scholarly journals Herpes Simplex Virus Type 1 Infection Leads to Loss of Serine-2 Phosphorylation on the Carboxyl-Terminal Domain of RNA Polymerase II

2005 ◽  
Vol 79 (17) ◽  
pp. 11323-11334 ◽  
Author(s):  
Kathryn A. Fraser ◽  
Stephen A. Rice
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Rna Polymerase Ii ◽  
Mrna Processing ◽  
Nuclear Speckles ◽  
Carboxyl Terminal ◽  
Rnap Ii ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACT Previous studies have shown that herpes simplex virus type 1 (HSV-1) infection alters the phosphorylation of the carboxyl-terminal domain (CTD) of RNA polymerase II (RNAP II), creating a new form of the enzyme known as RNAP III. However, the specific phosphorylation changes induced by HSV-1 have not been characterized. In this study, we used phospho-specific anti-CTD antibodies to probe the structure of the postinfection RNAP II. We find that RNAP III is phosphorylated on serine-5 (Ser-5) of the CTD consensus repeat but generally lacks phosphorylation on serine-2 (Ser-2). Since Ser-2 phosphorylation is normally associated with efficient transcriptional elongation and the recruitment of pre-mRNA processing factors, our results suggest that RNAP III may have altered elongation properties and decreased interactions with the mRNA processing machinery. The viral factors responsible for the reduction in Ser-2 CTD phosphorylation were studied. We found that viral immediate-early (IE) gene expression is required and sufficient, in the context of infection, for loss of Ser-2 phosphorylation. However, studies with viral mutants failed to implicate a single IE protein (among ICP0, ICP4, ICP22, and ICP27) in this process. Although most Ser-2-phosphorylated RNAP II is lost after infection, our immunofluorescence analyses identified a small subfraction that escapes loss and relocalizes to splicing antigen-rich nuclear speckles. A similar phenomenon is seen in uninfected cells after various treatments that inhibit RNAP II transcription. We hypothesize that the HSV-1-induced relocalization of residual Ser-2-phosphorylated RNAP II to nuclear speckles reflects a host response to the inhibition of cellular gene transcription.

scholarly journals Recruitment of the Transcriptional Coactivator HCF-1 to Viral Immediate-Early Promoters during Initiation of Reactivation from Latency of Herpes Simplex Virus Type 1

2009 ◽  
Vol 83 (18) ◽  
pp. 9591-9595 ◽  
Author(s):  
Zackary W. Whitlow ◽  
Thomas M. Kristie
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Rna Polymerase Ii ◽  
Virus Type ◽  
Herpes Simplex Virus Type ◽  
Immediate Early ◽  
Transcriptional Coactivator ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACT The transcriptional coactivator host cell factor 1 (HCF-1) is critical for the expression of immediate-early (IE) genes of the alphaherpesviruses herpes simplex virus type 1 (HSV-1) and varicella-zoster virus. HCF-1 may also be involved in the reactivation of these viruses from latency as it is sequestered in the cytoplasm of sensory neurons but is rapidly relocalized to the nucleus upon stimulation that results in reactivation. Here, chromatin immunoprecipitation assays demonstrate that HCF-1 is recruited to IE promoters of viral genomes during the initiation of reactivation, correlating with RNA polymerase II occupancy and IE expression. The data support the model whereby HCF-1 plays a pivotal role in the reactivation of HSV-1 from latency.

scholarly journals Herpes Simplex Virus Immediate-Early Protein ICP22 Triggers Loss of Serine 2-Phosphorylated RNA Polymerase II

2007 ◽  
Vol 81 (10) ◽  
pp. 5091-5101 ◽  
Author(s):  
Kathryn A. Fraser ◽  
Stephen A. Rice
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Immediate Early ◽  
Synthetic Activity ◽  
Transfected Cells ◽  
Rnap Ii ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACT During eukaryotic mRNA transcription, the synthetic activity and mRNA processing factor interactions of RNA polymerase II (RNAP II) are regulated by phosphorylation of its carboxyl-terminal domain (CTD), with modification occurring primarily on serines 2 and 5 of the CTD. We previously showed that herpes simplex virus type 1 (HSV-1) infection rapidly triggers the loss of RNAP II forms bearing serine 2 phosphorylation (Ser-2P RNAP II). Here we show that the HSV-1 immediate-early (IE) protein ICP22 is responsible for this effect during the IE phase of infection. This activity does not require the viral UL13 protein kinase, which is required for several other regulatory functions of ICP22. Additionally, we show that transient expression of ICP22 can trigger the loss of Ser-2P RNAP II in transfected cells. Thus, the ability of ICP22 to cause the loss of Ser-2 RNAP II does not require other viral factors or the context of the infected cell. Expression of the HSV-1 ICP22-related protein US1.5, which corresponds to residues 147 to 420 of ICP22, also triggers a loss of Ser-2P RNAP II in transfected cells, whereas expression of the varicella-zoster virus ICP22 homolog, ORF63, does not. Our study also provides evidence for a second, viral late gene-dependent pathway that triggers loss of Ser-2P RNAP II in infected cells, consistent with the recent work of Dai-Ju et al. (J. Q. Dai-Ju, L. Li, L. A. Johnson, and R. M. Sandri-Goldin, J. Virol. 80:3567-3581, 2006). Therefore, it appears that HSV-1 has evolved redundant mechanisms for triggering the loss of a specific phosphorylated form of RNAP II.

