scholarly journals The Polyserine Tract of Herpes Simplex Virus ICP4 Is Required for Normal Viral Gene Expression and Growth in Murine Trigeminal Ganglia

1998 ◽  
Vol 72 (9) ◽  
pp. 7115-7124 ◽  
Author(s):  
Patricia A. Bates ◽  
Neal A. DeLuca
Keyword(s):  
Gene Expression ◽  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Productive Infection ◽  
Wild Type Virus ◽  
Trigeminal Ganglia ◽  
Type Virus ◽  
Wild Type ◽  
Low Levels ◽  
Simplex Virus

ABSTRACT ICP4 of herpes simplex virus (HSV) is essential for productive infection due to its central role in the regulation of HSV transcription. This study identified a region of ICP4 that is not required for viral growth in culture or at the periphery of experimentally inoculated mice but is critical for productive growth in the trigeminal ganglia. This region of ICP4 encompasses amino acids 184 to 198 and contains 13 nearly contiguous serine residues that are highly conserved among the alphaherpesviruses. A mutant in which this region is deleted (ΔSER) was able to grow on the corneas of mice and be transported back to the trigeminal ganglia. ΔSER did not grow in the trigeminal ganglia but did express low levels of several immediate-early (ICP4 and ICP27) and early (thymidine kinase [tk] and UL42) genes. It expressed very low levels of the late gC gene and did not appear to replicate DNA. This pattern of gene expression was similar to that observed for a tk mutant,dlsptk. Both ΔSER and dlsptk expressed higher levels of the latency-associated transcript (LAT) per genome earlier in infected ganglia than did the wild-type virus, KOS. However, infected ganglia from all three viruses accumulated the same level of LAT per genome at 30 days postinfection (during latency). The data suggest that the polyserine tract of ICP4 provides an activity that is required for lytic infection in ganglia to progress to viral DNA synthesis and full lytic gene expression. In the absence of this activity, higher levels of LAT per genome accumulate earlier in infection than with wild-type virus.

scholarly journals Replication of Herpes Simplex Virus 1 Depends on the γ134.5 Functions That Facilitate Virus Response to Interferon and Egress in the Different Stages of Productive Infection

2004 ◽  
Vol 78 (14) ◽  
pp. 7653-7666 ◽  
Author(s):  
Xianghong Jing ◽  
Melissa Cerveny ◽  
Kui Yang ◽  
Bin He
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Productive Infection ◽  
Wild Type Virus ◽  
Compensatory Mutation ◽  
Type Virus ◽  
Ns1 Protein ◽  
Wild Type ◽  
Amino Terminal ◽  
Simplex Virus

ABSTRACT The ability of the γ134.5 protein to suppress the PKR response plays a crucial role in herpes simplex virus pathogenesis. In this process, the γ134.5 protein associates with protein phosphatase 1 to form a large complex that dephosphorylates eIF-2α and thereby prevents translation shutoff mediated by PKR. Accordingly, γ134.5 null mutants are virulent in PKR-knockout mice but not in wild-type mice. However, γ134.5 deletion mutants, with an extragenic compensatory mutation, inhibit PKR activity but remain avirulent, suggesting that the γ134.5 protein has additional functions. Here, we show that a substitution of the γ134.5 gene with the NS1 gene from influenza A virus renders viral resistance to interferon involving PKR. The virus replicates as efficiently as wild-type virus in SK-N-SH and CV-1 cells. However, in mouse 3T6 cells, the virus expressing the NS1 protein grows at an intermediate level between the wild-type virus and the γ134.5 deletion mutant. This decrease in growth, compared to that of the wild-type virus, is due not to an inhibition of viral protein synthesis but rather to a block in virus release or egress. Virus particles are predominantly present in the nucleus and cytoplasm. Notably, deletions in the amino terminus of the γ134.5 protein lead to a significant decrease in virus growth in mouse 3T6 cells, which is independent of eIF-2α dephosphorylation. In correlation, a series of deletions in the amino-terminal domain impair nuclear as well as cytoplasmic egress. These results indicate that efficient viral replication depends on the γ134.5 functions required to prevent the PKR response and to facilitate virus egress in the different stages during virus infection.

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 Failure of Thymidine Kinase-Negative Herpes Simplex Virus To Reactivate from Latency following Efficient Establishment

2004 ◽  
Vol 78 (1) ◽  
pp. 520-523 ◽  
Author(s):  
Shih-Heng Chen ◽  
Angela Pearson ◽  
Donald M. Coen ◽  
Shun-Hua Chen
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Thymidine Kinase ◽  
Wild Type Virus ◽  
Trigeminal Ganglia ◽  
Type Virus ◽  
Wild Type ◽  
Viral Loads ◽  
Simplex Virus

ABSTRACT Thymidine kinase-negative mutants of herpes simplex virus did not reactivate from latency in mouse trigeminal ganglia, even when their latent viral loads were comparable to those that permitted reactivation by wild-type virus. Thus, reduced establishment of latency does not suffice to account for the failure to reactivate.

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.

