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Bernard Roizman

Publications and source records attributed to Bernard Roizman.

At least 19 recordsLinked to original sources
PubMed2006

Mapping of key functions of the herpes simplex virus 1 U(S)3 protein kinase: the U(S)3 protein can form functional heteromultimeric structures derived from overlapping truncated polypeptides.

Earlier studies have shown that the herpes simplex virus (HSV) U(S)3 encodes two transcriptional units directing the synthesis of the U(S)3 (residues 1 to 481) and U(S)3.5 (residues 77 to 481) protein kinases. Both kinases phosphorylate histone deacetylase 1 (HDAC1) and HDAC2 and enable the expression of genes cotransduced into U2OS cells by recombinant baculoviruses, an activity designated the "helper function." The two kinases differ with respect to antiapoptotic activity. In the studies reported here, we made a series of FLAG-tagged amino- and carboxyl-terminal truncations of U(S)3 and these were tested for antiapoptotic activity, phosphorylation of HDAC1, and the helper function. We report the following. (i) HDAC1 phosphorylation and the helper function were expressed in cells transduced by the truncation encoding residues 182 to 481 but not in cells transduced by the truncation encoding residues 189 to 481 or the amino-terminal polypeptides encompassing the first 188 amino acids. (ii) The self-posttranslational modification requires residues 164 to 481. (iii) The antiapoptotic activity requires both the amino-terminal and the carboxyl-terminal domains, of which the truncated protein containing residues 1 to 163 and that containing residues 164 to 481, respectively, were the smallest fragments tested to be effective. The two domains need not be on the same molecule, but they must overlap. The smallest overlapping pair tested was the fragment containing residues 1 to 181 and that containing residues 164 to 481. Consistent with the hypothesis that the effective overlapping truncations form a heteromultimeric structure, antibody to FLAG coprecipitated untagged U(S)3 from lysates of cells cotransduced with FLAG-tagged, truncated U(S)3 constructs. Although U(S)3 has been reported to be a monomeric enzyme, the results indicate that it can form enzymatically active multimeric structures.

Animals↗
PubMed2006

ReVOLT: radiation-enhanced viral oncolytic therapy.

Viral oncolytic therapy has been pursued with renewed interest as the molecular basis of carcinogenesis and viral replication has been elucidated. Genetically engineered, attenuated viruses have been rationally constructed to achieve a therapeutic index in tumor cells compared with surrounding normal tissue. Many of these attenuated mutant viruses have entered clinical trials. Here we review the preclinical literature demonstrating the interaction of oncolytic viruses with ionizing radiation and provides a basis for future clinical trials.

Adenoviridae↗
PubMed2006

Glycoprotein M of herpes simplex virus 1 is incorporated into virions during budding at the inner nuclear membrane.

It is widely accepted that nucleocapsids of herpesviruses bud through the inner nuclear membrane (INM), but few studies have been undertaken to characterize the composition of these nascent virions. Such knowledge would shed light on the budding reaction at the INM and subsequent steps in the egress pathway. The present study focuses on glycoprotein M (gM), a type III integral membrane protein of herpes simplex virus 1 (HSV-1) that likely contains eight transmembrane domains. The results indicated that gM localized primarily at the perinuclear region, with especially bright staining near the nuclear membrane (NM). Immunogold electron microscopic analysis indicated that, like gB and gD (M. R. Torrisi et al., J. Virol. 66:554-561, 1992), gM localized within both leaflets of the NM, the envelopes of nascent virions that accumulate in the perinuclear space, and the envelopes of cytoplasmic and mature extracellular virus particles. Indirect immunofluorescence studies revealed that gM colocalized almost completely with a marker of the Golgi apparatus and partially with a marker of the trans-Golgi network (TGN), whether or not these markers were displaced to the perinuclear region during infection. gM was also located in punctate extensions and invaginations of the NM induced by the absence of a viral kinase encoded by HSV-1 U(S)3 and within virions located in these extensions. Our findings therefore support the proposition that gM, like gB and gD, becomes incorporated into the virion envelope upon budding through the INM. The localization of viral glycoproteins and Golgi and TGN markers to a perinuclear region may represent a mechanism to facilitate the production of infectious nascent virions, thereby increasing the amount of infectivity released upon cellular lysis.