scholarly journals ICP27 Interacts with the C-Terminal Domain of RNA Polymerase II and Facilitates Its Recruitment to Herpes Simplex Virus 1 Transcription Sites, Where It Undergoes Proteasomal Degradation during Infection

2006 ◽  
Vol 80 (7) ◽  
pp. 3567-3581 ◽  
Author(s):  
Jenny Q. Dai-Ju ◽  
Ling Li ◽  
Lisa A. Johnson ◽  
Rozanne M. Sandri-Goldin
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Proteasomal Degradation ◽  
Herpes Simplex Virus 1 ◽  
Transcription Sites ◽  
Rnap Ii ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACT Herpes simplex virus 1 (HSV-1) ICP27 has been shown to interact with RNA polymerase II (RNAP II) holoenzyme. Here, we show that ICP27 interacts with the C-terminal domain (CTD) of RNAP II and that ICP27 mutants that cannot interact fail to relocalize RNAP II to viral transcription sites, suggesting a role for ICP27 in RNAP II recruitment. Using monoclonal antibodies specific for different phosphorylated forms of the RNAP II CTD, we found that the serine-2 phosphorylated form, which is found predominantly in elongating complexes, was not recruited to viral transcription sites. Further, there was an overall reduction in phosphoserine-2 staining. Western blot analysis revealed that there was a pronounced decrease in the phosphoserine-2 form and in overall RNAP II levels in lysates from cells infected with wild-type HSV-1. There was no appreciable difference in cdk9 levels, suggesting that protein degradation rather than dephosphorylation was occurring. Treatment of infected cells with proteasome inhibitors MG-132 and lactacystin prevented the decrease in the phosphoserine-2 form and in overall RNAP II levels; however, there was a concomitant decrease in the levels of several HSV-1 late proteins and in virus yield. Proteasomal degradation has been shown to resolve stalled RNAP II complexes at sites of DNA damage to allow 3′ processing of transcripts. Thus, we propose that at later times of infection when robust transcription and DNA replication are occurring, elongating complexes may collide and proteasomal degradation may be required for resolution.

scholarly journals RNA Polymerase II Holoenzyme Modifications Accompany Transcription Reprogramming in Herpes Simplex Virus Type 1-Infected Cells

2001 ◽  
Vol 75 (20) ◽  
pp. 9872-9884 ◽  
Author(s):  
H. L. Jenkins ◽  
C. A. Spencer
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Gene Transcription ◽  
Herpes Simplex Virus Type ◽  
Large Subunit ◽  
Rnap Ii ◽  
Simplex Virus

ABSTRACT During lytic infection, herpes simplex virus type 1 (HSV-1) represses host transcription, recruits RNA polymerase II (RNAP II) to viral replication compartments, and alters the phosphorylation state of the RNAP II large subunit. Host transcription repression and RNAP II modifications require expression of viral immediate-early (IE) genes. Efficient modification of the RNAP II large subunit to the intermediately phosphorylated (IIi) form requires expression of ICP22 and the UL13 kinase. We have further investigated the mechanisms by which HSV-1 effects global changes in RNAP II transcription by analyzing the RNAP II holoenzyme. We find that the RNAP II general transcription factors (GTFs) remain abundant after infection and are recruited into viral replication compartments, suggesting that they continue to be involved in viral gene transcription. However, virus infection modifies the composition of the RNAP II holoenzyme, in particular triggering the loss of the essential GTF, TFIIE. Loss of TFIIE from the RNAP II holoenzyme requires viral IE gene expression, and viral IE proteins may be redundant in mediating this effect. Although viral IE proteins do not associate with the RNAP II holoenzyme, they interact with RNAP II in complexes of lower molecular mass. As the RNAP II holoenzyme containing TFIIE is necessary for activated transcription initiation and RNAP II large subunit phosphorylation in uninfected cells, virus-induced modifications to the holoenzyme may affect both of these processes, leading to aberrant phosphorylation of the RNAP II large subunit and repression of host gene transcription.

