scholarly journals The Two Functions of Herpes Simplex Virus 1 ICP0, Inhibition of Silencing by the CoREST/REST/HDAC Complex and Degradation of PML, Are Executed in Tandem

2008 ◽  
Vol 83 (1) ◽  
pp. 181-187 ◽  
Author(s):  
Haidong Gu ◽  
Bernard Roizman
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Binding Site ◽  
Nuclear Bodies ◽  
Wild Type Virus ◽  
Type Virus ◽  
Wild Type ◽  
Herpes Simplex Virus 1 ◽  
Simplex Virus ◽  
In Cells

ABSTRACT ICP0, an α (immediate-early) protein of herpes simplex virus 1, performs at least two key functions. It blocks inhibition of viral-gene expression by interferon, a function dependent on the degradation of the ND10 components PML and SP100 by the ubiquitin ligase expressed by the RING finger (RF), and it blocks silencing of viral DNA mediated by the HDAC1/2-CoREST-REST complex. In the latter case, a mutant CoREST lacking the HDAC1 binding site compensates totally or in part for the absence of ICP0 in a cell-type-dependent manner. Here, we compare the phenotypes of an ICP0 mutant containing disabling amino acid substitutions in the RF with those of a mutant with substitutions in the CoREST binding site (R8507). We report the following: (i) the onset of replication of both mutants was delayed, but the RF mutant yields did not reach wild-type virus levels even as late as 48 h after infection, and (ii) in infected cells, PML is rapidly degraded by wild-type virus, with some delay by the R8507 mutant, and is spared by the RF mutant. The translocation of ICP0 to the cytoplasm is impaired in cells infected with the RF mutant or delayed in cells infected with the R8507 mutant. Finally, in contrast to wild-type viruses, both mutants are inhibited by alpha or gamma interferon. The results indicate that both sets of events, the degradation of PML and the blocking of silencing, are interdependent and in large measure dependent on events in the ND10 nuclear bodies.

scholarly journals Posttranslational Processing of Infected Cell Proteins 0 and 4 of Herpes Simplex Virus 1 Is Sequential and Reflects the Subcellular Compartment in Which the Proteins Localize

2001 ◽  
Vol 75 (17) ◽  
pp. 7904-7912 ◽  
Author(s):  
Sunil J. Advani ◽  
Ryan Hagglund ◽  
Ralph R. Weichselbaum ◽  
Bernard Roizman
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Infected Cell ◽  
Wild Type Virus ◽  
Type Virus ◽  
Wild Type ◽  
Herpes Simplex Virus 1 ◽  
Simplex Virus ◽  
Made In ◽  
In Cells

ABSTRACT The herpes simplex virus 1 (HSV-1) infected cell proteins 0 and 4 (ICP0 and ICP4) are multifunctional proteins extensively posttranscriptionally processed by both cellular and viral enzymes. We examined by two-dimensional separations the posttranslational forms of ICP0 and ICP4 in HEp-2 cells and in human embryonic lung (HEL) fibroblasts infected with wild-type virus, mutant R325, lacking the sequences encoding the US1.5 protein and the overlapping carboxyl-terminal domain of ICP22, or R7914, in which the aspartic acid 199 of ICP0 was replaced by alanine. We report the following (i) Both ICP0 and ICP4 were sequentially posttranslationally modified at least until 12 h after infection. In HEL fibroblasts, the processing of ICP0 shifted from A+B forms at 4 h to D+G forms at 8 h and finally to G, E, and F forms at 12 h. The ICP4 progression was from the A′ form noted at 2 h to B′ and C′ forms noted at 4 h to the additional D′ and E′ forms noted at 12 h. The progression tended to be toward more highly charged forms of the proteins. (ii) Although the overall patterns were similar, the mobility of proteins made in HEp-2 cells differed from those made in HEL fibroblasts. (iii) The processing of ICP0 forms E and F was blocked in HEL fibroblasts infected with R325 or with wild-type virus and treated with roscovitine, a specific inhibitor of cell cycle-dependent kinases cdc2, cdk2, and cdk5. R325-infected HEp-2 cells lacked the D′ form of ICP4, and roscovitine blocked the appearance of the most highly charged E′ form of ICP4. (iv) A characteristic of ICP0 is that it is translocated into the cytoplasm of HEL fibroblasts between 5 and 9 h after infection. Addition of MG132 to the cultures late in infection resulted in rapid relocation of cytoplasmic ICP0 back into the nucleus. Exposure of HEL fibroblasts to MG132 late in infection resulted in the disappearance of the highly charged ICP0 G isoform. The G form of ICP0 was also absent in cells infected with R7914 mutant. In cells infected with this mutant, ICP0 is not translocated to the cytoplasm. (v) Last, cdc2 was active in infected cells, and this activity was inhibited by roscovitine. In contrast, the activity of cdk2 exhibited by immunoprecipitated protein was reduced and resistant to roscovitine and may represent a contaminating kinase activity. We conclude from these results that the ICP0 G isoform is the cytoplasmic form, that it may be phosphorylated by cdc2, consistent with evidence published earlier (S. J., Advani, R. R. Weichselbaum, and B. Roizman, Proc. Natl. Acad. Sci. USA 96:10996–11001, 2000), and that the processing is reversed upon relocation of the G isoform from the cytoplasm into the nucleus. The processing of ICP4 is also affected by R325 and roscovitine. The latter result suggests that ICP4 may also be a substrate of cdc2 late in infection. Last, additional modifications are superimposed by cell-type-specific enzymes.

