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Biomedical subjects

B Roizman

Publications and source records attributed to B Roizman.

At least 91 records · Page 5Linked to original sources
PubMed1997

Interaction of herpes simplex virus 1 alpha regulatory protein ICP0 with elongation factor 1delta: ICP0 affects translational machinery.

The herpes simplex virus 1 (HSV-1)-infected cell protein 0 (ICP0) is a promiscuous transactivator, and by necessity, its functions must be mediated through cellular gene products. In an attempt to identify cellular factors interacting with ICP0, we used the carboxyl-terminal domain of ICP0 as "bait" in the yeast (Saccharomyces cerevisiae) two-hybrid system. Our results were as follows: (i) All 43 cDNAs in positive yeast colonies were found to encode the same translation factor, elongation factor delta-1 (EF-1delta). (ii) Purified chimeric protein consisting of glutathione S-transferase (GST) fused to EF-1delta specifically formed complexes with ICP0 contained in HSV-1-infected cell lysate. (iii) Fractionation of infected HEp-2 cells and immunofluorescence studies revealed that ICP0 was localized both in the nucleus and in the cytoplasm. In primary human foreskin fibroblasts, ICP0 was localized predominantly in the cytoplasm throughout HSV-1 infection even early in infection. (iv) Addition of the chimeric protein GST-carboxyl-terminal domain of ICP0 to the rabbit reticulocyte lysate in vitro translation system resulted in a dose-dependent decrease in protein synthesis. In contrast, GST alone or GST fused to the amino-terminal domain of ICP0 had no effect on the in vitro translation system. (v) The predominant forms of EF-1delta on electrophoresis in denaturing gels have apparent Mrs of 38,000 and 40,000. The higher-Mr form is a minor species in mock-infected cells, whereas in human fibroblasts and Vero cells infected with HSV-1, this isoform becomes dominant. These results indicate that ICP0 is present and may have a significant role in the cytoplasm of infected cells, possibly by altering the efficiency of translation of viral mRNAs.

Animals↗
PubMed1997

Association of herpes simplex virus regulatory protein ICP22 with transcriptional complexes containing EAP, ICP4, RNA polymerase II, and viral DNA requires posttranslational modification by the U(L)13 proteinkinase.

The expression of herpes simplex virus 1 gamma (late) genes requires functional alpha proteins (gamma1 genes) and the onset of viral DNA synthesis (gamma2 genes). We report that late in infection after the onset of viral DNA synthesis, cell nuclei exhibit defined structures which contain two viral regulatory proteins (infected cell proteins 4 and 22) required for gamma gene expression, RNA polymerase II, a host nucleolar protein (EAP or L22) known to be associated with ribosomes and to bind small RNAs, including the Epstein-Barr virus small nuclear RNAs, and newly synthesized progeny DNA. The formation of these complexes required the onset of viral DNA synthesis. The association of infected cell protein 22, a highly posttranslationally processed protein, with these structures did not occur in cells infected with a viral mutant deleted in the genes U(L)13 and U(S)3, each of which specifies a protein kinase known to phosphorylate the protein.

DNA, Viral↗
PubMed1997

The range and distribution of murine central nervous system cells infected with the gamma(1)34.5- mutant of herpes simplex virus 1.