Cell Line↗
PubMed2006

ICP0 and the US3 protein kinase of herpes simplex virus 1 independently block histone deacetylation to enable gene expression.

SK-N-SH cells exposed to low ratios of ICP0-null (DeltaICP0) mutants of herpes simplex virus per cell express the viral alpha proteins, but the progression to beta and gamma gene expression does not ensue. In these restrictive cells, post-alpha gene expression can be induced after exposure of the infected cells to sodium butyrate, an indication that VP16 brought into cells by the virus and the alpha gene products made after infection cannot block the silencing of viral post-alpha genes by histone deacetylases (HDACs). This observation is consistent with evidence reported earlier that ICP0 dissociates HDAC1/2 from the CoREST/REST complex. In permissive U2OS cells, replication is independent of the ratio of DeltaICP0 mutant per cell. To determine whether other viral genes are involved in blocking HDACs, we used a surrogate system consisting of baculoviruses carrying viral or cellular genes driven by CMV immediate-early promoter. Expression of these genes requires blocking of histone deacetylation. We report that (i) cotransduced U(S)3 or U(S)3.5 protein kinase substitutes for sodium butyrate in enabling the expression of a reporter gene in restrictive cells and enhancing it in permissive cells; (ii) HDAC1 is phosphorylated concomitant with the expression of reporter genes; and (iii) the amounts and appearance of HDAC1 are altered in transduced cells expressing U(S)3 protein kinase in the absence of other viral proteins. We conclude that the U(S)3 protein kinase blocks histone deacetylation by a mechanism distinct from that of ICP0 and that debilitated histone deacetylation contributes to the permissiveness of U2OS cells for DeltaICP0 mutants.

Acetylation↗
PubMed2006

Construction and properties of a herpes simplex virus 1 designed to enter cells solely via the IL-13alpha2 receptor.

Current design of genetically engineered viruses for selective destruction of cancer cells is based on the observation that attenuated viruses replicate better in tumor cells than in normal cells. The ideal virus, however, is one that can infect only cancer cells by virtue of altered host range. Such a virus can be made more robust than the highly attenuated viruses used in clinical trials. Earlier, we reported the construction of a recombinant herpes simplex virus 1 (R5111) in which the capacity to bind heparan sulfate was disabled and which contained a chimeric IL-13-glycoprotein D that enabled the virus to infect cells expressing the IL-13alpha2 receptor (IL-13Ralpha2) commonly found on the surface of malignant glioblastomas or high-grade astrocytomas. In the earlier report, we showed that the recombinant R5111 was able to enter and infect cells via the interaction of the chimeric glycoprotein D with IL-13Ralpha2 but that the virus retained the capacity to bind and replicate in cells expressing the natural viral receptors HveA or nectin-1. Here, we report the construction of a recombinant virus (R5141) that can only enter and replicate in cells that express the IL-13Ralpha2. The recombinant R5141 does not depend on endocytosis to infect cells. It does not infect cells expressing HveA or nectin-1 receptors or cells expressing IL-13Ralpha2 that had been exposed to soluble IL-13 before infection. The studies described here show that the host range of herpes simplex viruses can be altered by genetic manipulation to specifically target cancer cells.

Animals↗
PubMed2006

The U(L)41 protein of herpes simplex virus 1 degrades RNA by endonucleolytic cleavage in absence of other cellular or viral proteins.

The herpes simplex virus 1 ORF U(L)41 encodes a protein (virion host shutoff or vhs) associated with selective degradation of mRNA early in infection. Some mRNAs, exemplified by GAPDH or beta-actin mRNAs, are degraded rapidly. Others, for example IEX-1 mRNA, are degraded in two stages: whereas the 3' domain disappears rapidly, a large 5' domain fragment of the mRNA lingers for several hours. Still a third, exemplified by tristetraprolin mRNA, is not degraded, allowing its protein product to accumulate in infected cells. Here we report the following: (i) a GST-vhs protein produced in Escherichia coli, solubilized and purified to homogeneity acts as bona fide endoribonuclease when tested on in vitro transcribed IEX-1 probes. A GST-vhs protein in which three key vhs amino acids were replaced with alanines, solubilized and purified by the same protocol, had no enzymatic activity. (ii) The number of fragments generated by cleavage of a truncated IEX-1 RNA by vhs appears to be small; the cleavage sites are centered at or near the AU-rich elements located at the 3' untranslated region of the mRNA. A truncated RNA containing only the IEX-1 coding domain was cleaved numerous times. (iii) In cells infected at high multiplicity and exposed to a large number of particles per cell, the vhs protein accumulated within 3 h after infection, in small uniform cytoplasmic granules raising the possibility that vhs colocalizes with tristerapolin, a protein induced after infection, in structures involved in degradation of RNA.