scholarly journals Cells Lacking NF-κB or in Which NF-κB Is Not Activated Vary with Respect to Ability To Sustain Herpes Simplex Virus 1 Replication and Are Not Susceptible to Apoptosis Induced by a Replication-Incompetent Mutant Virus

2004 ◽  
Vol 78 (21) ◽  
pp. 11615-11621 ◽  
Author(s):  
Brunella Taddeo ◽  
Weiran Zhang ◽  
Fred Lakeman ◽  
Bernard Roizman
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Viral Replication ◽  
Cell Lines ◽  
Wild Type Virus ◽  
Type Virus ◽  
Wild Type ◽  
Herpes Simplex Virus 1 ◽  
Simplex Virus ◽  
In Cells

ABSTRACT Earlier we reported that NF-κB is activated by protein kinase R (PKR) in herpes simplex virus 1-infected cells. Here we report that in PKR−/− cells the yields of wild-type virus are 10-fold higher than in PKR+/+ cells. In cells lacking NF-κB p50 (nfkb1), p65 (relA), or both p50 and p65, the yields of virus were reduced 10-fold. Neither wild-type nor mutant cells undergo apoptosis following infection with wild-type virus. Whereas PKR+/+ and NF-κB+/+ control cell lines undergo apoptosis induced by the d120 (Δα4) mutant of HSV-1, the mutant PKR−/− and NF-κB−/− cell lines were resistant. The evidence suggests that the stress-induced apoptosis resulting from d120 infection requires activation of NF-κB and that this proapoptotic pathway is blocked in cells in which NF-κB is not activated or absent. Activation of NF-κB in the course of viral infection may have dual roles of attempting to curtain viral replication by rendering the cell susceptible to apoptosis induced by the virus and by inducing the synthesis of proteins that enhance viral replication.

scholarly journals Herpes Simplex Virus 1 Induces Cytoplasmic Accumulation of TIA-1/TIAR and both Synthesis and Cytoplasmic Accumulation of Tristetraprolin, Two Cellular Proteins That Bind and Destabilize AU-Rich RNAs

2004 ◽  
Vol 78 (16) ◽  
pp. 8582-8592 ◽  
Author(s):  
Audrey Esclatine ◽  
Brunella Taddeo ◽  
Bernard Roizman
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Mutant Virus ◽  
Wild Type Virus ◽  
Type Virus ◽  
Wild Type ◽  
Herpes Simplex Virus 1 ◽  
Infected Cells ◽  
Cytoplasmic Accumulation ◽  
Simplex Virus

