Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.5.1.18 (glutathione S-transferase)
22,582 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

During lytic infections, the virion host shutoff (Vhs) protein of herpes simplex virus accelerates the degradation of both host and viral mRNAs. In so doing, it helps redirect the cell from host to viral protein synthesis and facilitates the sequential expression of different viral genes. Vhs interacts with the cellular translation initiation factor eIF4H, and several point mutations that abolish its mRNA degradative activity also abrogate its ability to bind eIF4H. In addition, a complex containing bacterially expressed Vhs and a glutathione S-transferase (GST)-eIF4H fusion protein has RNase activity. eIF4H shares a region of sequence homology with eIF4B, and it appears to be functionally similar in that both stimulate the RNA helicase activity of eIF4A, a component of the mRNA cap-binding complex eIF4F. We show that eIF4H interacts physically with eIF4A in the yeast two-hybrid system and in GST pull-down assays and that the two proteins can be coimmunoprecipitated from mammalian cells. Vhs also interacts with eIF4A in GST pull-down and coimmunoprecipitation assays. Site-directed mutagenesis of Vhs and eIF4H revealed residues of each that are important for their mutual interaction, but not for their interaction with eIF4A. Thus, Vhs, eIF4H, and eIF4A comprise a group of proteins, each of which is able to interact directly with the other two. Whether they interact simultaneously as a tripartite complex or sequentially is unclear. The data suggest a mechanism for linking the degradation of an mRNA to its translation and for targeting Vhs to mRNAs and to regions of translation initiation.
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PMID:mRNA decay during herpes simplex virus (HSV) infections: protein-protein interactions involving the HSV virion host shutoff protein and translation factors eIF4H and eIF4A. 1601 27

The herpes simplex virus 1 ORF U(L)41 encodes a protein (virion host shutoff or vhs) associated with selective degradation of mRNA early in infection. Some mRNAs, exemplified by GAPDH or beta-actin mRNAs, are degraded rapidly. Others, for example IEX-1 mRNA, are degraded in two stages: whereas the 3' domain disappears rapidly, a large 5' domain fragment of the mRNA lingers for several hours. Still a third, exemplified by tristetraprolin mRNA, is not degraded, allowing its protein product to accumulate in infected cells. Here we report the following: (i) a GST-vhs protein produced in Escherichia coli, solubilized and purified to homogeneity acts as bona fide endoribonuclease when tested on in vitro transcribed IEX-1 probes. A GST-vhs protein in which three key vhs amino acids were replaced with alanines, solubilized and purified by the same protocol, had no enzymatic activity. (ii) The number of fragments generated by cleavage of a truncated IEX-1 RNA by vhs appears to be small; the cleavage sites are centered at or near the AU-rich elements located at the 3' untranslated region of the mRNA. A truncated RNA containing only the IEX-1 coding domain was cleaved numerous times. (iii) In cells infected at high multiplicity and exposed to a large number of particles per cell, the vhs protein accumulated within 3 h after infection, in small uniform cytoplasmic granules raising the possibility that vhs colocalizes with tristerapolin, a protein induced after infection, in structures involved in degradation of RNA.
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PMID:The U(L)41 protein of herpes simplex virus 1 degrades RNA by endonucleolytic cleavage in absence of other cellular or viral proteins. 1647 41