3' Untranslated Regions↗
PubMed2006

Herpes simplex virus 1 precludes replenishment of the short-lived receptor of tumor necrosis factor alpha by virion host shutoff-dependent degradation of its mRNA.

Cell function is tightly regulated by surface receptors. Earlier reports showed that herpes simplex virus 1 regulates by diverse mechanisms the presentation of antigenic peptides, downregulates the signaling pathways associated with receptor tyrosine kinases, and posttranslationally modifies members of the Src family of protein kinases. Here we report that the receptor for tumor necrosis factor alpha (TNF-R1) rapidly disappears from both the cell surface and total cell lysates in cells infected with wild-type virus or a variety of mutants but not in cells infected with the mutant DeltaU(L)41, which lacks the U(L)41 gene, the virion host shutoff gene. The half-life of TNF-R1 appears to be less than 30 min in both mock-infected and infected cells. The disappearance of TNF-R1 correlates with the disappearance of cytoplasmic TNF-R1 mRNA in wild-type-virus-infected cells. The results suggest that by degrading the TNFR1 mRNA, the virus precludes the replenishment of naturally decaying TNF-R1.

Cytoplasm↗
PubMed2006

The virion host shutoff protein (UL41) of herpes simplex virus 1 is an endoribonuclease with a substrate specificity similar to that of RNase A.

Earlier, our laboratory reported that purified glutathione S-transferase-virion host shutoff (GST-vhs) protein exhibited endoribonucleolytic activity in in vitro assays using as substrates in vitro-transcribed regions of IEX-1 mRNA. Here, we report that studies of the cleavage patterns of synthetic RNA oligonucleotides defined the activity of GST-vhs as being similar to that of RNase A. Thus, GST-vhs cleaved the RNA at the 3' end of single-stranded cytidine and uridine residues. Since the GST-mvhs nuclease-defective mutant protein failed to cleave the synthetic RNAs, the results unambiguously attribute the activity to vhs.

Base Sequence↗
PubMed2006

Activated MEK suppresses activation of PKR and enables efficient replication and in vivo oncolysis by Deltagamma(1)34.5 mutants of herpes simplex virus 1.

Herpes simplex virus mutants lacking the gamma(1)34.5 gene are not destructive to normal tissues but are potent cytolytic agents in human tumor cells in which the activation of double-stranded RNA-dependent protein kinase (PKR) is suppressed. Thus, replication of a Deltagamma(1)34.5 mutant (R3616) in 12 genetically defined cancer cell lines correlates with suppression of PKR but not with the genotype of RAS. Extensive analyses of two cell lines transduced with either dominant negative MEK (dnMEK) or constitutively active MEK (caMEK) indicated that in R3616 mutant-infected cells dnMEK enabled PKR activation and decreased virus yields, whereas caMEK suppressed PKR and enabled better viral replication and cell destruction in transduced cells in vitro or in mouse xenografts. The results indicate that activated MEK mediates the suppression of PKR and that the status of MEK predicts the ability of Deltagamma(1)34.5 mutant viruses to replicate in and destroy tumor cells.

Animals↗
PubMed2006

Protein kinase B/Akt is present in activated form throughout the entire replicative cycle of deltaU(S)3 mutant virus but only at early times after infection with wild-type herpes simplex virus 1.