scholarly journals ICP22 and the UL13 Protein Kinase Are both Required for Herpes Simplex Virus-Induced Modification of the Large Subunit of RNA Polymerase II

1999 ◽  
Vol 73 (7) ◽  
pp. 5593-5604 ◽  
Author(s):  
Melissa C. Long ◽  
Vivian Leong ◽  
Priscilla A. Schaffer ◽  
Charlotte A. Spencer ◽  
Stephen A. Rice
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Protein Kinase ◽  
Rna Polymerase Ii ◽  
Large Subunit ◽  
Viral Gene ◽  
Viral Gene Product ◽  
Rnap Ii ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACT Herpes simplex virus type 1 (HSV-1) infection alters the phosphorylation of the large subunit of RNA polymerase II (RNAP II), resulting in the depletion of the hypophosphorylated and hyperphosphorylated forms of this polypeptide (known as IIa and IIo, respectively) and induction of a novel, alternatively phosphorylated form (designated IIi). We previously showed that the HSV-1 immediate-early protein ICP22 is involved in this phenomenon, since induction of IIi and depletion of IIa are deficient in cells infected with 22/n199, an HSV-1 ICP22 nonsense mutant (S. A. Rice, M. C. Long, V. Lam, P. A. Schaffer, and C. A. Spencer, J. Virol. 69:5550–5559, 1995). However, depletion of IIo still occurs in 22/n199-infected cells. This suggests either that another viral gene product affects the RNAP II large subunit or that the truncated ICP22 polypeptide encoded by 22/n199 retains residual activity which leads to IIo depletion. To distinguish between these possibilities, we engineered an HSV-1 ICP22 null mutant, d22-lacZ, and compared it to 22/n199. The two mutants are indistinguishable in their effects on the RNAP II large subunit, suggesting that an additional viral gene product is involved in altering RNAP II. Two candidates are UL13, a protein kinase which has been implicated in ICP22 phosphorylation, and the virion host shutoff (Vhs) factor, the expression of which is positively regulated by ICP22 and UL13. To test whether UL13 is involved, a UL13-deficient viral mutant,d13-lacZ, was engineered. This mutant was defective in IIi induction and IIa depletion, displaying a phenotype very similar to that of d22-lacZ. In contrast, a Vhs mutant had effects that were indistinguishable from wild-type HSV-1. Therefore, UL13 but not the Vhs function plays a role in modifying the RNAP II large subunit. To study the potential role of UL13 in viral transcription, we carried out nuclear run-on transcription analyses in infected human embryonic lung cells. Infections with either UL13 or ICP22 mutants led to significantly reduced amounts of viral genome transcription at late times after infection. Together, our results suggest that ICP22 and UL13 are involved in a common pathway that alters RNAP II phosphorylation and that in some cell lines this change promotes viral late transcription.

scholarly journals Enhanced Phosphorylation of Transcription Factor Sp1 in Response to Herpes Simplex Virus Type 1 Infection Is Dependent on the Ataxia Telangiectasia-Mutated Protein

2007 ◽  
Vol 81 (18) ◽  
pp. 9653-9664 ◽  
Author(s):  
Satoko Iwahori ◽  
Noriko Shirata ◽  
Yasushi Kawaguchi ◽  
Sandra K. Weller ◽  
Yoshitaka Sato ◽  
...  
Keyword(s):  
Dna Damage ◽  
Transcription Factor ◽  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Herpes Simplex Virus Type ◽  
Ataxia Telangiectasia ◽  
Ataxia Telangiectasia Mutated ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACT The ataxia telangiectasia-mutated (ATM) protein, a member of the related phosphatidylinositol 3-like kinase family encoded by a gene responsible for the human genetic disorder ataxia telangiectasia, regulates cellular responses to DNA damage and viral infection. It has been previously reported that herpes simplex virus type 1 (HSV-1) infection induces activation of protein kinase activity of ATM and hyperphosphorylation of transcription factor, Sp1. We show that ATM is intimately involved in Sp1 hyperphosphorylation during HSV-1 infection rather than individual HSV-1-encoded protein kinases. In ATM-deficient cells or cells silenced for ATM expression by short hairpin RNA targeting, hyperphosphorylation of Sp1 was prevented even as HSV-1 infection progressed. Mutational analysis of putative ATM phosphorylation sites on Sp1 and immunoblot analysis with phosphopeptide-specific Sp1 antibodies clarified that at least Ser-56 and Ser-101 residues on Sp1 became phosphorylated upon HSV-1 infection. Serine-to-alanine mutations at both sites on Sp1 considerably abolished hyperphosphorylation of Sp1 upon infection. Although ATM phosphorylated Ser-101 but not Ser-56 on Sp1 in vitro, phosphorylation of Sp1 at both sites was not detected at all upon infection in ATM-deficient cells, suggesting that cellular kinase(s) activated by ATM could be involved in phosphorylation at Ser-56. Upon viral infection, Sp1-dependent transcription in ATM expression-silenced cells was almost the same as that in ATM-intact cells, suggesting that ATM-dependent phosphorylation of Sp1 might hardly affect its transcriptional activity during the HSV-1 infection. ATM-dependent Sp1 phosphorylation appears to be a global response to various DNA damage stress including viral DNA replication.