Wild-type herpes simplex virus 1 (HSV-1) multiplies, spreads, and rapidly destroys cells of the murine central nervous system (CNS). In contrast, mutants lacking both copies of the gamma(1)34.5- gene have been shown to be virtually lacking in virulence even after direct inoculation of high-titered virus into the CNS of susceptible mice (J. Chou, E. R. Kern, R. J. Whitley, and B. Roizman, Science 250:1262-1266, 1990). To investigate the host range and distribution of infected cells in the CNS of mice, 4- to 5-week-old mice were inoculated stereotaxically into the caudate/putamen with 3 x 10(5) PFU of the gamma(1)34.5- virus R3616. Four-micrometer-thick sections of mouse brains removed on day 3, 5, or 7 after infection were reacted with a polyclonal antibody directed primarily to structural proteins of the virus and with antibodies specific for neurons, astrocytes, or oligodendrocytes. This report shows the following: (i) most of the tissue damage caused by R3616 was at the site of injection, (ii) the virus spread by retrograde transport from the site of infection to neuronal cell nuclei at distant sites and to ependymal cells by cerebrospinal fluid, (iii) the virus infected neurons, astrocytes, oligodendrocytes, and ependymal cells and hence did not discriminate among CNS cells, (iv) viral replication in some neurons could be deduced from the observation of infected astrocytes and oligodendrocytes at distant sites, and (v) infected cells were being efficiently cleared from the nervous system by day 7 after infection. We conclude that the gamma(1)34.5- attenuation phenotype is reflected in a gross reduction in the ability of the virus to replicate and spread from cell to cell and is not due to a restricted host range. The block in viral replication appears to be a late event in viral replication.

Animals↗
PubMed1997

Suppression of the phenotype of gamma(1)34.5- herpes simplex virus 1: failure of activated RNA-dependent protein kinase to shut off protein synthesis is associated with a deletion in the domain of the alpha47 gene.

Earlier studies have shown that infection of human cells by herpes simplex virus 1 (HSV-1) results in the activation of RNA-dependent protein kinase (PKR) but that the alpha subunit of eIF-2 is not phosphorylated and that protein synthesis is unaffected. In the absence of the viral gamma(1)34.5 gene, eIF-2alpha is phosphorylated and protein synthesis is prematurely shut off (J. Chou, J. J. Chen, M. Gross, and B. Roizman, Proc. Natl. Acad. Sci. USA 92:10516-10520, 1995). A second recent paper reported the selection of second-site suppressor mutants characterized by near-wild-type protein synthesis in cells infected with gamma(1)34.5- mutants (I. Mohr and Y. Gluzman, EMBO J. 15:4759-4766, 1996). Here, we report the properties of the spontaneous HSV-1 suppressor mutant Sup-1, which is characterized by spontaneous deletion of 503 bp encompassing the domain of the alpha47 gene and junction with the inverted repeats flanking the unique short (U(S)) sequence of the HSV-1 DNA resulting in the juxtaposition of the alpha47 promoter to the coding domain of the U(S)11 gene. This mutant does not exhibit the shutoff of protein synthesis characteristic of the gamma(1)34.5- virus. Specifically, Sup-1 in SK-N-SH human neuroblastoma cells (i) did not exhibit the function of the alpha47 gene characterized by a reduction in the transport of peptides across the endoplasmic reticulum of permealized cells consistent with the absence of alpha47 gene sequences, (ii) accumulated U(S)11 protein at levels analogous to those of the wild-type parent but the protein was made at earlier times after infection, as would be expected from a change in the promoter, and (iii) activated PKR like that of the parent, gamma(1)34.5- virus, but (iv) did not cause premature shutoff of protein synthesis and therefore was similar to the wild-type parent virus rather than the gamma(1)34.5- virus from which it was derived. We conclude that the mechanism by which Sup-1 blocks the shutoff of protein synthesis associated with phosphorylation of eIF-2alpha by the activated PKR is not readily explainable by a secondary mutation characterized by a deletion.

Animals↗
PubMed1996

The UL 16 gene product of herpes simplex virus 1 is a virion protein that colocalizes with intranuclear capsid proteins.

The UL16 gene of herpes simplex virus maps within the intron of the UL 15 gene. This report shows the following: (i) A polyclonal antiserum directed against a bacterial fusion protein containing glutathione S-transferase fused to the C-terminus of the UL 16 gene reacted with an apparent M(r) 40,000 protein in HSV-1 infected cell lysates. (ii) The protein encoded by UL 16 was dependent on viral DNA synthesis for accumulation to detectable levels. (iii) In immunofluorescence studies, the polyclonal UL 16/GST-specific antiserum was shown to stain the nucleus of infected cells at 18 hr after infection in areas containing high concentrations of HSV capsid proteins. These nuclear compartments have been described previously as viral assemblons (Ward et al., J. Virol. 70, 4623-4631, 1996) and are distinct from compartments containing replicating DNA. Localization within assemblons argues for a role of UL 16 encoded protein in capsid assembly or maturation. (iv) At 22 hr after infection, UL 16-specific immunofluorescence was present in both the nucleus and the cytoplasm. (v) Consistent with the change in localization at late times after infection, the UL 16 protein was found to be a component of purified virions.

