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J A Blaho

Publications and source records attributed to J A Blaho.

At least 19 recordsLinked to original sources
PubMed2003

Viral oncoapoptosis of human tumor cells.

Many cancer cells refractory to radiation treatment and chemotherapy proliferate because of loss of intrinsic programmed cell death (apoptosis) regulation. Consequently, the resolution of these cancers are many times outside the management capabilities of conventional therapeutics. We now report that replication-defective delta27 herpes simplex virus (rd delta27) triggers apoptosis in three representative transformed human cell lines. Susceptibility to virus-induced cell death is dependent on the abundance and distribution of modified p53 protein in the tumor cells indicating specific targeting of the treatment. Primary human and mouse fibroblast cells that produce modified p53 are resistant to rd delta27 killing but not to apoptosis induced by nonviral environmental factors. These results suggest that induction of apoptosis by nonreplicating virus is a feasible genetic therapy approach for killing human cancer cells. Our findings may have important implications in designing novel virus-based anticancer strategies in appropriate animal model systems.

Animals↗
PubMed2001

Assessment of the subcellular localization of the herpes simplex virus structural protein VP22 in the absence of other viral gene products.

We previously demonstrated that the herpes simplex virus type 1 (HSV-1) structural protein VP22 exists in the cytoplasm early in infection and migrates to and accumulates in the nucleus late in infection (J. Virol. 73(8) (1999) 6769). The goal of this study is to document the behavior of VP22 in cells in the absence of other viral polypeptides. We characterized the effects of various indirect immunofluorescence sample preparation conditions on the localization of VP22 in cells and have determined the following. (i) Fixing with formaldehyde and permeabilizing with acetone maintains the structure of microtubules in cells, in as much as we observed classic microtubule organizing centers. (ii) Acetone or methanol alone did not completely fix the cells. (iii) Triton X-100 decreased tubulin immunofluorescence signals in our system. (iv) VP22 predominated in the nucleus of cells that were fixed with formaldehyde. Based on our results, we conclude the following. (v) Due to the partial fixation by acetone or methanol alone, microtubules form diffuse irregular shapes. (vi) VP22 is detected in the cytoplasm of cells fixed with acetone or methanol only due to its seepage from the nucleus. Taken together, these findings indicate that (vii) the nuclear localization of VP22 does not require additional viral factors.

Animals↗
PubMed2001

Modulation of apoptosis during herpes simplex virus infection in human cells.

Human herpes simplex virus (HSV) is cytolytic and has profound impacts on its host cells. Consequences of HSV infection include the induction of apoptosis and the concomitant synthesis of proteins which act to block this process. We review recent evidence showing how this important human pathogen modulates the fundamental cell death process.

Apoptosis↗
PubMed2001

Microtubule reorganization during herpes simplex virus type 1 infection facilitates the nuclear localization of VP22, a major virion tegument protein.

Full-length VP22 is necessary for efficient spread of herpes simplex virus type 1 (HSV-1) from cell to cell during the course of productive infection. VP22 is a virion phosphoprotein, and its nuclear localization initiates between 5 and 7 h postinfection (hpi) during the course of synchronized infection. The goal of this study was to determine which features of HSV-1 infection function to regulate the translocation of VP22 into the nucleus. We report the following. (i) HSV-1(F)-induced microtubule rearrangement occurred in infected Vero cells by 13 hpi and was characterized by the loss of obvious microtubule organizing centers (MtOCs). Reformed MtOCs were detected at 25 hpi. (ii) VP22 was observed in the cytoplasm of cells prior to microtubule rearrangement and localized in the nucleus following the process. (iii) Stabilization of microtubules by the addition of taxol increased the accumulation of VP22 in the cytoplasm either during infection or in cells expressing VP22 in the absence of other viral proteins. (iv) While VP22 localized to the nuclei of cells treated with the microtubule depolymerizing agent nocodazole, either taxol or nocodazole treatment prevented optimal HSV-1(F) replication in Vero cells. (v) VP22 migration to the nucleus occurred in the presence of phosphonoacetic acid, indicating that viral DNA and true late protein synthesis were not required for its translocation. Based on these results, we conclude that (iv) microtubule reorganization during HSV-1 infection facilitates the nuclear localization of VP22.

Animals↗
PubMed2001

Accumulation of herpes simplex virus type 1 early and leaky-late proteins correlates with apoptosis prevention in infected human HEp-2 cells.

