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 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 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 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 Requirements for the Nuclear-Cytoplasmic Translocation of Infected-Cell Protein 0 of Herpes Simplex Virus 1

2001 ◽  
Vol 75 (8) ◽  
pp. 3832-3840 ◽  
Author(s):  
Pascal Lopez ◽  
Charles Van Sant ◽  
Bernard Roizman
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Regulatory Protein ◽  
Wild Type Virus ◽  
Cell Protein ◽  
Type Virus ◽  
Wild Type ◽  
Infected Cells ◽  
Infected Cell Protein ◽  
Simplex Virus

ABSTRACT Earlier studies have shown that wild-type infected-cell protein 0 (ICP0), a key herpes simplex virus regulatory protein, translocates from the nucleus to the cytoplasm of human embryonic lung (HEL) fibroblasts within several hours after infection (Y. Kawaguchi, R. Bruni, and B. Roizman, J. Virol. 71:1019–1024, 1997). Translocation of ICP0 was also observed in cells infected with thed120 mutant, in which both copies of the gene encoding ICP4, the major regulatory protein, had been deleted (V. Galvan, R. Brandimarti, J. Munger, and B. Roizman, J. Virol. 74:1931–1938, 2000). Furthermore, a mutant (R7914) carrying the D199A substitution in ICP0 does not bind or stabilize cyclin D3 and is retained in the nucleus (C. Van Sant, P. Lopez, S. J. Advani, and B. Roizman, J. Virol. 75:1888–1898, 2001). Studies designed to elucidate the requirements for the translocation of ICP0 between cellular compartments revealed the following. (i) Translocation of ICP0 to the cytoplasm in productive infection maps to the D199 amino acid, inasmuch as wild-type ICP0 delivered in trans to cells infected with an ICP0 null mutant was translocated to the cytoplasm whereas the D199A-substituted mutant ICP0 was not. (ii) Translocation of wild-type ICP0 requires a function expressed late in infection, inasmuch as phosphonoacetate blocked the translocation of ICP0 in wild-type virus-infected cells but not in d120 mutant-infected cells. Moreover, whereas in d120 mutant-infected cells ICP0 was translocated rapidly from the cytoplasm to the nucleus at approximately 5 h after infection, the translocation of ICP0 in wild-type virus-infected cells extended from 5 to at least 9 h after infection. (iii) In wild-type virus-infected cells, the MG132 proteasomal inhibitor blocked the translocation of ICP0 to the cytoplasm early in infection, but when added late in infection, it caused ICP0 to be relocated back to the nucleus from the cytoplasm. (iv) MG132 blocked the translocation of ICP0 in d120 mutant-infected cells early in infection but had no effect on the ICP0 aggregated in vesicle-like structures late in infection. However, ind120 mutant-infected cells treated with MG132 at late times, proteasomes formed a shell-like structure around the aggregated ICP0. These structures were not seen in wild-type virus or R7914 mutant-infected cells. The results indicate the following. (i) In the absence of β or γ protein synthesis, ICP0 dynamically associates with proteasomes and is translocated to the cytoplasm. (ii) In cells productively infected beyond α gene expression, ICP0 is retained in the nucleus until after the onset of viral DNA synthesis and the synthesis of γ2 proteins. (iii) Late in infection, ICP0 is actively sequestered in the cytoplasm by a process mediated by proteasomes, inasmuch as interference with proteasomal function causes rapid relocation of ICP0 to the nucleus.

scholarly journals Role of Cellular Phosphatase cdc25C in Herpes Simplex Virus 1 Replication

2008 ◽  
Vol 82 (9) ◽  
pp. 4527-4532 ◽  
Author(s):  
Benjamin A. Smith-Donald ◽  
Lizette O. Durand ◽  
Bernard Roizman
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Cyclin B1 ◽  
Wild Type Virus ◽  
Physical Interaction ◽  
Wild Type ◽  
Herpes Simplex Virus 1 ◽  
Infected Cells ◽  
Simplex Virus

ABSTRACT Earlier studies have shown that in herpes simplex virus 1-infected cells, ICP22 upregulates the accumulation of a subset of γ2 proteins exemplified by the products of the UL38, UL41, and US11 genes. The ICP22-dependent process involves degradation of cyclins A and B1, the stabilization and activation of cdc2, physical interaction of activated cdc2 with the UL42 DNA synthesis processivity factor, and recruitment and phosphorylation of topoisomerase IIα by the cdc2/UL42 complex. Activation of cdc2, the first step in the process, is a key function of the mitotic phosphatase cdc25C. To define the role of cdc25C, we probed some features of the ICP22-dependent pathway of upregulation of γ2 genes in cdc25C−/− cells and in cdc25C+/+ cells derived from sibling mice. We report that cyclin B1 turned over in cdc25C+/+ or cdc25C−/− cells at the same rate, that cdc2 increased in amount, and that US11 and UL38 proteins and infectious virus accumulated in smaller amounts than in wild-type infected cells. The reduction in UL38 protein accumulation and virus was greater in cdc25C−/− cells infected with virus lacking ICP22 than in cells infected with wild-type virus. We conclude that cdc25C phosphatase plays a role in viral replication and that this role extends beyond its function of activating cdc2 for initiation of the ICP22-dependent cascade for upregulation of γ2 gene expression.

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 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 The Herpes Simplex Virus Type 1 US11 Protein Binds the Coterminal UL12, UL13, and UL14 RNAs and Regulates UL13 Expression In Vivo

2002 ◽  
Vol 76 (16) ◽  
pp. 8090-8100 ◽  
Author(s):  
Helen L. Attrill ◽  
Sarah A. Cumming ◽  
J. Barklie Clements ◽  
Sheila V. Graham
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Wild Type Virus ◽  
Type Virus ◽  
Wild Type ◽  
Infected Cells ◽  
Simplex Virus ◽  
Us11 Protein ◽  
Hsv 1

ABSTRACT The US11 protein of herpes simplex virus type 1 (HSV-1) is a small, highly basic phosphoprotein expressed at late times during infection. US11 localizes to the nucleolus in infected cells, can associate with ribosomes, and has been shown to bind RNA. The RNA substrates of US11 identified thus far have no apparent role in the virus lytic cycle, so we set out to identify a novel, biologically relevant RNA substrate(s) for this protein in HSV-1-infected cells. We designed a reverse transcriptase PCR-based protocol that allowed specific selection of a 600-bp RNA binding partner for US11. This RNA sequence, designated 12/14, is present in the coterminal HSV-1 mRNAs UL12, UL13, and UL14. We show that the binding of US11 to 12/14 is sequence-specific and mediated by the C-terminal domain of the protein. To elucidate the role of US11 in the virus life cycle, we infected cells with wild-type virus, a cosmid-reconstructed US11 HSV-1 null mutant, and a cosmid-reconstructed wild-type virus and analyzed expression of UL12, -13, and -14 during a time course of infection. These experiments revealed that this interaction has biological activity; at early times of infection, US11 down-regulates UL13 protein kinase mRNA and protein.

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.

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