Animals↗
PubMed1996

Partial substitution of the functions of the herpes simplex virus 1 U(L)13 gene by the human cytomegalovirus U(L)97 gene.

The predicted amino acid sequence of the human cytomegalovirus U(L)97 protein bears partial homology to the herpes simplex virus 1 U(L)13 protein, especially in regions that are homologous to conserved domains characteristic of protein kinases. Earlier studies showed that U(L)13 mediated the posttranslational processing of several herpes simplex virus 1 proteins including ICP22. Whereas no kinase activity has been specifically attributed to U(L)13, it has been shown that U(L)97 can phosphorylate ganciclovir. To examine whether U(L)97 can substitute for U(L)13, we constructed a herpes simplex virus 1 recombinant virus, R4970, in which U(L)13 was replaced by U(L)97 and in addition, the thymidine kinase gene was deleted. Characterization of this recombinant virus showed the following: (1) The recombinant virus grew as well as the wild-type virus in BHKTK+ cells, which restricted the growth of the U(L)13 virus. (2) U(L)97 could partially mediate the posttranslational modification of HSV-1 ICP22. This modification correlated with the restoration of the amounts of ICP0 and U(S)11 proteins, which were down regulated in the U(L)13- virus-infected cells. (3) The recombinant virus was sensitive to ganciclovir in Vero- and KHOS-infected cells but not in the 143 thymidine kinase minus cells derived from KHOS cells. Vero cells infected with this recombinant virus phosphorylated ganciclovir. We conclude that U(L)97 partially compensates for U(L)13 functions.

Animals↗
PubMed1996

Alternatively spliced mRNAs predicted to yield frame-shift proteins and stable intron 1 RNAs of the herpes simplex virus 1 regulatory gene alpha 0 accumulate in the cytoplasm of infected cells.

The infected cell protein no. 0 (ICP0), the product of the alpha 0 gene, and an important herpes simplex virus 1 regulatory protein is encoded by three exons. We report that intron 1 forms a family of four stable nonpolyadenylylated cytoplasmic RNAs sharing a common 5' end but differing in 3' ends. The 5' and 3' ends correspond to the accepted splice donor and four splice acceptor sites within the mapped intron domain. The most distant splice acceptor site yields the mRNA encoding the 775-aa protein known as ICP0. The mRNAs resulting from the use of alternative splice acceptor sites were also present in the cytoplasm of infected cells and would be predicted to encode proteins of 152 (ICP0-B), 87 (ICP0-C), and 90 (ICP0-D) amino acids, respectively. Both the stability of the alpha 0 mRNA and the utilization of at least one splice acceptor site was regulated by ICP22 and or US1.5 protein inasmuch as cells infected with a mutant from which these genes had been deleted accumulated smaller amounts of alpha 0 mRNA than would be predicted from the amounts of accumulated intron RNAs. In addition, one splice acceptor site was at best underutilized. These results indicate that both the splicing pattern and longevity of alpha 0 mRNA are regulated. These and other recent examples indicate that herpes simplex virus 1 regulates its own gene expression and that of the infected cells through control of mRNA splicing and longevity.

Alternative Splicing↗
PubMed1996

The function of herpes simplex virus genes: a primer for genetic engineering of novel vectors.

Herpes simplex virus vectors are being developed for delivery and expression of human genes to the central nervous system, selective destruction of cancer cells, and as carriers for genes encoding antigens that induce protective immunity against infectious agents. Vectors constructed to meet these objectives must differ from wild-type virus with respect to host range, reactivation from latency, and expression of viral genes. The vectors currently being developed are (i) helper free amplicons, (ii) replication defective viruses, and (iii) genetically engineered replication competent viruses with restricted host range. Whereas the former two types of vectors require stable, continuous cell lines expressing viral genes for their replication, the replication competent viruses will replicate on approved primary human cell strains.