ABSTRACT Herpes simplex virus 1 causes a shutoff of cellular protein synthesis through the degradation of RNA that is mediated by the virion host shutoff (Vhs) protein encoded by the UL41 gene. We reported elsewhere that the Vhs-dependent degradation of RNA is selective, and we identified RNAs containing AU-rich elements (AREs) that were upregulated after infection but degraded by deadenylation and progressive 3′-to-5′ degradation. We also identified upregulated RNAs that were not subject to Vhs-dependent degradation (A. Esclatine, B. Taddeo, L. Evans, and B. Roizman, Proc. Natl. Acad. Sci. USA 101:3603-3608, 2004). Among the latter was the RNA encoding tristetraprolin, a protein that binds AREs and is known to be associated with the degradation of RNAs containing AREs. Prompted by this observation, we examined the status of the ARE binding proteins tristetraprolin and TIA-1/TIAR in infected cells. We report that tristetraprolin was made and accumulated in the cytoplasm of wild-type virus-infected human foreskin fibroblasts as early as 2 h and in HEp-2 cells as early as 6 h after infection. The amounts of tristetraprolin that accumulated in the cytoplasm of cells infected with a mutant virus lacking UL41 were significantly lower than those in wild-type virus-infected cells. The localization of tristetraprolin was not modified in cells infected with a mutant lacking the gene encoding infected cell protein 4 (ICP4). TIA-1 and TIAR are two other proteins that are associated with the regulation of ARE-containing RNAs and that normally reside in nuclei. In infected cells, they started to accumulate in the cytoplasm after 6 h of infection. In cells infected with the mutant virus lacking UL41, TIA-1/TIAR accumulated in the cytoplasm in granular structures reminiscent of stress granules in a significant percentage of the cells. In addition, an antibody to tristetraprolin coprecipitated the Vhs protein from lysates of cells late in infection. The results indicate that the Vhs-dependent degradation of ARE-containing RNAs correlates with the transactivation, cytoplasmic accumulation, and persistence of tristetraprolin in infected cells.

scholarly journals Translocation and Colocalization of ICP4 and ICP0 in Cells Infected with Herpes Simplex Virus 1 Mutants Lacking Glycoprotein E, Glycoprotein I, or the Virion Host Shutoff Product of the UL41 Gene

2007 ◽  
Vol 82 (4) ◽  
pp. 1701-1713 ◽  
Author(s):  
Maria Kalamvoki ◽  
Jianguo Qu ◽  
Bernard Roizman
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Wild Type Virus ◽  
Type Virus ◽  
Wild Type ◽  
Herpes Simplex Virus 1 ◽  
Glycoprotein E ◽  
Glycoprotein I ◽  
Infected Cells ◽  
Simplex Virus

ABSTRACT In wild-type herpes simplex virus 1-infected cells, the major regulatory protein ICP4 resides in the nucleus whereas ICP0 becomes dynamically associated with proteasomes and late in infection is translocated and dispersed in the cytoplasm. Inhibition of proteasomal function results in retention or transport of ICP0 to the nucleus. We report that in cells infected with mutants lacking glycoprotein E (gE), glycoprotein I (gI), or the product of the UL41 gene, both ICP4 and ICP0 are translocated to the cytoplasm and coaggregate in small dense structures that, in the presence of proteasomal inhibitor MG132, also contain proteasomal components. Gold particle-conjugated antibody to ICP0 reacted in thin sections with dense protein aggregates in the cytoplasm of mutant virus-infected cells. Similar aggregates were present in the nuclei but not in the cytoplasm of wild-type virus-infected cells. Exposure of cells early in infection to MG132 does not result in retention of ICP0 as in wild-type virus-infected cells. The results suggest that the retention of ICP4 and ICP0 in the nucleus is a dynamic process that involves the function of other viral proteins that may include the Fc receptor formed by the gE/gI complex and is not merely the consequence of expression of a nuclear localization signal. It is noteworthy that in ΔUL41-infected cells gE is retained in the trans-Golgi network and is not widely dispersed in cellular membranes.

scholarly journals Herpes Simplex Virus 1 Open Reading Frames O and P Are Not Necessary for Establishment of Latent Infection in Mice