We previously reported that a recombinant ICP27-null virus stimulated, but did not prevent, apoptosis in human HEp-2 cells during infection (M. Aubert and J. A. Blaho, J. Virol. 73:2803-2813, 1999). In the present study, we used a panel of 15 recombinant ICP27 mutant viruses to determine which features of herpes simplex virus type 1 (HSV-1) replication are required for the apoptosis-inhibitory activity. Each virus was defined experimentally as either apoptotic, partially apoptotic, or nonapoptotic based on infected HEp-2 cell morphologies, percentages of infected cells with condensed chromatin, and patterns of specific cellular death factor processing. Viruses d27-1, d1-5, d1-2, M11, M15, M16, n504R, n406R, n263R, and n59R are apoptotic or partially apoptotic in HEp-2 cells and severely defective for growth in Vero cells. Viruses d2-3, d3-4, d4-5, d5-6, and d6-7 are nonapoptotic, demonstrating that ICP27 contains a large amino-terminal region, including its RGG box RNA binding domain, which is not essential for apoptosis prevention. Accumulations of viral TK, VP16, and gD but not gC, ICP22, or ICP4 proteins correlated with prevention of apoptosis during the replication of these viruses. Of the nonapoptotic viruses, d4-5 did not produce gC, indicating that accumulation of true late gene products is not necessary for the prevention process. Analyses of viral DNA synthesis in HEp-2 cells indicated that apoptosis prevention by HSV-1 requires that the infection proceeds to the stage in which viral DNA replication takes place. Infections performed in the presence of the drug phosphonoacetic acid confirmed that the process of viral DNA synthesis and the accumulation of true late (gamma(2)) proteins are not required for apoptosis prevention. Based on our results, we conclude that the accumulation of HSV-1 early (beta) and leaky-late (gamma(1)) proteins correlates with the prevention of apoptosis in infected HEp-2 cells.

Animals↗
PubMed2000

Assembly of infectious Herpes simplex virus type 1 virions in the absence of full-length VP22.

VP22, the 301-amino-acid phosphoprotein product of the herpes simplex virus type 1 (HSV-1) U(L)49 gene, is incorporated into the tegument during virus assembly. We previously showed that highly modified forms of VP22 are restricted to infected cell nuclei (L. E. Pomeranz and J. A. Blaho, J. Virol. 73:6769-6781, 1999). VP22 packaged into infectious virions appears undermodified, and nuclear- and virion-associated forms are easily differentiated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (J. A. Blaho, C. Mitchell, and B. Roizman, J. Biol. Chem. 269:17401-17410, 1994). As VP22 packaging-associated undermodification is unique among HSV-1 tegument proteins, we sought to determine the role of VP22 during viral replication. We now show the following. (i) VP22 modification occurs in the absence of other viral factors in cell lines which stably express its gene. (ii) RF177, a recombinant HSV-1 strain generated for this study, synthesizes only the amino-terminal 212 amino acids of VP22 (Delta212). (iii) Delta212 localizes to the nucleus and incorporates into virions during RF177 infection of Vero cells. Thus, the carboxy-terminal region is not required for nuclear localization of VP22. (iv) RF177 synthesizes the tegument proteins VP13/14, VP16, and VHS (virus host shutoff) and incorporates them into infectious virions as efficiently as wild-type virus. However, (v) the loss of VP22 in RF177 virus particles is compensated for by a redistribution of minor virion components. (vi) Mature RF177 virions are identical to wild-type particles based on electron microscopic analyses. (vii) Single-step growth kinetics of RF177 in Vero cells are essentially identical to those of wild-type virus. (viii) RF177 plaque size is reduced by nearly 40% compared to wild-type virus. Based on these results, we conclude that VP22 is not required for tegument formation, virion assembly/maturation, or productive HSV-1 replication, while the presence of full-length VP22 in the tegument is needed for efficient virus spread in Vero cell monolayers.

Animals↗
PubMed1999

Induction and prevention of apoptosis in human HEp-2 cells by herpes simplex virus type 1.