The varicella-zoster virus (VZV) ORF9 protein is a member of the herpesvirus UL49 gene family but shares limited identity and similarity with the UL49 prototype, herpes simplex virus type 1 VP22. ORF9 mRNA is the most abundantly expressed message during VZV infection; however, little is known concerning the functions of the ORF9 protein. We have found that the VZV major transactivator IE62 and the ORF9 protein can be coprecipitated from infected cells. Yeast two-hybrid analysis localized the region of the ORF9 protein required for interaction with IE62 to the middle third of the protein encompassing amino acids 117 to 186. Protein pull-down assays with GST-IE62 fusion proteins containing N-terminal IE62 sequences showed that amino acids 1 to 43 of the acidic transcriptional activation domain of IE62 can bind recombinant ORF9 protein. Confocal microscopy of transiently transfected cells showed that in the absence of other viral proteins, the ORF9 protein was localized in the cytoplasm while IE62 was localized in the nucleus. In VZV-infected cells, the ORF9 protein was localized to the cytoplasm whereas IE62 exhibited both nuclear and cytoplasmic localization. Cotransfection of plasmids expressing ORF9, IE62, and the viral ORF66 kinase resulted in significant colocalization of ORF9 and IE62 in the cytoplasm. Coimmunoprecipitation experiments with antitubulin antibodies indicate the presence of ORF9-IE62-tubulin complexes in infected cells. Colocalization of ORF9 and tubulin in transfected cells was visualized by confocal microscopy. These data suggest a model for ORF9 protein function involving complex formation with IE62 and possibly other tegument proteins in the cytoplasm at late times in infection.
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PMID:The varicella-zoster virus (VZV) ORF9 protein interacts with the IE62 major VZV transactivator. 1707 4

The DP71L protein of African swine fever virus (ASFV) shares sequence similarity with the herpes simplex virus ICP34.5 protein over a C-terminal domain. We showed that the catalytic subunit of protein phosphatase 1 (PP1) interacts specifically with the ASFV DP71L protein in a yeast two-hybrid screen. The chimeric full-length DP71L protein, from ASFV strain Badajoz 71 (BA71V), fused to glutathione S-transferase (DP71L-GST) was expressed in Escherichia coli and shown to bind specifically to the PP1-alpha catalytic subunit expressed as a histidine fusion protein (6xHis-PP1alpha) in E. coli. The functional effects of this interaction were investigated by measuring the levels of PP1 and PP2A in ASFV-infected Vero cells. This showed that infection with wild-type ASFV strain BA71V activated PP1 between two- and threefold over that of mock-infected cells. This activation did not occur in cells infected with the BA71V isolate in which the DP71L gene had been deleted, suggesting that expression of DP71L leads to PP1 activation. In contrast, no effect was observed on the activity of PP2A following ASFV infection. We showed that infection of cells with wild-type BA71V virus resulted in decreased phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF-2alpha). ICP34.5 recruits PP1 to dephosphorylate the alpha subunit of eukaryotic translational initiation factor 2 (also known as eIF-2alpha); possibly the ASFV DP71L protein has a similar function.
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PMID:The MyD116 African swine fever virus homologue interacts with the catalytic subunit of protein phosphatase 1 and activates its phosphatase activity. 1721 79

The US3 protein kinase of herpes simplex virus 1 blocks apoptosis induced by replication-incompetent virus mutants, proapoptotic members of the Bcl-2 family of proteins, and by a variety of other agents that act at the premitochondrial level in the proapoptotic cascade. To define the role of US3 in blocking apoptosis at the postmitochondrial level, we investigated the US3 protein kinase in transduced cells that were either transfected with a plasmid encoding procaspase 3 or superinfected with a proapoptotic mutant virus lacking the gene encoding the infected cell protein no. 4. (i) We show that US3 blocks the proteolytic cleavage that generates active caspase 3 from the transfected zymogen procaspase 3, concomitant with inhibition of apoptosis. (ii) Studies based on detection of fluorescence emitted upon cleavage of a synthetic caspase 3 substrate showed that expression of the US3 kinase and appearance of the cleaved substrate were mutually exclusive. (iii) An affinity-purified glutathione S-transferase (GST)-US3 fusion protein, but not the inactive GST-US3(K220N) protein, phosphorylated procaspase 3 in vitro. The studies published earlier on the effect of US3 on the upstream regulatory proteins and current studies suggest that the US3 protein kinase may act on several proteins in the proapoptotic cascade to enable the virus to complete its replication.
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PMID:In transduced cells, the US3 protein kinase of herpes simplex virus 1 precludes activation and induction of apoptosis by transfected procaspase 3. 1763 20