2000 ◽  
Vol 74 (19) ◽  
pp. 9019-9027 ◽  
Author(s):  
Glenn Randall ◽  
Michael Lagunoff ◽  
Bernard Roizman
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Productive Infection ◽  
Wild Type Virus ◽  
Type Virus ◽  
Wild Type ◽  
Herpes Simplex Virus 1 ◽  
Methionine Codon ◽  
Simplex Virus

ABSTRACT Open reading frame (ORF) O and ORF P partially overlap and are located antisense to the γ134.5 gene within the domain transcribed during latency. In wild-type virus-infected cells, ORF O and ORF P are completely repressed during productive infection by ICP4, the major viral transcriptional activator/repressor. In cells infected with a mutant in which ORF P was derepressed there was a significant delay in the appearance of the viral α-regulatory proteins ICP0 and ICP22. The ORF O protein binds to and inhibits ICP4 binding to its cognate DNA site in vitro. These characteristics suggested a role for ORF O and ORF P in the establishment of latency. To test this hypothesis, two recombinant viruses were constructed. In the first, R7538(P−/O−), the ORF P initiator methionine codon, which also serves as the initiator methionine codon for ORF O, was replaced and a diagnostic restriction endonuclease was introduced upstream. In the second, R7543(P−/O−)R, the mutations were repaired to restore the wild-type virus sequences. We report the following. (i) The R7538(P−/O−) mutant failed to express ORF O and ORF P proteins but expressed a wild-type γ134.5 protein. (ii) R7538(P−/O−) yields were similar to that of the wild type following infection of cell culture or following infection of mice by intracerebral or ocular routes. (iii) R7538(P−/O−) virus reactivated from latency following explanation and cocultivation of murine trigeminal ganglia with Vero cells at a frequency similar to that of the wild type, herpes simplex virus 1(F). (iv) The amount of latent R7538(P−/O−) virus as assayed by quantitative PCR is eightfold less than that of the repair virus. The repaired virus could not be differentiated from the wild-type parent in any of the assays done in this study. We conclude that ORF O and ORF P are not essential for the establishment of latency in mice but may play a role in determining the quantity of latent virus maintained in sensory neurons.

scholarly journals Herpes Simplex Virus 1-Encoded Protein Kinase UL13 Phosphorylates Viral Us3 Protein Kinase and Regulates Nuclear Localization of Viral Envelopment Factors UL34 and UL31

2006 ◽  
Vol 80 (3) ◽  
pp. 1476-1486 ◽  
Author(s):  
Akihisa Kato ◽  
Mayuko Yamamoto ◽  
Takashi Ohno ◽  
Michiko Tanaka ◽  
Tetsutaro Sata ◽  
...  
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Protein Kinase ◽  
Electrophoretic Mobility ◽  
Wild Type Virus ◽  
Type Virus ◽  
Wild Type ◽  
Herpes Simplex Virus 1 ◽  
Infected Cells ◽  
Simplex Virus

ABSTRACT UL13 and Us3 are protein kinases encoded by herpes simplex virus 1. We report here that Us3 is a physiological substrate for UL13 in infected cells, based on the following observations. (i) The electrophoretic mobility, in denaturing gels, of Us3 isoforms from Vero cells infected with wild-type virus was slower than that of isoforms from cells infected with a UL13 deletion mutant virus (ΔUL13). After treatment with phosphatase, the electrophoretic mobility of the Us3 isoforms from cells infected with wild-type virus changed, with one isoform migrating as fast as one of the Us3 isoforms from ΔUL13-infected cells. (ii) A recombinant protein containing a domain of Us3 was phosphorylated by UL13 in vitro. (iii) The phenotype of ΔUL13 resembles that of a recombinant virus lacking the Us3 gene (ΔUs3) with respect to localization of the viral envelopment factors UL34 and UL31, whose localization has been shown to be regulated by Us3. UL34 and UL31 are localized in a smooth pattern throughout the nuclei of cells infected with wild-type virus, whereas their localization in ΔUL13- and ΔUs3-infected cells appeared as nuclear punctate patterns. These results indicate that UL13 phosphorylates Us3 in infected cells and regulates UL34 and UL31 localization, either by phosphorylating Us3 or by a Us3-independent mechanism.