Cultured human epithelial cells infected with an ICP27 deletion strain of herpes simplex virus type 1 (HSV-1) show characteristic features of apoptotic cells including cell shrinkage, nuclear condensation, and DNA fragmentation. These cells do not show such apoptotic features when infected with a wild-type virus unless the infections are performed in the presence of a protein synthesis inhibitor. Thus, both types of virus induce apoptosis, but the ICP27-null virus is unable to prevent this process from killing the cells. In this report, we show that this ICP27-deficient virus induced apoptosis in human HEp-2 cells through a pathway which involved the activation of caspase-3 and the processing of the death substrates DNA fragmentation factor and poly(ADP-ribose) polymerase. The induction of apoptosis by wild-type HSV-1 occurred prior to 6 h postinfection (hpi), and de novo viral protein synthesis was not required to induce the process. The ability of the virus to inhibit apoptosis was shown to be effective between 3 to 6 hpi. Wild-type HSV-1 infection was also able to block the apoptosis induced in cells by the addition of cycloheximide, staurosporine, and sorbitol. While U(S)3- and ICP22-deficient viruses showed a partial prevention of apoptosis, deletion of either the U(L)13 or vhs gene products did not affect the ability of HSV-1 to prevent apoptosis in infected cells. Finally, we demonstrate that in UV-inactivated viruses, viral binding and entry were not sufficient to induce apoptosis. Taken together, these results suggest that either gene expression or another RNA metabolic event likely plays a role in the induction of apoptosis in HSV-1-infected human cells.

Animals↗
PubMed1999

The herpes simplex virus type 1 regulatory protein ICP27 is required for the prevention of apoptosis in infected human cells.

The herpes simplex virus type 1 (HSV-1) ICP27 protein is an immediate-early or alpha protein which is essential for the optimal expression of late genes as well as the synthesis of viral DNA in cultures of Vero cells. Our specific goal was to characterize the replication of a virus incapable of synthesizing ICP27 in cultured human cells. We found that infection with an HSV-1 ICP27 deletion virus of at least three separate strains of human cells did not produce immediate-early or late proteins at the levels observed following wild-type virus infections. Cell morphology, chromatin condensation, and genomic DNA fragmentation measurements demonstrated that the human cells died by apoptosis after infection with the ICP27 deletion virus. These features of the apoptosis were identical to those which occur during wild-type infections of human cells when total protein synthesis has been inhibited. Vero cells infected with the ICP27 deletion virus did not exhibit any of the features of apoptosis. Based on these results, we conclude that while HSV-1 infection likely induced apoptosis in all cells, viral evasion of the response differed among the cells tested in this study.

Animals↗
PubMed1999

Modified VP22 localizes to the cell nucleus during synchronized herpes simplex virus type 1 infection.

The UL49 gene product (VP22) of herpes simplex virus types 1 and 2 (HSV-1 and HSV-2) is a virion phosphoprotein which accumulates inside infected cells at late stages of infection. We previously (J. A. Blaho, C. Mitchell, and B. Roizman, J. Biol. Chem. 269:17401-17410, 1994) discovered that the form of VP22 packaged into infectious virions differed from VP22 extracted from infected-cell nuclei in that the virion-associated form had a higher electrophoretic mobility in denaturing gels. Based on these results, we proposed that VP22 in virions was "undermodified" in some way. The goal of this study is to document the biological and biochemical properties of VP22 throughout the entire course of a productive HSV-1 infection. We now report the following. (i) VP22 found in infected cells is distributed in at least three distinct subcellular localizations, which we define as cytoplasmic, diffuse, and nuclear, as measured by indirect immunofluorescence. (ii) Using a synchronized infection system, we determined that VP22 exists predominantly in the cytoplasm early in infection and accumulates in the nucleus late in infection. (iii) While cytoplasmic VP22 colocalizes with the HSV-1 glycoprotein D early in infection, the nuclear form of VP22 is not restricted to replication compartments which accumulate ICP4. (iv) VP22 migrates as at least three unique electrophoretic species in denaturing sodium dodecyl sulfate-DATD-polyacrylamide gels. VP22a, VP22b, and VP22c have high, intermediate, and low mobility, respectively. (v) The relative distribution of the various forms of VP22 derived from infected whole-cell extracts varies during the course of infection such that low-mobility species predominate at early times and high-mobility forms accumulate later. (vi) The highest-mobility forms of VP22 partition with the cytoplasmic fraction of infected cells, while the lowest-mobility forms are associated with the nuclear fraction. (vii) Finally, full-length VP22 which partitions in the nucleus incorporates radiolabel from [32P]orthophosphate whereas cytoplasmic VP22 does not. Based on these results, we conclude that modification of VP22 coincides with its appearance in the nucleus during the course of productive HSV-1 infection.

Animals↗
PubMed1998

Cosolvents facilitate DNA synthesis in the herpes simplex virus 1 unique short (Us) inverted repeat.

DNA synthesis under standard conditions is not successful within a portion of the Us1 gene of HSV-1 which is juxtaposed to an 86% G + C-containing tract in the Us inverted repeat sequence. We report that the independent addition of specific amounts of at least three different types of cosolvents is capable of facilitating DNA synthesis within this G + C-rich region. In addition, this strategy was used to successfully place a specific site-directed mutation in the Us1 gene. Consideration of these observations should enable future site-specific mutational analyses of portions of the HSV-1 genome which have traditionally been unamenable to genetic manipulations.

