scholarly journals The US3 Protein Kinase Blocks Apoptosis Induced by the d120 Mutant of Herpes Simplex Virus 1 at a Premitochondrial Stage

2001 ◽  
Vol 75 (12) ◽  
pp. 5491-5497 ◽  
Author(s):  
Joshua Munger ◽  
Ana V. Chee ◽  
Bernard Roizman
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Protein Kinase ◽  
Protein Kinase R ◽  
Herpes Simplex Virus 1 ◽  
Reading Frame ◽  
Dna Fragment ◽  
Simplex Virus ◽  
Apoptotic Cascade ◽  
In Cells

ABSTRACT Earlier studies have shown that the d120 mutant of herpes simplex virus 1, which lacks both copies of the α4 gene, induces caspase-3-dependent apoptosis in HEp-2 cells. Apoptosis was also induced by the α4 rescuant but was blocked by the complementation of rescuant with a DNA fragment encoding the US3 protein kinase (R. Leopardi and B. Roizman, Proc. Natl. Acad. Sci. USA 93:9583–9587, 1996, and R. Leopardi, C. Van Sant, and B. Roizman, Proc. Natl. Acad. Sci. USA 94:7891–7896, 1997). To investigate its role in the apoptotic cascade, the US3 open reading frame was cloned into a baculovirus (Bac-US3) under the control of the human cytomegalovirus immediate-early promoter. We report the following. (i) Bac-US3 blocks processing of procaspase-3 to active caspase. Procaspase-3 levels remained unaltered if superinfected with Bac-US3 at 3 h afterd120 mutant infection, but significant amounts of procaspase-3 remained in cells superinfected with Bac-Us3 at 9 h postinfection with d120 mutant. (ii) The US3 protein kinase blocks the proapoptotic cascade upstream of mitochondrial involvement inasmuch as Bac-US3 blocks release of cytochrome c in cells infected with thed120 mutant. (iii) Concurrent infection of HEp-2 cells with Bac-US3 and the d120 mutant did not alter the pattern of accumulation or processing of ICP0, -22, or -27, and therefore US3 does not appear to block apoptosis by targeting these proteins.

scholarly journals Glycoprotein D or J Delivered in transBlocks Apoptosis in SK-N-SH Cells Induced by a Herpes Simplex Virus 1 Mutant Lacking Intact Genes Expressing Both Glycoproteins

2000 ◽  
Vol 74 (24) ◽  
pp. 11782-11791 ◽  
Author(s):  
Guoying Zhou ◽  
Veronica Galvan ◽  
Gabriella Campadelli-Fiume ◽  
Bernard Roizman
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Dna Sequences ◽  
De Novo ◽  
Cell Protein ◽  
Herpes Simplex Virus 1 ◽  
Simplex Virus ◽  
Apoptotic Cascade ◽  
Made In ◽  
In Cells

ABSTRACT We have made two stocks of a herpes simplex virus 1 mutant lacking intact US5 and US6 open reading frames encoding glycoproteins J (gJ) and D (gD), respectively. The stock designated gD−/+, made in cells carrying US6 and expressing gD, was capable of productively infecting cells, whereas the stock designated gD−/−, made in cells lacking viral DNA sequences, was known to attach but not initiate infection. We report the following. (i) Both stocks of virus induced apoptosis in SK-N-SH cells. Thus, annexin V binding to cell surfaces was detected as early as 8 h after infection. (ii) US5 or US6 cloned into the baculovirus under the human cytomegalovirus immediate-early promoter was expressed in SK-N-SH cells and blocked apoptosis in cells infected with either gD−/+ or gD−/− virus, whereas glycoprotein B, infected cell protein 22, or the wild-type baculovirus did not block apoptosis. (iii) In SK-N-SH cells, internalized, partially degraded virus particles were detected at 30 min after exposure to gD−/− virus but not at later intervals. (iv) Concurrent infection of cells with baculoviruses did not alter the failure of gD−/− virus from expressing its genes or, conversely, the expression of viral genes by gD−/+ virus. These results underscore the capacity of herpes simplex virus to initiate the apoptotic cascade in the absence of de novo protein synthesis and indicate that both gD and gJ independently, and most likely at different stages in the reproductive cycle, play a key role in blocking the apoptotic cascade leading to cell death.

scholarly journals The UL3 Protein of Herpes Simplex Virus 1 Is Translated Predominantly from the Second In-Frame Methionine Codon and Is Subject to at Least Two Posttranslational Modifications

1999 ◽  
Vol 73 (10) ◽  
pp. 8010-8018 ◽  
Author(s):  
Nancy S. Markovitz ◽  
Felix Filatov ◽  
Bernard Roizman
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Protein Kinase ◽  
Posttranslational Modifications ◽  
Wild Type Virus ◽  
Herpes Simplex Virus 1 ◽  
Methionine Codon ◽  
Simplex Virus ◽  
In Cells

