scholarly journals Mapping of Key Functions of the Herpes Simplex Virus 1 US3 Protein Kinase: the US3 Protein Can Form Functional Heteromultimeric Structures Derived from Overlapping Truncated Polypeptides

2006 ◽  
Vol 81 (4) ◽  
pp. 1980-1989 ◽  
Author(s):  
Alice P. W. Poon ◽  
Bernard Roizman
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Herpes Simplex Virus 1 ◽  
Histone Deacetylase 1 ◽  
Amino Terminal ◽  
Helper Function ◽  
Antiapoptotic Activity ◽  
Carboxyl Terminal ◽  
Simplex Virus ◽  
In Cells

ABSTRACT Earlier studies have shown that the herpes simplex virus (HSV) US3 encodes two transcriptional units directing the synthesis of the US3 (residues 1 to 481) and US3.5 (residues 77 to 481) protein kinases. Both kinases phosphorylate histone deacetylase 1 (HDAC1) and HDAC2 and enable the expression of genes cotransduced into U2OS cells by recombinant baculoviruses, an activity designated the “helper function.” The two kinases differ with respect to antiapoptotic activity. In the studies reported here, we made a series of FLAG-tagged amino- and carboxyl-terminal truncations of US3 and these were tested for antiapoptotic activity, phosphorylation of HDAC1, and the helper function. We report the following. (i) HDAC1 phosphorylation and the helper function were expressed in cells transduced by the truncation encoding residues 182 to 481 but not in cells transduced by the truncation encoding residues 189 to 481 or the amino-terminal polypeptides encompassing the first 188 amino acids. (ii) The self-posttranslational modification requires residues 164 to 481. (iii) The antiapoptotic activity requires both the amino-terminal and the carboxyl-terminal domains, of which the truncated protein containing residues 1 to 163 and that containing residues 164 to 481, respectively, were the smallest fragments tested to be effective. The two domains need not be on the same molecule, but they must overlap. The smallest overlapping pair tested was the fragment containing residues 1 to 181 and that containing residues 164 to 481. Consistent with the hypothesis that the effective overlapping truncations form a heteromultimeric structure, antibody to FLAG coprecipitated untagged US3 from lysates of cells cotransduced with FLAG-tagged, truncated US3 constructs. Although US3 has been reported to be a monomeric enzyme, the results indicate that it can form enzymatically active multimeric structures.

scholarly journals cdc2 Cyclin-Dependent Kinase Binds and Phosphorylates Herpes Simplex Virus 1 UL42 DNA Synthesis Processivity Factor

2001 ◽  
Vol 75 (21) ◽  
pp. 10326-10333 ◽  
Author(s):  
Sunil J. Advani ◽  
Ralph R. Weichselbaum ◽  
Bernard Roizman
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Kinase Activity ◽  
Dominant Negative ◽  
Cdc2 Kinase ◽  
Herpes Simplex Virus 1 ◽  
Amino Terminal ◽  
Processivity Factor ◽  
Carboxyl Terminal ◽  
Simplex Virus

ABSTRACT Earlier studies have shown that cdc2 kinase is activated during herpes simplex virus 1 infection and that its activity is enhanced late in infection even though the levels of cyclin A and B are decreased below levels of detection. Furthermore, activation of cdc2 requires the presence of infected cell protein no. 22 and the UL13 protein kinase, the same gene products required for optimal expression of a subset of late genes exemplified by US11, UL38, and UL41. The possibility that the activation of cdc2 and expression of this subset may be connected emerged from the observation that dominant negative cdc2 specifically blocked the expression of US11 protein in cells infected and expressing dominant negative cdc2. Here we report that in the course of searching for a putative cognate partner for cdc2 that may have replaced cyclins A and B, we noted that the DNA polymerase processivity factor encoded by the UL42 gene contains a degenerate cyclin box and has been reported to be structurally related to proliferating cell nuclear antigen, which also binds cdk2. Consistent with this finding, we report that (i) UL42 is able to physically interact with cdc2 at both the amino-terminal and carboxyl-terminal domains, (ii) the carboxyl-terminal domain of UL42 can be phosphorylated by cdc2, (iii) immunoprecipitates obtained with anti UL42 antibody contained a roscovitine-sensitive kinase activity, (iv) kinase activity associated with UL42 could be immunodepleted by antibody to cdc2, and (v) UL42 transfected into cells associates with a nocodazole-enhanced kinase. We conclude that UL42 can associate with cdc2 and that the kinase activity has the characteristic traits of cdc2 kinase.