DNA Restriction Enzymes↗
PubMed1997

The nucleotidylylation of herpes simplex virus 1 regulatory protein alpha22 by human casein kinase II.

The products of the alpha genes of herpes simplex virus 1, the infected cells proteins (ICP) 0, 4, 22, and 27 perform regulatory functions, are nucleotidylylated, and share the signaling or recognition sequence (RR(A/T)(P/S)R) that correctly predicted the nucleotidylylation of viral proteins encoded by UL21, UL31, UL49, and UL47 genes expressed later in infection. Extracts from uninfected HeLa cells or casein kinase II purified from sea star nucleotidylylated the ICP22 moiety of a glutathione S-transferase-ICP22 (GST22P) fusion protein with [alpha-32P]ATP or [2-3H]ATP. We report that: (i) Purified HeLa cell casein kinase II specifically labeled a glutathione S-transferase fusion protein containing the amino-terminal 151 amino acids of ICP22 with [2-3H]ATP. (ii) Nucleotidylylation of GST-ICP22 by purified enzyme exhibited positive cooperativity (Hill coefficient of 2 and a K' of 3.7 microM) and a Km = 37.7 microM for ATP. (iii) Nucleotidylylation was inhibited by heparin, casein, or ATPalphaS but not by ATPgammaS. (iv) Mutation of the signaling sequence from RRAPRR to LKAPEK abolished nucleotidylylation. We conclude that nucleotidylylation of proteins by casein kinase II requires the presence of the signaling or recognition sequence, involves the cleavage of the phosphodiester bond between the alpha and beta phosphate, and need not be preceded by phosphorylation.

Adenosine Triphosphate↗
PubMed1997

Tyrosine phosphorylation of the herpes simplex virus type 1 regulatory protein ICP22 and a cellular protein which shares antigenic determinants with ICP22.

At least eight herpes simplex virus type 1 (HSV-1) and five HSV-2 proteins were tyrosine phosphorylated in infected cells. The first viral tyrosine phosphoprotein identified was the HSV-1 regulatory protein ICP22. Also, two novel phosphotyrosine proteins were bound by anti-ICP22 antibodies. H(R22) is a cellular protein, while the F(R10) protein is observed only in HSV-1-infected cells.

3T3 Cells↗
PubMed1994

Casein kinase II specifically nucleotidylylates in vitro the amino acid sequence of the protein encoded by the alpha 22 gene of herpes simplex virus 1.

An earlier report has shown that eight viral proteins with a common amino acid sequence (R/P)RA(P/S)R are nucleotidylyated in vitro by nuclear extracts from cells infected with herpes simplex virus 1. One, the product of the alpha 22 gene, is nucleotidylylated in the absence of viral proteins made late in infection. A chimeric protein (GST22P) consisting of amino acids 50-200 of the alpha 22 coding sequence fused to the C terminus of the glutathione S-transferase was nucleotidylylated by enzymes in nuclear extracts of infected or mock-infected cells and also by a casein kinase II enzyme purified from the sea star. The enzyme did not nucleotidylylate common casein kinase II substrates (casein, phosvitin) and the reaction was inhibited by heparin. The results are consistent with the hypothesis that nucleotidylylation of the eight viral proteins involves casein kinase II.

Adenosine Triphosphate↗
PubMed1994

An amino acid sequence shared by the herpes simplex virus 1 alpha regulatory proteins 0, 4, 22, and 27 predicts the nucleotidylylation of the UL21, UL31, UL47, and UL49 gene products.

Earlier reports showed that at least 3 herpes simplex virus 1 proteins were adenylylated and guanylylated in addition to the infected cell proteins (ICPs) 0, 4, 22, and 27. ICP22 and 27 share the amino acid sequence R/PRAP/SR which is also predicted to be in ICPs 4 and 0 and in the products of the HSV-1 genes UL21, UL31, UL47, and UL49. ICPs 0, 4, 22, and 27 are regulatory proteins, UL21 is dispensable for growth in cultured cells, UL31 co-fractionates with the nuclear matrix, and UL47 may interact stoichiometrically with the alpha trans-inducing factor (VP16, the product of the UL48 gene). To avoid bias, the genes encoding the additional nucleotidylylated proteins were mapped, initially by analyses of intertypic recombinant viruses, and subsequently by analyses of the products encoded by the mapped genome domains. We conclude that (i) the products of UL21, UL31, UL47, and UL49 are nucleotidylylated by [alpha-32P]GTP or [alpha-32P]ATP in isolated nuclei, (ii) this modification does not involve the viral protein kinases encoded by the UL13 and US3 gene products and, (iii) UL49 is a virion protein which is labeled in cells with [32P]orthophosphate and also is ADP-ribosylated.