The product of the herpes simplex virus 1 (HSV-1) US3 gene is a multifunctional serine-threonine protein kinase that can block apoptosis induced by proapoptotic cellular proteins, exogenous agents, or replication-defective viruses. Earlier studies showed that the U(S)3 kinase activates and functionally overlaps cellular protein kinase A (PKA). In this study we examined the status of phosphatidylinositol 3-kinase [PI3K] and of its effector, protein kinase B/Akt (PKB/Akt), a component of a major pathway of mammalian antiapoptotic signaling systems. We report the following. (i) Infection of target cells with HSV-1 induces transient phosphorylation of serine 473 of PKB/Akt early in infection, with a mechanism that is dependent on PI3K. Inhibition of PI3K induced apoptosis in mock-infected or deltaU(S)3 mutant-virus-infected but not in wild-type-virus-infected cells and reduced the accumulation of specific viral gene products, including the U(S)3 protein kinase, but had a marginal effect on virus yields. (ii) At later times after infection, the total amounts of PKB/Akt decreased and phosphorylated PKB/Akt forms disappeared in a U(S)3-dependent and protein phosphatase 2A-independent manner. (iii) Activation of PKA by forskolin did not mediate significant dephosphorylation of PKB/Akt. Our results are consistent with the model that PKB/Akt is activated early in infection and acts to block apoptosis in infected cells prior to the accumulation of U(S)3 protein kinase and that it persists and continues to function as an antiapoptotic protein in the absence of U(S)3 but becomes redundant or even inimical once U(S)3 protein kinase accumulates in effective amounts.

Apoptosis↗
PubMed2006

State and role of SRC family kinases in replication of herpes simplex virus 1.

An earlier report showed that infected cell protein no. 0 (ICP0) of herpes simplex virus 1 (HSV-1) interacts with the SH3 domains of a recently discovered adaptor protein, CIN85. Here, we report the following. (i) ICP0 also interacts with other SH3 domain-containing proteins and, in particular, with nonneuronal members of the Src kinase family. (ii) HSV-1 infection enhanced the activating phosphorylation of Tyr416 of the members of the Src kinase family, modestly enhanced the kinase activity of Src, and posttranslationally modified at least one additional member of the Src kinase family by phosphorylation in a manner dependent on the viral gene products ICP0, unique short 3 (U(S)3), and unique long 13 (U(L)13). (iii) To define the roles of Src kinase family members, we examined the accumulation of viral proteins, DNA, and mRNA and virus yields from wild-type mouse embryo fibroblasts and sibling cells lacking Src, Fyn, and Yes (SYF-); a mutant cell line, +Src, in which Src was restored to SYF- cells; and the mutant cell line (CSK-) lacking the negative regulator Csk gene of the Src kinase family. Representative alpha, beta, and gamma2 proteins accumulated in the largest amounts in SYF- cells and the smallest amounts in +Src compared to wild-type cells. The CSK- cells yielded smaller amounts of the gamma2 protein and at least 10-fold less virus than wild-type cells. We conclude that HSV-1 proteins regulate the activities of Src family kinases to achieve optimal viral yields in the course of viral replication.

Animals↗
PubMed2006

U(S)3 and U(S)3.5 protein kinases of herpes simplex virus 1 differ with respect to their functions in blocking apoptosis and in virion maturation and egress.

Previously, we reported that the U(S)3 protein kinase blocks apoptosis, that it activates protein kinase A (PKA), that activation of PKA blocks apoptosis in cells infected with a U(S)3 deletion mutant, and that an overlapping transcriptional unit encodes a truncated kinase designated U(S)3.5. Here, we report the properties of the kinases based on comparisons of herpes simplex virus and baculoviruses expressing U(S)3 or U(S)3.5 kinase. Specifically, we report the following. (i) Both kinases mediate the phosphorylation of HDAC1, HDAC2, and the PKA regulatory IIalpha subunit in the absence of other viral proteins. (ii) Both enzymes mediate the phosphorylation of largely identical sets of proteins carrying the phosphorylation consensus site of PKA, but only U(S)3 blocks apoptosis, suggesting that it is U(S)3 and not PKA that is responsible for the phosphorylation of the proteins bearing the shared consensus phosphorylation site and the antiapoptotic activity. (iii) Both kinases cofractionate with mitochondria. Immune depletion of the U(S)3 and U(S)3.5 kinases from the cytoplasm removed the kinases from the supernatant fraction, but not from the mitochondrial fraction, and therefore, if the antiapoptotic activity of the U(S)3 kinase is expressed in mitochondria, the localization signal and the antiapoptotic functions are located on different parts of the protein. (iv) The U(S)3 protein kinase is required for the translocation of virus particles from the nucleus. Although the U(L)31 protein is phosphorylated in cells infected with the mutant expressing U(S)3.5 kinase, the release of virus particles from nuclei was impeded in some cells, suggesting that the U(S)3 kinase affects the modification of the nuclear membrane more efficiently than the U(S)3.5 kinase.

