of viral antigens and electron microscopic analysis of structural elements indicated that a larger number of cord blood monocytes and placental macrophages were abortively infected than were productively infected. These results indicate that monocytes and macrophages from human neonates do not show the enhanced permissiveness for HSV demonstrated in newborn mice and suggest that dissemination of herpetic infection in human newborns cannot be explained by increased neonatal monocyte permissiveness for HSV.

DNA Replication↗

PubMed1988

Role for DNA-protein interaction in activation of the herpes simplex virus glycoprotein D gene.

On the basis of experiments with mutant virus and transfection with isolated genes, the herpes simplex virus immediate-early gene product ICP4 is known to positively regulate the transcription of viral early and late genes and negatively regulate expression from its own promoter. Binding of ICP4 to DNA sequences in several viral genes has been reported, yet the significance of ICP4-DNA interaction in transcriptional activation remains unclear. We have studied this problem by using the early glycoprotein D (gD) gene, which possesses a binding site at approximately -100 relative to the RNA initiation site. We linked this promoter and various mutant constructs to the chloramphenicol acetyltransferase gene in order to measure promoter activity in transient transfections both in the presence and in the absence of an ICP4-encoding plasmid. The natural promoter was activated 3.3-fold, and a deletion construct lacking the binding site was activated minimally (1.7-fold). Constructs containing multiple tandem repeats of the binding site (three or five inserts) demonstrated higher expression in the presence of ICP4 than did the natural promoter while retaining low levels of expression when unstimulated. Gel mobility shift assays and DNase I footprinting analyses indicated that ICP4 associated with multiple binding sites. In vitro transcription from a gD promoter construct containing multiple binding sites showed increased RNA synthesis in the presence of partially purified ICP4. These data provide the first direct evidence that binding of ICP4 to a specific DNA sequence in the gD gene contributes to activation of transcription.

Animals↗

PubMed1987

A mammalian cell line designed to test the mutagenic activity of anti-herpes nucleosides.

The herpes simplex virus (HSV) thymidine kinase (tk) gene was transfected into Chinese hamster ovary (CHO) 51-11 gly- cells to test its effect on the cytotoxic and mutagenic activity of anti-herpetic nucleoside analogues. Insertion of the viral tk was verified by Southern blot analysis, by sensitivity to acyclovir, and by elevated in vitro thymidine kinase (TK) activity. TK activity was increased by superinfection with a tk- virus and inhibited by antibody to viral TK. Acyclovir (ACV) was somewhat more cytotoxic in the 51-D3 cell line that expresses the viral TK than in the 51-11 parent line. Growth in ACV did not increase over background mutations at the hprt locus. FIAC (2'-fluoro-5-iodio-aracytosine) was slightly cytotoxic to the parent 51-11 line and the tk-containing clone 51-D3. FMAU (2'-fluoro-5-methyl-arauracil) had pronounced cytotoxicity in both cell lines: the 50% survival points were 1.0 microM for 51-11 cells and 0.2 microM for 51-D3. The clone 51-D3 was more sensitive than 51-11 to low concentrations of FIAU (2'-fluoro-5-iodo-arauracil), and when treated with FIAU 51-D3 had a mutation frequency to glycine independence 5 times greater than that of 51-11 cells. With both cell lines the mutation frequency at the hprt locus did not increase after growth in the presence of FIAC or FIAU. A 7-fold increase in mutation frequency at the hprt locus was detected after 51-D3 cells were grown with iododeoxyuridine. Trifluorothymidine was more toxic to 51-D3 than to 51-11 cells and increased the mutation frequency 2-fold. Cytosine-beta-D-arabinofuranoside showed no differential cytotoxicity on the two cell lines and did not increase the mutation frequency at the hprt locus.

Acyclovir↗

PubMed1986

Herpes simplex virus immediate early infected-cell polypeptide 4 binds to DNA and promotes transcription.

In herpes simplex virus (HSV)-infected cells, there is a sequential expression of viral genes. In vivo experiments have implicated the Mr 175,000 immediate early protein ICP4 (infected-cell polypeptide 4) in the regulation of viral RNA synthesis, but the mechanism whereby ICP4 regulates transcription of viral genes is at present unknown. In this report we describe experiments with an in vitro transcription system and a purified preparation of ICP4 (estimated 5% of total protein). Using DNA from the HSV glycoprotein D gene (gD) as the template, we have observed that specific binding occurs between ICP4 and DNA sequences adjacent to the gD gene promoter and ICP4 stimulates initiation of transcription from the gD gene. The degree of stimulation depends on the amount of ICP4 present in the incubation. The kinetics of RNA synthesis demonstrate that the protein acts at the initiation step of transcription. These results identify ICP4 as a viral transcription factor whose presence on DNA facilitates the formation of transcription complexes.

Animals↗

PubMed1986

Regulation of transcription in vitro from herpes simplex virus genes.

In vitro transcription assays were carried out by using as templates DNAs cut from the herpes simplex virus early glycoprotein D gene, the late glycoprotein C gene, the late VP5 gene, and the immediate-early ICP22 gene. Nuclear extracts from suspension cultures of uninfected HeLa cells effectively synthesized RNAs from genes of the immediate-early and delayed-early classes. To a lesser extent, the extracts also used DNAs cut from the late genes as templates. Transcription from the immediate-early gene was inhibited in extracts prepared from infected cells. Analysis of the proteins in infected-cell extracts by gel electrophoresis, transfer to nitrocellulose, and probing with specific antibody demonstrated the presence of the viral regulatory protein ICP4. Chromatographic fractionation of nuclear extract from infected cells yielded a mixture of proteins (fraction VIII) enriched in ICP4 (S.W. Faber and K.W. Wilcox, Nucleic Acids Res., 14:6067-6083, 1986). Addition of fraction VIII to the in vitro assay affected transcription. Depending on the DNA in the assay, an inhibitory or stimulatory effect was observed. Inhibition of RNA synthesis was found when DNA from the immediate-early gene was used as a template, and stimulation was found when DNA from the early or late gene was used.