Genes, Viral↗
PubMed1996

The application of genetically engineered herpes simplex viruses to the treatment of experimental brain tumors.

Due to lack of effective therapy, primary brain tumors are the focus of intense investigation of novel experimental approaches that use vectors and recombinant viruses. Therapeutic approaches have been both indirect, whereby vectors are used, or direct to allow for direct cell killing by the introduced virus. Genetically engineered herpes simplex viruses are currently being evaluated as an experimental approach to eradicate malignant human gliomas. Initial studies with gamma (1)34.5 mutants, R3616 (from which both copies of the gamma (1)34.5 gene have been deleted) and R4009 (a construct with two stop codons inserted into the gamma (1)34.5 gene), have been assessed. In a syngeneic scid mouse intracranial tumor model, recombinant herpes simplex virus can be experimentally used for the treatment of brain tumors. These viruses and additional engineered viruses were subsequently tested in human glioma cells both in vitro and in vivo. Using a xenogeneic scid mouse intracranial glioma model, R4009 therapy of established tumors significantly prolonged survival. Most importantly, long-term survival was achieved, with histologic evidence that R4009 eradicated intracranial tumors in this model. Furthermore, the opportunity to evaluate gamma (1)34.5 mutants that have enhanced oncolytic activity, e.g., R8309 where the carboxyl terminus of the gamma (1)34.5 gene has been replaced by the murine homologue, MyD116, are considered.

Adenoviridae↗
PubMed1996

Open reading frame P--a herpes simplex virus gene repressed during productive infection encodes a protein that binds a splicing factor and reduces synthesis of viral proteins made from spliced mRNA.

The open reading frame P (ORF P) is located in the domain and on the DNA strand of the herpes simplex virus 1 transcribed during latent infection. ORF P is not expressed in productively infected cells as a consequence of repression by the binding of the major viral regulatory protein to its high-affinity binding site. In cells infected with a mutant virus carrying a derepressed gene, ORF P protein is extensively posttranslationally processed. We report that ORF P interacts with a component of the splicing factor SF2/ASF, pulls down a component of the SM antigens, and colocalizes with splicing factors in nuclei of infected cells. The hypothesis that ORF P protein may act to regulate viral gene expression, particularly in situations such as latently infected sensory neurons in which the major regulatory protein is not expressed, is supported by the evidence that in cells infected with a mutant in which the ORF P gene was derepressed, the products of the regulatory genes alpha 0 and alpha 22 are reduced in amounts early in infection but recover late in infection. The proteins encoded by these genes are made from spliced mRNAs, and the extent of recovery of these proteins late in infection correlates with the extent of accumulation of post-translationally processed forms of ORF P protein.

Carcinoma, Hepatocellular↗
PubMed1996

The herpes simplex virus major regulatory protein ICP4 blocks apoptosis induced by the virus or by hyperthermia.

Cells infected with herpes simplex virus 1 (HSV-1) undergo productive or latent infection without exhibiting features characteristic of apoptosis. In this report, we show that HSV-1 induces apoptosis but has evolved a function that blocks apoptosis induced by infection as well as by other means. Specifically, (i) Vero cells infected with a HSV-1 mutant deleted in the regulatory gene alpha 4 (that encodes repressor and transactivating functions), but not those infected with wild-type HSV-1(F), exhibit cytoplasmic blebbing, chromatin condensation, and fragmented DNA detected as a ladder in agarose gels or by labeling free DNA ends with terminal transferase; (ii) Vero cells infected with wild-type HSV-1(F) or cells expressing the alpha 4 gene and infected with the alpha 4- virus did not exhibit apoptosis; (iii) fragmentation of cellular DNA was observed in Vero cells that were mock-infected or infected with the alpha 4- virus and maintained at 39.5 degrees C, but not in cells infected with wild-type virus and maintained at the same temperature. Wild-type strains of HSV-1 with limited extrahuman passages, such as HSV-1 (F), carry a temperature-sensitive lesion in the alpha 4 gene and at 39.5 degrees C only alpha genes are expressed. These results indicate that the product of the alpha 4 gene is able to suppress apoptosis induced by the virus as well by other means.