The putative DNA encapsidation genes encoded by open reading frames (ORFs) 25, 26, 30, 34, 43, 45/42 and 54 were cloned from Varicella-zoster virus (VZV) strain Ellen. Sequencing revealed that the Ellen ORFs were highly conserved at the amino acid level when compared to those of 19 previously published VZV isolates. Additionally, RT-PCR provided the first evidence that ORF45/42 was expressed as a spliced transcript in VZV-infected cells. All seven ORFs were expressed in vitro and full length products were identified using a C-terminal V5 epitope tag. The in vitro products of the putative VZV terminase subunits encoded by ORFs 30 and 45/42 proved useful in protein-protein interaction assays. Previous studies have reported the formation of a heterodimeric terminase complex involved in DNA encapsidation for both herpes simplex virus-type 1 (HSV-1) and human cytomegalovirus (HCMV). Here we report that the C-terminal portion of exon II of ORF45/42 (ORF42-C269) interacted in GST-pull down experiments with in vitro synthesized ORF30 and ORF45/42. The interactions were maintained in the presence of anionic detergents and in buffers of increasing ionic strength. Cells transiently transfected with epitope tagged ORF45/42 or ORF30 showed primarily cytoplasmic staining. In contrast, an antiserum directed to the N-terminal portion of ORF45 showed nearly exclusive nuclear localization of the ORF45/42 gene product in infected cells. An ORF30 specific antiserum detected an 87 kDa protein in both the cytoplasmic and nuclear fractions of VZV infected cells. The results were consistent with the localization and function of herpesviral terminase subunits. This is the first study aimed at the identification and characterization of the VZV DNA encapsidation gene products.
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PMID:The Varicella-zoster virus DNA encapsidation genes: Identification and characterization of the putative terminase subunits. 1786 47

Herpes simplex virus type 1 (HSV-1) virions contain a proteinaceous layer termed the tegument that lies between the nucleocapsid and viral envelope. The mechanisms underlying tegumentation remain largely undefined for all herpesviruses. Using glutathione S-transferase (GST) pulldowns and coimmunoprecipitation studies, we have identified a domain of the tegument protein VP22 that facilitates interaction with VP16. This region of VP22 (residues 165-225) overlaps the glycoprotein E (gE) binding domain of VP22 (residues 165-270), which is sufficient to mediate VP22 packaging into assembling virus particles. To ascertain the contribution of the VP16 and gE binding activities of VP22 to its virion incorporation, a transfection/infection based virion incorporation assay, using point mutants that discern between the two binding activities, was utilized. Our results suggest that interaction with VP16 is not required for incorporation of VP22 into virus particles and that binding to the cytoplasmic tail of gE is sufficient to facilitate packaging.
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PMID:Incorporation of the herpes simplex virus type 1 tegument protein VP22 into the virus particle is independent of interaction with VP16. 1788 78

Herpes simplex virus type 1 glycoprotein K (gK) and the UL20 protein (UL20p) are coordinately transported to the trans-Golgi network (TGN) and cell surfaces and are required for cytoplasmic virion envelopment at the TGN. In addition, cell surface expression of gK and UL20p is required for virus-induced cell fusion. Previously, confocal microscopy colocalization and intracellular transport experiments strongly suggested direct protein-protein interactions between gK and UL20p. Direct protein-protein interactions between gK and UL20p were demonstrated through reciprocal coimmunoprecipitation experiments, as well as with glutathione S-transferase (GST) pull-down experiments. A fusion protein consisting of the amino-terminal 66 amino acids of UL20p fused in-frame with GST was expressed in Escherichia coli and purified via glutathione column chromatography. Precipitation of GST-UL20p from mixtures of GST-UL20p fusion protein with cellular extracts containing gK specifically coprecipitated gK but not other viral glycoproteins. The purified UL20p-GST fusion protein reacted with all gK-associated protein species. It was concluded that the amino terminus of UL20p, most likely, interacted with gK domain III, which is predicted to lie intracellularly. UL20p-gK domain-specific interactions must serve important functions in the coordinate transport of UL20p and gK to the TGN, because retention of UL20p in the endoplasmic reticulum (ER) via the addition of an ER retention signal at the carboxyl terminus of UL20p forced the ER retention of gK and drastically inhibited intracellular virion envelopment and virus-induced cell fusion.
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PMID:Functional and physical interactions of the herpes simplex virus type 1 UL20 membrane protein with glycoprotein K. 1843 1