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 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 Functional Comparison of Herpes Simplex Virus 1 (HSV-1) and HSV-2 ICP27 Homologs Reveals a Role for ICP27 in Virion Release

2014 ◽  
Vol 89 (5) ◽  
pp. 2892-2905 ◽  
Author(s):  
Donglim Park ◽  
Joseph Lalli ◽  
Lenka Sedlackova-Slavikova ◽  
Stephen A. Rice
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Infected Cell ◽  
Regulatory Protein ◽  
Late Gene Expression ◽  
Herpes Simplex Virus 1 ◽  
Amino Terminal ◽  
Virion Release ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACTNumerous studies have focused on the regulatory functions of ICP27, an immediate-early (IE) protein of herpes simplex virus 1 (HSV-1). However, its homolog in HSV-2, termed ICP27t2, has been little studied. Here, we used two different approaches to functionally compare ICP27t2 and ICP27. In transfection-based assays, ICP27t2 closely resembled ICP27 in its capacity to enhance HSV-1 late gene expression, suppress the splicing of a viral intron, and complement the growth of an HSV-1 ICP27 null mutant. To study ICP27t2 in the context of viral infection, we engineered K2F1, an HSV-1 mutant that encodes ICP27t2 in place of ICP27. In Vero cells, K2F1 replicated with wild-type (WT) kinetics and yields, expressed delayed-early and late proteins normally, and was fully capable of activating several cellular signal transduction pathways that are ICP27 dependent. Thus, we conclude that ICP27t2 and ICP27 are functionally very similar and that ICP27t2 can mediate all ICP27 activities that are required for HSV-1 replication in cell culture. Surprisingly, however, we found that K2F1 forms plaques that are morphologically different from those of WT HSV-1. Investigation of this trait demonstrated that it results from the decreased release of progeny virions into the culture medium. This appears to be due to a reduction in the detachment of K2F1 progeny from the extracellular surface of the infected cell. We identified two HSV-1 ICP27 amino-terminal deletion mutants with a similar release defect. Together, these results demonstrate that ICP27 plays a heretofore-unappreciated role in modulating the efficiency of progeny virion release.IMPORTANCEICP27 is an essential, multifunctional regulatory protein that has a number of critical roles in the HSV-1 life cycle. Although ICP27 homologs are encoded by all known members of theHerpesviridae, previous work with several of these homologs has shown that they cannot substitute for ICP27 in the context of HSV-1-infected cells. Here, we identify ICP27t2 as the first homolog that can efficiently replace ICP27 in HSV-1 infection. Unexpectedly, our results also reveal that the sequence of the ICP27 gene can affect the release of HSV-1 progeny virions from the infected cell. Thus, our comparative study has revealed a novel function for ICP27 in the regulation of virus release.

scholarly journals Characterization of the triplet repeats in the central domain of the γ 134·5 protein of herpes simplex virus 1

2005 ◽  
Vol 86 (9) ◽  
pp. 2411-2419 ◽  
Author(s):  
Xianghong Jing ◽  
Bin He
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Triplet Repeats ◽  
Herpes Simplex Virus 1 ◽  
Central Domain ◽  
Amino Terminal ◽  
Terminal Domain ◽  
Different Strains ◽  
Simplex Virus ◽  
Hsv 1

The γ 134·5 protein of herpes simplex virus 1 (HSV-1) consists of an amino-terminal domain, a central domain with triplet repeats (Ala–Thr–Pro) and a carboxyl-terminal domain. The triplet repeats are a unique feature of the γ 134·5 protein encoded by HSV-1, but the number of repeats varies among different strains. Notably, the central domain containing the triplet repeats is implicated in neuroinvasion. In this report, it has been shown that partial or full deletion of triplet repeats, i.e. from ten to either three or zero, in the γ 134·5 protein has no effect on the virus response to interferon. The triplet deletion mutants replicate efficiently in CV-1 and mouse 10T1/2 cells. However, in mouse 3T6 cells, these mutants grow with delayed growth kinetics. This decrease in growth, compared with wild-type HSV-1(F), does not result from failure of the virus to suppress the RNA-dependent protein kinase response, but rather from a delay in virus release or egress. Accordingly, these mutant viruses are predominantly present within infected cells. These results indicate that deletions in the central domain of the γ 134·5 protein impair virus egress, but not virus response to interferon.

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.

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