Animals↗
PubMed1996

Functional interaction and colocalization of the herpes simplex virus 1 major regulatory protein ICP4 with EAP, a nucleolar-ribosomal protein.

The herpes simplex virus 1 infected cell protein 4 (ICP4) binds to DNA and regulates gene expression both positively and negatively. EAP (Epstein-Barr virus-encoded small nuclear RNA-associated protein) binds to small nonpolyadenylylated nuclear RNAs and is found in nucleoli and in ribosomes, where it is also known as L22. We report that EAP interacts with a domain of ICP4 that is known to bind viral DNA response elements and transcriptional factors. In a gel-shift assay, a glutathione S-transferase (GST)-EAP fusion protein disrupted the binding of ICP4 to its cognate site on DNA in a dose-dependent manner. This effect appeared to be specifically due to EAP binding to ICP4 because (i) GST alone did not alter the binding of ICP4 to DNA, (ii) GST-EAP did not bind to the probe DNA, and (iii) GST-EAP did not influence the binding of the alpha gene trans-inducing factor (alphaTIF or VP16) to its DNA cognate site. Early in infection, ICP4 was dispersed throughout the nucleoplasm, whereas EAP was localized to the nucleoli. Late in infection, EAP was translocated from nucleoli and colocalized with ICP4 in small, dense nuclear structures. The formation of dense structures and the colocalization of EAP and ICP4 did not occur if virus DNA synthesis and late gene expression were prevented by the infection of cells at the nonpermissive temperature with a mutant virus defective in DNA synthesis, or in cells infected and maintained in the presence of phosphonoacetate, which is an inhibitor of viral DNA synthesis. These results suggest that the translocation of EAP from the nucleolus to the nucleoplasm is a viral function and that EAP plays a role in the regulatory functions expressed by ICP4.

Base Sequence↗
PubMed1996

The promoter and transcriptional unit of a novel herpes simplex virus 1 alpha gene are contained in, and encode a protein in frame with, the open reading frame of the alpha 22 gene.

The herpes simplex virus type 1 genome encodes a set of genes (alpha genes) expressed in the absence of de novo viral protein synthesis. Earlier studies have shown that the product of the alpha 22 gene, a member of this set, is nucleotidylylated by casein kinase II and phosphorylated by viral protein kinases encoded by UL13 and US3. Mutants lacking the carboxyl-terminal domain starting with amino acid 200 exhibit reduced capacity to replicate in primary human cell strains or in cells of rodent derivation and also exhibit reduced expression of a subset of gamma or late genes. We report that the domain of the alpha 22 gene is transcribed by two 3'-coterminal mRNAs. The longer transcript reported encodes the 420-amino-acid alpha 22 protein, whereas the shorter transcript reported here encodes a protein containing the carboxyl-terminal 273 amino acids of the alpha 22 protein. The shorter gene is designated US1.5. The US1.5 mRNA is synthesized in cells infected and maintained in the presence of cycloheximide and under other conditions which restrict viral gene expression to alpha genes. In-frame insertion of linkers encoding 18, 21, or 22 amino acids after codon 200 or 240 of the alpha 22 protein did not affect the known functions or phenotype associated with the wild-type alpha 22 gene or its product. Earlier studies have placed the nucleotidylylated sequences in the amino-terminal portion of the protein. The results of these studies indicate that the US1.5 gene encodes the functions associated with replication in human primary or rodent cells and optimal expression of alpha 0 and gamma genes. This finding brings the number of genes known to map in the unique short region of the herpes simplex virus type 1 DNA to 14 and the total number of different genes to 78.

Amino Acid Sequence↗
PubMed1996

The carboxyl terminus of the murine MyD116 gene substitutes for the corresponding domain of the gamma(1)34.5 gene of herpes simplex virus to preclude the premature shutoff of total protein synthesis in infected human cells.