Adenosine Diphosphate↗
PubMed1993

Guanylylation and adenylylation of the alpha regulatory proteins of herpes simplex virus require a viral beta or gamma function.

Herpes simplex virus genes form several groups whose expression is coordinately regulated and sequentially ordered in a cascade fashion. Most of the products of the first group, the alpha genes, appear to have regulatory functions. We report that the alpha proteins, infected cell proteins 4, 0, 22, and 27 of herpes simplex virus 1 and 4, 0, and 27 of herpes simplex virus 2, were labeled in the isolated nuclei of infected HeLa cells with [alpha-32P]GTP or [alpha-32P]ATP late in infection and that these proteins represent the largest group of virus-specific proteins labeled in this fashion. Studies with [2-3H]ATP, in which the label is in the purine ring, showed that a portion of the label in alpha proteins and in at least one other infected cell protein is due to nucleotidylylation. Analyses of the labeling reactions in nuclei of (i) cells infected with temperature-sensitive mutants at nonpermissive temperatures, (ii) cells infected with wild-type virus and harvested at different times postinfection, and (iii) cells treated with inhibitors of protein synthesis or of synthesis of viral DNA led to the conclusion that viral gene functions expressed after the synthesis of alpha proteins are required for the labeling of the alpha proteins with [alpha-32P]GTP. We conclude that several of the alpha proteins are extensively posttranslationally modified and that these modifications include nucleotidylylation.

Adenosine Triphosphate↗
PubMed1992

Differences in the poly(ADP-ribosyl)ation patterns of ICP4, the herpes simplex virus major regulatory protein, in infected cells and in isolated nuclei.

Infected-cell protein 4 (ICP4), the major regulatory protein in herpes simplex viruses 1 and 2, was previously reported to accept 32P from [32P]NAD in isolated nuclei. This modification was attributed to poly(ADP-ribosyl)ation (C. M. Preston and E. L. Notarianni, Virology 131:492-501, 1983). We determined that an antibody specific for poly(ADP-ribose) reacts with ICP4 extracted from infected cells, electrophoretically separated in denaturing gels, and electrically transferred to nitrocellulose. Our results indicate that all forms of ICP4 observed in one-dimensional gel electrophoresis are poly(ADP-ribosyl)ated. Poly(ADP-ribose) on ICP4 extracted from infected cells was resistant to cleavage by purified poly(ADP-ribose) glycohydrolase unless ICP4 was in a denatured state. Poly(ADP-ribose) added to ICP4 in isolated nuclei was sensitive to this enzyme. This result indicates that the two processes are distinct and may involve different sites on the ICP4 molecule.

Adenosine Monophosphate↗
PubMed1991

ICP4, the major regulatory protein of herpes simplex virus, shares features common to GTP-binding proteins and is adenylated and guanylated.

Infected cell protein 4 (ICP4), the product of the alpha 4 gene, regulates herpes simplex virus 1 and herpes simplex virus 2 gene expression at the transcriptional level both positively and negatively. Previous studies have shown that ICP4 is extensively modified posttranslationally. We report that ICP4 was labeled in isolated nuclei of infected cells by [alpha-32P]GTP or [alpha-32P]ATP. The labeling of ICP4 by [alpha-32P]GTP or [alpha-32P]ATP required excess GTP, ATP, GDP, and ADP and occurred also in the presence of excess GTP(gamma)S. While GDP and ADP activated the labeling process, only GTP and ATP labeled ICP4. Accumulation of labeled ICP4 was favored at temperatures from 15 to 27 degrees C and in the presence of okadaic acid. The conditions for labeling ICP4 with [alpha-32P]GTP or [alpha-32P]ATP and the stability of the labeled protein were different from those of ICP4 labeled with [gamma-32P]ATP. Labeling studies with tritiated ATP and GTP showed that ICP4 is nucleotidylated, and chemical degradation of ICP4 labeled with [alpha-32P]GTP yielded ribose-5-phosphate. Pulse-chase experiments indicated that the adenylation and guanylation are independent processes. These results, and the observation that ICP4 contains four regions which possess consensus GTP-binding elements, suggest that ICP4 may belong to a class of GTP-binding proteins which function in transcriptional transactivation.

Adenosine Triphosphate↗