The molecular mechanisms responsible for the addition of tegument proteins into nascent herpesvirus particles are poorly understood. To better understand the tegumentation process of herpes simplex virus type 1 (HSV-1) virions, we initiated studies that showed the tegument protein pUL46 (VP11/12) has a similar cellular localization to the membrane-associated tegument protein VP22. Using membrane flotation analysis we found that pUL46 associates with membranes in both the presence and absence of other HSV-1 proteins. However, when purified virions were stripped of their envelope, the majority of pUL46 was found to associate with the capsid fraction. This strong affinity of pUL46 for capsids was confirmed by an in vitro capsid pull-down assay in which purified pUL46-GST was able to interact specifically with capsids purified from the nuclear fraction of HSV-1 infected cells. These results suggest that pUL46 displays a dynamic interaction between cellular membranes and capsids.
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PMID:The HSV-1 tegument protein pUL46 associates with cellular membranes and viral capsids. 1845 63

Previous results indicated that the U(L)34 protein (pU(L)34) of herpes simplex virus 1 (HSV-1) is targeted to the nuclear membrane and is essential for nuclear egress of nucleocapsids. The normal localization of pU(L)34 and virions requires the U(S)3-encoded kinase that phosphorylates U(L)34 and lamin A/C. Moreover, pU(L)34 was shown to interact with lamin A in vitro. In the present study, glutathione S-transferase/pU(L)34 was shown to specifically pull down lamin A and lamin B1 from cellular lysates. To determine the role of these interactions on viral infectivity and pU(L)34 targeting to the inner nuclear membrane (INM), the localization of pU(L)34 was determined in LmnA(-/-) and LmnB1(-/-) mouse embryonic fibroblasts (MEFs) by indirect immunofluorescence and immunogold electron microscopy in the presence or absence of U(S)3 kinase activity. While pU(L)34 INM targeting was not affected by the absence of lamin B1 in MEFs infected with wild-type HSV as viewed by indirect immunofluorescence, it localized in densely staining scalloped-shaped distortions of the nuclear membrane in lamin B1 knockout cells infected with a U(S)3 kinase-dead virus. Lamin B1 knockout cells were relatively less permissive for viral replication than wild-type MEFs, with viral titers decreased at least 10-fold. The absence of lamin A (i) caused clustering of pU(L)34 in the nuclear rim of cells infected with wild-type virus, (ii) produced extensions of the INM bearing pU(L)34 protein in cells infected with a U(S)3 kinase-dead mutant, (iii) precluded accumulation of virions in the perinuclear space of cells infected with this mutant, and (iv) partially restored replication of this virus. The latter observation suggests that lamin A normally impedes viral infectivity and that U(S)3 kinase activity partially alleviates this impediment. On the other hand, lamin B1 is necessary for optimal viral replication, probably through its well-documented effects on many cellular pathways. Finally, neither lamin A nor B1 was absolutely required for targeting pU(L)34 to the INM, suggesting that this targeting is mediated by redundant functions or can be mediated by other proteins.
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PMID:Effects of lamin A/C, lamin B1, and viral US3 kinase activity on viral infectivity, virion egress, and the targeting of herpes simplex virus U(L)34-encoded protein to the inner nuclear membrane. 1852 19


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