The herpes simplex virus 1 mutants from which both copies of the gamma(1)34.5 gene had been deleted trigger total shutoff of protein synthesis in human neuroblastoma cells and human foreskin fibroblasts but not in African green monkey (Vero) cells. The carboxyl-terminal 64 amino acids of gamma(1)34.5 are homologous to the corresponding domain of MyD116, a murine myeloid differentiation primary responsive gene. The carboxyl-terminal domain of gamma(1)34.5 is required to preclude the shutoff of protein synthesis (J. Chou and B. Roizman, Proc. Natl. Acad. Sci. USA 91:5247-5251, 1994). We report that in-frame substitution of the carboxyl terminus of gamma(1)34.5 with the corresponding domain of MyD116 in the context of the viral genome restored the ability of gamma(1)34.5 to preclude premature shutoff of protein synthesis in both neuroblastoma cells and in human foreskin fibroblasts. The results suggest that (i) in the course of its evolution, the virus "borrowed" a gene fragment to preclude a cell response to infection and (ii) the carboxyl terminus of MyD116 and its family of genes known as GADD34 may have a similar function(s) in cells stressed by growth arrest, DNA damage, and differentiation and in herpes simplex virus infection.

Animals↗
PubMed1996

Characterization of the products of the U(L)43 gene of herpes simplex virus 1: potential implications for regulation of gene expression by antisense transcription.

The products, RNA or proteins, of the herpes simplex virus 1 open reading frame U(L)43 have not been previously identified. The expression of an open reading frame antisense to U(L)43, U(L)43.5 (P. L. Ward, D. E. Barker, and B. Roizman, J. Virol. 70:2684-2690, 1996), has been reported. We report the existence of a transcript corresponding to the domain of the U(L)43 open reading frame extending approximately 30 bp from the predicted TATA box to the predicted polyadenylation signal. The RNA of the U(L)43 open reading frame accumulates to higher levels in the presence of phosphonoacetic acid, an inhibitor of viral DNA synthesis, than in its absence, whereas the U(L)43.5 transcript accumulates in larger amounts in the absence of phosphonoacetic acid. The open reading frame tagged with a sequence encoding a 20-amino-acid epitope yielded a protein with an apparent Mr of 32,000, i.e., considerably lower than that predicted from the size of the open reading frame. The discovery of a pair of antisense genes expressed during productive infection raises the possibilities that additional antisense genes exist and that the antisense arrangement provides still another mechanism for regulation of gene expression.

Animals↗
PubMed1996

Phenotypic properties of herpes simplex virus 1 containing a derepressed open reading frame P gene.

Open reading frame P (ORF P) maps in the viral DNA sequences transcribed during latency and is located antisense to the gamma 1 34.5 gene. Earlier studies have shown that the expression of ORF P is repressed by an infected cell protein no. 4 binding site straddling the transcription initiation site. We have made monospecific polyclonal antibodies to the protein and constructed a virus, designated ORF P++, in which the infected cell protein no. 4 binding site has been mutagenized, thereby allowing full expression of an unmodified ORF P gene from its natural promoter. We report the following findings. (i) The native protein forms multiple bands on denaturing polyacrylamide gels suggestive of extensive processing and aggregation of the protein; (ii) the protein accumulates in the nucleus in rod-shaped structures perpendicular to the axis of attachment of the infected cell to the solid matrix; (iii) the virus was highly attenuated on inoculation into mice by the intracerebral or ocular route, and virus was not recovered upon explantation of trigeminal ganglia; (iv) although protein synthesis was not prematurely shut off in the human neuroblastoma cell line SK-N-SH, gamma 1 34.5 protein was not detected in immunoblasts. Analyses of electrophoretically separated denatured RNAs indicated that in cells infected with the ORF P++ virus, there was a large increase in the amount of ORF P RNA and a corresponding decrease in the amount of gamma 1 34.5 RNA. We conclude that either the overproduction of ORF P protein blocks the expression of some herpes simplex virus 1 genes or derepression of the transcription of ORF P has a negative effect on the transcription of the antisense gamma 1 34.5 RNA.

Animals↗