Animals↗
PubMed2005

Ionizing radiation activates late herpes simplex virus 1 promoters via the p38 pathway in tumors treated with oncolytic viruses.

Ionizing radiation potentiates the oncolytic activity of attenuated herpes simplex viruses in tumors exposed to irradiation at specific time intervals by inducing higher virus yields. Cell culture studies have shown that an attenuated virus lacking the viral gamma(1)34.5 genes underproduces late proteins whose synthesis depends on sustained synthesis of viral DNA. Here we report that ionizing radiation enhances gene expression from late viral promoters in transduced cells in the absence of other viral gene products. Consistent with this result, we show that in tumors infected with the attenuated virus, ionizing radiation increases 13.6-fold above baseline the gene expression from a late viral promoter as early as 2 hours after virus infection, an interval too short to account for viral DNA synthesis. The radiation-dependent up-regulation of late viral genes is mediated by the p38 pathway, inasmuch as the enhancement is abolished by p38 inhibitors or a p38 dominant-negative construct. The p38 pathway is not essential for wild-type virus gene expression. The results suggest that ionizing radiation up-regulates late promoters active in the course of viral DNA synthesis and provide a rationale for use of radiation to up-regulate cytotoxic genes introduced into tumor cells by viral vectors for cytoreductive therapy.

Animals↗
PubMed2005

The first 30 minutes in the life of a virus: unREST in the nucleus.

On entry of viral DNA into cells, a competition ensues between cellular factors that silence viral genes and transcriptional factors that block silencing and transcribe the DNA. In the case of herpes simplex virus 1, the first set of genes expressed after infection-are induced by a viral protein (alphaTIF or VP16). Expression of later (beta,gamma) genes in cells infected at low ratios of virus per cells requires a transcriptional activator (ICP4) that cannot block silencing and a protein, ICP0, hitherto characterized as a promiscuous transactivator. Recent studies indicate that ICP0 blocks silencing of viral DNA by dissociating HDACs 1 and 2 from the CoREST/REST repressor complex. HDACs 1/2 are phosphorylated and translocated to the nucleus. The findings have broad implications regarding silencing of the viral genome during latency and, potentially, the expression of genes encoded by other DNA viruses as well.

Cell Nucleus↗
PubMed2005

Components of the REST/CoREST/histone deacetylase repressor complex are disrupted, modified, and translocated in HSV-1-infected cells.

The infected cell protein (ICP)0 enables gene expression and the replication of herpes simplex virus (HSV)-1 in cells infected at low multiplicities and enhances the expression of genes introduced into cells by transfection or infection. We report that a short sequence of ICP0 is similar to a sequence in the amino terminus of CoREST, a corepressor that exists in complexes with the repressor REST and histone deacetylases (HDACs) 1 or 2 to repress cellular gene expression. In wild-type-virus-infected cells, HDAC1 dissociates from the CoREST/REST complex, CoREST and HDAC1 are phosphorylated by a process mediated by viral protein kinases, and CoREST and HDAC1 are partially translocated to the cytoplasm. In cells infected with a virus mutant (DeltaICP4), in which ICP0 accumulates, but post-alpha gene expression is blocked, HDAC1 is dissociated from the CoREST/REST complex, but translocation to the cytoplasm does not occur. After infection with a mutant virus from which ICP0 is deleted, the complex remains intact, but, under conditions of productive infection, the complex is partially translocated to the cytoplasm. These results suggest that, at low multiplicities of infection, ICP0 blocks CoREST-mediated silencing of viral genes by dissociation of HDAC1, whereas subsequent modifications and translocation of the components of the complex are the functions of other viral gene products made later in infection.

Cell Fractionation↗
PubMed2005

Herpes simplex virus 1 infected cell protein 0 forms a complex with CIN85 and Cbl and mediates the degradation of EGF receptor from cell surfaces.