Capsid↗

PubMed1985

Different forms of membrane-associated herpes simplex virus glycoproteins induce functionally distinct subsets of herpes simplex virus-specific suppressor T cells.

Previous studies have shown that two types of virus-specific suppressor T cells (Ts) are induced in mice made tolerant with herpes simplex virus (HSV)-infected spleen cells (SC). One type of Ts blocks the afferent phase of the delayed hypersensitivity response to HSV (Ts-aff), and the other blocks the efferent or effector phase (Ts-eff). In this report we show that the induction requirements for these suppressor populations differ. Injection of SC infected for 6 h with HSV at a multiplicity of infection of 5 or less or treated with heat-inactivated virus induced only Ts-aff. Similar results were seen with SC incubated for 90 min in virus-free preparations containing only viral proteins. In contrast, the Ts-eff population was induced only by SC treated for 6 h with infectious HSV at a multiplicity of infection of 10. Collectively, these data indicate that Ts-aff are induced by adsorbed HSV antigens on SC, whereas Ts-eff are induced by nascent HSV antigens expressed on infected SC. In addition to their induction requirements, the two types of regulatory cells differ in their expression of effector function. Ts-eff but not Ts-aff require a cyclophosphamide-sensitive target cell in the immune recipient for suppressor function. The possible identity of this target cell and the significance of the different induction requirements between the two types of Ts are discussed.

Animals↗

PubMed1985

Sequences upstream of c-mos(rat) that block RNA accumulation in mouse cells do not inhibit in vitro transcription.

An unusual feature of the c-mos oncogene is the lack of expression in mouse tissues. Recombinant plasmids that contain the strong adenovirus late promoter and different amounts of cellular DNA 5' to c-mos(rat) were constructed and tested in transfection and transcription assays. The cellular sequences inhibit RNA accumulation in mouse but not human cells and do not inhibit in vitro transcription of the plasmid DNAs.

Adenoviridae↗

PubMed1985

Delayed hypersensitivity and immune protection against herpes simplex virus: suppressor T cells that regulate the induction of delayed hypersensitivity effector T cells also regulate the induction of protective T cells.

We have been studying delayed hypersensitivity (DH) to herpes simplex virus (HSV) in order to examine the role of this response in host defense against acute and recurrent HSV infections. In previous reports the basic parameters of DH to HSV have been characterized by using a murine ear swelling model, and also the regulation of DH to HSV induced by i.v. injection of the virus. In this paper, we describe a murine protection system and our use of the ability to specifically regulate DH to HSV to examine the correlation between T cells that transfer DH (TDH) and cells that transfer protection from acute HSV infection. Both DH and protection can be transferred with lymph node cells from mice immunized subcutaneously 4 days previously. The effector cell appears to be a T cell, because serum from these donors confers no protection and treatment of immune cells with anti-Thy-1.2 plus complement reduced their ability to protect. Tolerance of DH to HSV was induced by i.v. injection 7 days before subcutaneous immunization. Tolerized mice were unable to generate protective cells. Furthermore, tolerized mice contained suppressor T cells that suppressed not only DH but also the development of protective cells. Regulation of protective cells was shown to be virus specific, because mice tolerized with vesicular stomatitis virus (VSV) were not impaired in their ability to generate T cells that protected from HSV infection. The correlation between the TDH cell and cells that transfer protection from acute HSV infection is discussed.

Acute Disease↗

PubMed1984

Herpes simplex virus adsorption to and survival within placental lymphoid populations.

Neonatal herpes simplex virus (HSV) infections are frequently characterized by widespread dissemination and high morbidity and mortality. To assess the potential role of the lymphocyte in permitting or promoting dissemination of HSV, we studied lymphoid populations of placental and adult blood with respect to HSV survival. Adsorption of HSV to the lymphocyte surface did not differ between placental and adult cells. Placental blood permitted greater survival of HSV than adult lymphocytes. T-cell-enriched lymphocytes appeared to be slightly more permissive than T-cell-depleted lymphocytes, but these differences were not statistically significant and apparently less than adult-placental blood differences. These findings lend support to the hypothesis that intracellular changes common to various neonatal lymphoid populations may permit or promote dissemination of HSV in the neonate.

Adsorption↗

PubMed1983

Tolerance and suppression of immunity to herpes simplex virus: different presentations of antigens induce different types of suppressor cells.

In this report, we examine tolerance (hyporesponsiveness) and suppression of delayed hypersensitivity (DH) to herpes simplex virus (HSV) in mice, using two different forms of tolerogen: HSV particles and HSV-infected spleen cells. The intravenous injection of mice with either HSV particles or spleen cells 7 days before subcutaneous immunization with virus induced a profound state of unresponsiveness. This unresponsive state was mediated, at least in part, by suppressor T cells (Ts), which were demonstrated by passive transfer to naive recipients. However, different types of Ts were induced depending on the form of the tolerogen. The injection of HSV particles induced Ts which suppressed the induction but not the expression of DH. On the other hand, the injection of HSV spleen cells induced two types of Ts: one which inhibited the induction of the DH response and one which inhibited the expression of DH to HSV. Both tolerance and Ts are virus specific (i.e., the DH response to an unrelated virus was not inhibited) but not type specific for HSV type 1 and HSV type 2. Since both virus particles and virus-infected cells may be present in the blood during HSV infection, the induction of this type of immune regulation may influence the outcome of both acute and latent HSV infections.

Animals↗