scholarly journals Herpes Simplex Virus Type 1 Evades the Effects of Antibody and Complement In Vivo

2002 ◽  
Vol 76 (18) ◽  
pp. 9232-9241 ◽  
Author(s):  
John M. Lubinski ◽  
Ming Jiang ◽  
Lauren Hook ◽  
Yueh Chang ◽  
Chad Sarver ◽  
...  
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Virus Type ◽  
Immune Evasion ◽  
Herpes Simplex Virus Type ◽  
Complement Components ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACT Herpes simplex virus type 1 (HSV-1) encodes a complement-interacting glycoprotein, gC, and an immunoglobulin G (IgG) Fc binding glycoprotein, gE, that mediate immune evasion by affecting multiple aspects of innate and acquired immunity, including interfering with complement components C1q, C3, C5, and properdin and blocking antibody-dependent cellular cytotoxicity. Previous studies evaluated the individual contributions of gC and gE to immune evasion. Experiments in a murine model that examines the combined effects of gC and gE immune evasion on pathogenesis are now reported. Virulence of wild-type HSV-1 is compared with mutant viruses defective in gC-mediated C3 binding, gE-mediated IgG Fc binding, or both immune evasion activities. Eliminating both activities greatly increased susceptibility of HSV-1 to antibody and complement neutralization in vitro and markedly reduced virulence in vivo as measured by disease scores, virus titers, and mortality. Studies with C3 knockout mice indicated that other activities attributed to these glycoproteins, such as gC-mediated virus attachment to heparan sulfate or gE-mediated cell-to-cell spread, do not account for the reduced virulence of mutant viruses. The results support the importance of gC and gE immune evasion in vivo and suggest potential new targets for prevention and treatment of HSV disease.

scholarly journals Nucleolin Is Required for Efficient Nuclear Egress of Herpes Simplex Virus Type 1 Nucleocapsids

2009 ◽  
Vol 84 (4) ◽  
pp. 2110-2121 ◽  
Author(s):  
Ken Sagou ◽  
Masashi Uema ◽  
Yasushi Kawaguchi
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Nuclear Membrane ◽  
Herpes Simplex Virus Type ◽  
Viral Dna ◽  
Nuclear Egress ◽  
Infected Cells ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACT Herpesvirus nucleocapsids assemble in the nucleus and must cross the nuclear membrane for final assembly and maturation to form infectious progeny virions in the cytoplasm. It has been proposed that nucleocapsids enter the perinuclear space by budding through the inner nuclear membrane, and these enveloped nucleocapsids then fuse with the outer nuclear membrane to enter the cytoplasm. Little is known about the mechanism(s) for nuclear egress of herpesvirus nucleocapsids and, in particular, which, if any, cellular proteins are involved in the nuclear egress pathway. UL12 is an alkaline nuclease encoded by herpes simplex virus type 1 (HSV-1) and has been suggested to be involved in viral DNA maturation and nuclear egress of nucleocapsids. Using a live-cell imaging system to study cells infected by a recombinant HSV-1 expressing UL12 fused to a fluorescent protein, we observed the previously unreported nucleolar localization of UL12 in live infected cells and, using coimmunoprecipitation analyses, showed that UL12 formed a complex with nucleolin, a nucleolus marker, in infected cells. Knockdown of nucleolin in HSV-1-infected cells reduced capsid accumulation, as well as the amount of viral DNA resistant to staphylococcal nuclease in the cytoplasm, which represented encapsidated viral DNA, but had little effect on these viral components in the nucleus. These results indicated that nucleolin is a cellular factor required for efficient nuclear egress of HSV-1 nucleocapsids in infected cells.

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