ABSTRACT The UL3 open reading frame (ORF) has the coding capacity of 235 codons. The proteins reacting with the anti-UL3 antibody form in denaturing polyacrylamide gel bands with apparent M rs of 34,000, 35,000, 38,000, 40,000, 41,000, and 42,000 and designated 1 to 6, respectively. Studies on their origins revealed the following. (i) The UL3 proteins forming all six bands were present in lysates of cells infected with wild-type virus and treated with tunicamycin or monensin or in cells infected with the mutant lacking the gene encoding the US3 protein kinase. (ii) The proteins contained in the slower-migrating bands were absent from cells infected with the mutant lacking the UL13 protein kinase. Bands 1 and 3, however were phosphorylated in cells infected with this mutant. (iii) Band 2 protein was absent from cells transfected with a plasmid carrying a substitution of the predicted first methionine codon of the UL3 ORF and superinfected with the UL3− mutant. (iv) Band 1 and 3 proteins were absent from lysates of cells transfected with a plasmid carrying a substitution of the second (M12) methionine codon of the UL3 ORF and superinfected with the UL3− mutant. (v) Cells superinfected with mutants lacking both methionine codons did not accumulate any of the proteins contained in the six bands. (vi) In vitro transcription-translation studies indicated that the translation of band 1 protein was initiated from the second (M12) methionine codon and that band 3 protein represented a UL13-independent, posttranslationally processed form of these proteins. The results indicate that the UL3 protein of herpes simplex virus 1 is translated predominantly from the second in-frame methionine codon and is subject to at least two posttranslational modifications.

scholarly journals Herpes Simplex Virus 1 ICP22 Regulates the Accumulation of a Shorter mRNA and of a Truncated US3 Protein Kinase That Exhibits Altered Functions

2005 ◽  
Vol 79 (13) ◽  
pp. 8470-8479 ◽  
Author(s):  
Alice P. W. Poon ◽  
Bernard Roizman
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Open Reading Frame ◽  
Wild Type ◽  
Gene Encoding ◽  
Herpes Simplex Virus 1 ◽  
Reading Frame ◽  
Infected Cells ◽  
Simplex Virus ◽  
In Cells

ABSTRACT The US3 open reading frame of herpes simplex virus 1 (HSV-1) was reported to encode two mRNAs each directing the synthesis of the same protein. We report that the US3 gene encodes two proteins. The predominant US3 protein is made in wild-type HSV-1-infected cells. The truncated mRNA and a truncated protein designated US3.5 and initiating from methionine 77 were preeminent in cells infected with a mutant lacking the gene encoding ICP22. Both the wild-type and truncated proteins also accumulated in cells transduced with a baculovirus carrying the entire US3 open reading frame. The US3.5 protein accumulating in cells infected with the mutant lacking the gene encoding ICP22 mediated the phosphorylation of histone deacetylase 1, a function of US3 protein, but failed to block apoptosis of the infected cells. The US3.5 and US3 proteins differ with respect to the range of functions they exhibit.

scholarly journals Eukaryotic Elongation Factor 1δ Is Hyperphosphorylated by the Protein Kinase Encoded by the UL13 Gene of Herpes Simplex Virus 1

1998 ◽  
Vol 72 (3) ◽  
pp. 1731-1736 ◽  
Author(s):  
Yasushi Kawaguchi ◽  
Charles Van Sant ◽  
Bernard Roizman
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Protein Kinase ◽  
Elongation Factor ◽  
Vero Cells ◽  
Cell Protein ◽  
Herpes Simplex Virus 1 ◽  
Infected Cells ◽  
Simplex Virus ◽  
In Cells

ABSTRACT The translation elongation factor 1δ (EF-1δ) consists of two forms, a hypophosphorylated form (apparent M r, 38,000) and a hyperphosphorylated form (apparentM r, 40,000). Earlier Y. Kawaguchi, R. Bruni, and B. Roizman (J. Virol. 71:1019–1024, 1997) reported that whereas mock-infected cells accumulate the hypophosphorylated form, the hyperphosphorylated form of EF-1δ accumulates in cells infected with herpes simplex virus 1. We now report that the accumulation of the hyperphosphorylated EF-1δ is due to phosphorylation by UL13 protein kinase based on the following observations. (i) The relative amounts of hypo- and hyperphosphorylated EF-1δ in Vero cells infected with mutant virus lacking the UL13 gene could not be differentiated from those of mock-infected cells. In contrast, the hyperphosphorylated EF-1δ was the predominant form in Vero cells infected with wild-type viruses, a recombinant virus in which the deleted UL13 sequences were restored, or with a virus lacking the US3 gene, which also encodes a protein kinase. (ii) The absence of the hyperphosphorylated EF-1δ in cells infected with the UL13 deletion mutant was not due to failure of posttranslational modification of infected-cell protein 22 (ICP22)/US1.5 or of interaction with ICP0, inasmuch as preferential accumulation of hyperphosphorylated EF-1δ was observed in cells infected with viruses from which the genes encoding ICP22/US1.5 or ICP0 had been deleted. (iii) Both forms of EF-1δ were labeled by 32Pi in vivo, but the prevalence of the hyperphosphorylated EF-1δ was dependent on the presence of the UL13 protein. (iv) EF-1δ immunoprecipitated from uninfected Vero cells was phosphorylated by UL13 precipitated by the anti-UL13 antibody from lysates of wild-type virus-infected cells, but not by complexes formed by the interaction of the UL13 antibody with lysates of cells infected with a mutant lacking the UL13 gene. This is the first evidence that a viral protein kinase targets a cellular protein. Together with evidence that ICP0 also interacts with EF-1δ reported in the paper cited above, these data indicate that herpes simplex virus 1 has evolved a complex strategy for optimization of infected-cell protein synthesis.

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