Infected cell protein 0 (ICP0) is a 775-residue multifunctional herpes simplex virus protein associated with numerous functions related to transactivation of gene expression and repression of host defenses to infection. We report that an uncharted domain of ICP0 located between residues 245 and 510 contains multiple SH3 domain binding motifs similar to those required for binding to CIN85, the M(r) 85,000 protein that interacts with Cbl. CIN85 and Cbl are involved in endocytosis and negative regulation of numerous receptor tyrosine kinases. We report that ICP0 binds CIN85 in a reciprocal manner and that the complexes pulled down by ICP0 also contain Cbl. We tested the role of ICP0 in the down-regulation of receptor tyrosine kinases by using epidermal growth factor receptor (EGFR) as a prototypic receptor. In transfection assays, ICP0, in the absence of other viral genes, down-regulated EGF-dependent expression of a reporter gene (luciferase). ICP0 also down-regulated both total and cell surface levels of EGFR in EGF-independent manner. In wild-type virus-infected cells, the surface levels of EGFR were also decreased in the absence of EGF stimulation. Stimulation by EGF enhanced the decrease in surface EGFR. We conclude that ICP0 encodes SH3 domain binding sites that function to down-regulate signaling pathways associated with receptor tyrosine kinases. The results suggest that ICP0 precludes signaling to the infected cells through the receptor tyrosine kinases.

Adaptor Proteins, Signal Transducing↗
PubMed2005

Modulation of vascular remodeling induced by a brief intraluminal exposure to the recombinant R7020 strain of Herpes simplex-1.

OBJECTIVE: Vascular remodeling in response to injury or low shear stress (or both) is characterized by neointimal hyperplasia and luminal contraction. When profound, the response leads to restenosis after percutaneous endovascular intervention as well as to de novo stenosis in vein grafts. It has recently been reported that exposure of vein patches to neurovirulence-attenuated Herpes simplex virus-1 (HSV-1) decreases neointimal hyperplasia and increases luminal area. This experiment tested the hypothesis that R7020, a more highly attenuated mutant of HSV-1, would modulate the vascular remodeling response of experimental vein grafts chronically exposed to low shear stress. METHODS: The external jugular veins of 31 New Zealand white rabbits were clamped and intraluminally exposed to vehicle (phospate-buffered saline solution, n = 11), R7020 2.5 x 10(8) plaque forming units [PFU]/mL (n = 8), or R7020 2.5 x 10(9) PFU/mL (n = 12) for 10 or 30 minutes at an average pressure of 80 mm Hg. After exposure, an end-to-side distal external jugular-to-common carotid artery anastomosis was created, resulting in a widely patent arteriovenous fistula. The external jugular was suture-ligated just proximal to the thoracic inlet, distal to a small 10- to 50-microm venous tributary, creating a reversed vein "graft" segment immediately and abruptly exposed to arterial pressure (48 +/- 3 mm Hg) and low shear stress (0.12 +/- .02 dyne/cm(2)). In the 29 animals (N = 31) that survived to harvest, 26 grafts were found to be patent and were analyzed further. Nine grafts were harvested within the first week after operation, snap frozen in liquid nitrogen, and assayed for the presence of the Herpes viral immediate-response protein ICP0 by Western blot analysis. The 17 remaining grafts were perfusion-fixed, excised, stained, and analyzed morphometrically by digital planimetry. RESULTS: In patent grafts, the hemodynamic environment of low shear stress was maintained (shear stress at harvest, 0.26 +/- .06 dyne/cm(2)). Western blot analysis revealed the presence of ICP0 in R7020-exposed vein grafts after 2, 3, 7, and 14 days; ICP0 was not detected in unexposed vein grafts or adjacent carotid arteries. After 4 weeks, vein grafts exposed to R7020 exhibited a statistically significantly increased ratio of luminal radius to wall thickness, indicating altered remodeling (vehicle, 6.7 +/- 1.3; R7020 2.5 x 10(8), 9.1 +/- 1.3; R7020 2.5 x 10(9) ratio, 11.3 +/- 1.4; P < .05 for high dose compared with vehicle). CONCLUSION: A brief exposure of the neurovirulence-attenuated HSV-1 strain R7020 results in an increased ratio of luminal radius to wall thickness in experimental vein grafts chronically exposed to low shear stress.

Animals↗