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 Competition and complementation between thymidine kinase-negative and wild-type herpes simplex virus during co-infection of mouse trigeminal ganglia

2006 ◽  
Vol 87 (12) ◽  
pp. 3495-3502 ◽  
Author(s):  
Shih-Heng Chen ◽  
Yu-Wen Lin ◽  
Anthony Griffiths ◽  
Wen-Yen Huang ◽  
Shun-Hua Chen
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Thymidine Kinase ◽  
Antiviral Drug ◽  
Wild Type Virus ◽  
Trigeminal Ganglia ◽  
Wild Type ◽  
Viral Genomes ◽  
Simplex Virus

Laboratory strains of herpes simplex virus lacking thymidine kinase (TK) cannot replicate acutely to detectable levels in mouse trigeminal ganglia and do not reactivate from latency. However, many pathogenic clinical isolates that are resistant to the antiviral drug acyclovir are heterogeneous populations of TK-negative (TK−) and TK-positive (TK+) viruses. To recapitulate this in vivo, mice were infected with mixtures of wild-type virus and a recombinant TK− mutant in various ratios. Following co-infection, the replication, number of latent viral genomes and reactivation efficiency of TK+ virus in trigeminal ganglia were reduced in a manner related to the amount of TK− virus in the inoculum. TK+ virus did not always complement the acute replication or increase the number of latent viral genomes of TK− mutant in mouse ganglia. Even so, TK+ virus could still confer the pathogenic phenotype to a TK− mutant, somehow providing sufficient TK activity in trans to permit a TK− mutant to reactivate from latently infected ganglia.

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 Accumulation of Viral Transcripts and DNA during Establishment of Latency by Herpes Simplex Virus

1998 ◽  
Vol 72 (2) ◽  
pp. 1177-1185 ◽  
Author(s):  
Martha F. Kramer ◽  
Shun-Hua Chen ◽  
David M. Knipe ◽  
Donald M. Coen
Keyword(s):  
Gene Expression ◽  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Thymidine Kinase ◽  
Latent Infection ◽  
Acute Infection ◽  
Mutant Virus ◽  
Wild Type Virus ◽  
Wild Type ◽  
Simplex Virus

ABSTRACT Latent infection of mice with wild-type herpes simplex virus is established during an acute phase of ganglionic infection in which there is abundant viral replication and productive-cycle gene expression. Thymidine kinase-negative mutants establish latent infections but are severely impaired for acute ganglionic replication and productive-cycle gene expression. Indeed, by in situ hybridization assays, acute infection by these mutants resembles latency. To assess events during establishment of latency by wild-type and thymidine kinase-negative viruses, we quantified specific viral nucleic acid sequences in mouse trigeminal ganglia during acute ganglionic infection by using sensitive PCR-based assays. Through 32 h postinfection, viral DNA and transcripts representative of the three kinetic classes of productive-cycle genes accumulated to comparable levels in wild-type- and mutant-infected ganglia. At 48 and 72 h, although latency-associated transcripts accumulated to comparable levels in ganglia infected with wild-type or mutant virus, levels of DNA accumulating in wild-type-infected ganglia exceeded those in mutant-infected ganglia by 2 to 3 orders of magnitude. Coincident with this increase in DNA, wild-type-infected ganglia exhibited abundant expression of productive-cycle genes and high titers of infectious progeny. Nevertheless, the levels of productive-cycle RNAs expressed by mutant virus during acute infection greatly exceeded those expressed by wild-type virus during latency. The results thus distinguish acute infection of ganglia by a replication-compromised mutant from latent infection and may have implications for mechanisms of latency.

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 Recombinant Herpes Simplex Virus Type 1 Expressing Murine Interleukin-4 Is Less Virulent than Wild-Type Virus in Mice

2001 ◽  
Vol 75 (19) ◽  
pp. 9029-9036 ◽  
Author(s):  
Homayon Ghiasi ◽  
Yanira Osorio ◽  
Guey-Chuen Perng ◽  
Anthony B. Nesburn ◽  
Steven L. Wechsler
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Herpes Simplex Virus Type ◽  
Interleukin 4 ◽  
Wild Type Virus ◽  
Type Virus ◽  
Wild Type ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACT The effect of interleukin-4 (IL-4) on herpes simplex virus type 1 (HSV-1) infection in mice was evaluated by construction of a recombinant HSV-1 expressing the gene for murine IL-4 in place of the latency-associated transcript (LAT). The mutant virus (HSV-IL-4) expressed high levels of IL-4 in cultured cells. The replication of HSV-IL-4 in tissue culture and in trigeminal ganglia was similar to that of wild-type virus. In contrast, HSV-IL-4 appeared to replicate less well in mouse eyes and brains. Although BALB/c mice are highly susceptible to HSV-1 infection, ocular infection with HSV-IL-4 resulted in 100% survival. Furthermore, 57% of the mice survived coinfection with a mixture of HSV-IL-4 and a lethal dose of wild-type McKrae, compared with only 10% survival following infection with McKrae alone. Similar to wild-type BALB/c mice, 100% of IL-4−/− mice also survived HSV-IL-4 infection. T-cell depletion studies suggested that protection against HSV-IL-4 infection was mediated by a CD4+-T-cell response.

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