scholarly journals Engagement of the Lysine-Specific Demethylase/HDAC1/CoREST/REST Complex by Herpes Simplex Virus 1

2009 ◽  
Vol 83 (9) ◽  
pp. 4376-4385 ◽  
Author(s):  
Haidong Gu ◽  
Bernard Roizman
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Nuclear Bodies ◽  
Wild Type Virus ◽  
Viral Dna ◽  
Herpes Simplex Virus 1 ◽  
Lysine Specific Demethylase ◽  
Repressor Complex ◽  
Infected Cells ◽  
Simplex Virus

ABSTRACT Among the early events in herpes simplex virus 1 replication are localization of ICP0 in ND10 bodies and accumulation of viral DNA-protein complexes in structures abutting ND10. ICP0 degrades components of ND10 and blocks silencing of viral DNA, achieving the latter by dislodging HDAC1 or -2 from the lysine-specific demethylase 1 (LSD1)/CoREST/REST repressor complex. The role of this process is apparent from the observation that a dominant-negative CoREST protein compensates for the absence of ICP0 in a cell-dependent fashion. HDAC1 or -2 and the CoREST/REST complex are independently translocated to the nucleus once viral DNA synthesis begins. The focus of this report is twofold. First, we report that in infected cells, LSD1, a key component of the repressor complex, is partially degraded or remains stably associated with CoREST and is ultimately also translocated, in part, to the cytoplasm. Second, we examined the distribution of the components of the repressor complex and ICP8 early in infection in wild-type-virus- and ICP0 mutant virus-infected cells. The repressor component and ultimately ICP8 localize in structures that abut the ND10 nuclear bodies. There is no evidence that the two compartments fuse. We propose that ICP0 must dynamically interact with both compartments in order to accomplish its functions of degrading PML and SP100 and suppressing silencing of viral DNA through its interactions with CoREST. In turn, the remodeling of the viral DNA-protein complex enables recruitment of ICP8 and initiation of formation of replication compartments.

scholarly journals Virucidal Activity of a GT-Rich Oligonucleotide against Herpes Simplex Virus Mediated by Glycoprotein B

2006 ◽  
Vol 80 (10) ◽  
pp. 4740-4747 ◽  
Author(s):  
Benjamin Shogan ◽  
Lori Kruse ◽  
Gilbert B. Mulamba ◽  
André Hu ◽  
Donald M. Coen
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Resistant Mutant ◽  
Glycoprotein B ◽  
Wild Type Virus ◽  
Type Virus ◽  
Virucidal Activity ◽  
Wild Type ◽  
Phosphorothioate Oligonucleotide ◽  
Simplex Virus

ABSTRACT We have investigated the antiviral mechanism of a phosphorothioate oligonucleotide, ISIS 5652, which has activity against herpes simplex virus (HSV) in the low micromolar range in plaque reduction assays. We isolated a mutant that is resistant to this compound. Marker rescue and sequencing experiments showed that resistance was due to at least one of three mutations in the UL27 gene which result in amino acid changes in glycoprotein B (gB). Because gB has a role in attachment and entry of HSV, we tested the effects of ISIS 5652 at these stages of infection. The oligonucleotide potently inhibited attachment of virus to cells at 4°C; however, the resistant mutant did not exhibit resistance at this stage. Moreover, a different oligonucleotide with little activity in plaque reduction assays was as potent as ISIS 5652 in inhibiting attachment. Similarly, ISIS 5652 was able to inhibit entry of preattached virions into cells at 37°C, but the mutant did not exhibit resistance in this assay. The mutant did not attach to or enter cells more quickly than did wild-type virus. Strikingly, incubation of wild-type virus with 1 to 2 μM ISIS 5652 at 37°C led to a time-dependent, irreversible loss of infectivity (virucidal activity). No virucidal activity was detected at 4°C or with an unrelated oligonucleotide at 37°C. The resistant mutant and a marker-rescued derivative containing its gB mutations exhibited substantial resistance to this virucidal activity of ISIS 5652. We hypothesize that the GT-rich oligonucleotide induces a conformational change in gB that results in inactivation of infectivity.

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