The Epstein Barr Virus Genome Encodes Deoxythymidine Kinase Activity in a Nested Internal Open Reading Frame

Holton, Ronald H.; Gentry, Glenn A.

doi:10.1159/000150528

Cited by 7 publications

(12 citation statements)

References 9 publications

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“…We note that the N-terminal regions of gammaherpesviruses contain a domain of several hundred amino acids which have no homology to other herpesvirus TKs (17). These domains are not closely related between EBV and HHV8, and computer search reveals no recognizable motifs (11,44), although Pro, Gly, and Ser constitute 33% of the various domains.…”

Section: Hhv8 Orf 21 Has Tk Activity In Mammalian Cells To Determinementioning

confidence: 82%

Human Herpesvirus 8 Open Reading Frame 21 Is a Thymidine and Thymidylate Kinase of Narrow Substrate Specificity That Efficiently Phosphorylates Zidovudine but Not Ganciclovir

Gustafson

Schinazi

Fingeroth

2000

J Virol

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Human herpesvirus 8 (HHV8) open reading frame (ORF) 21 is predicted to encode a protein similar to the thymidine kinase (TK) enzyme of other herpesviruses. Expressed in mammalian cells, ORF 21 was found to have low TK activity, based on poor growth in media containing hypoxanthine-aminopterin-thymidine (HAT) and low incorporation of [3 H]thymidine into high-molecular-weight DNA. Kinetic analysis using HHV8 TK as a purified glutathione S-transferase (GST) fusion protein showed that the enzyme has a comparatively high K m for thymidine (dThd) of ϳ33.2 M. Nearly 50% of the phosphorylated product of the reaction with dThd was thymidylate. This monophosphate kinase activity was more pronounced with 3-azido-3-deoxythymidine (AZT), in which 78% of the reaction product was AZT diphosphate. Thymidine analogs competitively inhibited dThd phosphorylation by HHV8 TK, while 2-deoxyguanosine, 2-deoxyadenosine, 2-deoxycytidine, and corresponding analogs did not. Further competition experiments revealed that the nucleoside analog ganciclovir (GCV), at up to 1,000-fold molar excess, could not significantly inhibit dThd phosphorylation by the enzyme. In support of these data, 143B TK ؊ cells expressing HHV8 TK phosphorylated GCV very poorly and were not susceptible to GCV toxicity compared to parental cells. Phosphorylation of [ 3 H]GCV by a purified GST-HHV8 TK fusion protein was not detected by high-pressure liquid chromatography analysis. Structural features of HHV8 TK substrate recognition were investigated. Therapeutic implications of these findings are discussed.Human herpesvirus type 8 (HHV8) is the second human herpesvirus classified in the gamma subfamily. HHV8 has been causally associated with Kaposi's sarcoma (KS) (6, 31), body cavity-based lymphoma (BCBL), also known as primary effusion lymphoma (5), and multicentric Castleman's disease (45). Though the virus is in a tightly latent state in the majority of infected cells (43, 51), it has been suggested that in KS lesions, lytic cycle gene products may be involved in the proliferation of neighboring cells, contributing to pathogenesis (46). A reduction in the number of virions would both directly and indirectly decrease the likelihood of transmission to cellular targets with the capacity to proliferate uncontrollably upon infection. Prophylactic antiviral therapy to limit in vivo lytic HHV8 replication, particularly in the T-cell-deficient host, may decrease the incidence of KS and other virus-associated diseases. Therefore, conventional antiviral strategies are being examined, both retrospectively and prospectively, as a way to prevent development of HHV8-associated diseases (2,9,14,20,21,28,39).A few studies have examined the effect of a limited number of antiviral drugs on HHV8 replication in vitro (22,29,33). Of the nucleosides that require a viral thymidine kinase (TK) for activation, ganciclovir (GCV), an acyclic guanine analog, was the most effective at inhibiting viral replication, while penciclovir (PCV) and acyclovir (ACV), also acyclic guanine analogs, and t...

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Section: Hhv8 Orf 21 Has Tk Activity In Mammalian Cells To Determinementioning

confidence: 82%

Human Herpesvirus 8 Open Reading Frame 21 Is a Thymidine and Thymidylate Kinase of Narrow Substrate Specificity That Efficiently Phosphorylates Zidovudine but Not Ganciclovir

Gustafson

Schinazi

Fingeroth

2000

J Virol

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“…In the case of Epstein-Barr virus (EBV), it has been shown that the presence of the N-terminal domain in vitro does not significantly alter the ability of the C-terminal enzyme to phosphorylate thymidine (20). Although all gammaherpesvirus TKs contain N-terminal domains with high G, S, and P content (21), the similarity between these domains is otherwise modest (4,15,23,30). Furthermore these N termini do not resemble other known proteins in current databases.…”

mentioning

confidence: 95%

Functional Divergence of Kaposi's Sarcoma-Associated Herpesvirus and Related Gamma-2 Herpesvirus Thymidine Kinases: Novel Cytoplasmic Phosphoproteins That Alter Cellular Morphology and Disrupt Adhesion

Gill

Murphy

Fingeroth

2005

J Virol

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The nucleoside kinase encoded by Kaposi's sarcoma-associated herpesvirus (KSHV) is a relatively inefficient enzyme with substrate specificity for thymidine alone, unlike alphaherpesvirus thymidine kinases (TKs). Similar to all gammaherpesvirus TKs, KSHV TK is composed of two distinct domains, a conserved C-terminal kinase and a novel and uncharacterized N terminus. Ectopic expression of KSHV TK in adherent cells induced striking morphological changes and anchorage independence although cells survived, a property shared with the related rhadinovirus TKs of rhesus monkey rhadinovirus and herpesvirus saimiri. To determine whether KSHV TK served alternate functions relevant to the rhadinovirus life cycle and to reveal the contribution of the N terminus, an enhanced green fluorescent protein-tagged fusion protein and serial mutants were generated for investigation of intracellular localization and cell biology. Analysis of truncation mutants showed that a proline-rich region located within the N terminus cooperated with the conserved C-terminal kinase to tether KSHV TK to a reticular network in the cytoplasm and to induce morphological change. Fusion of the KSHV N terminus to herpes simplex virus type 1 TK, a nucleus-localized enzyme, similarly resulted in cytoplasmic redistribution of the chimeric protein but did not alter cell shape or adhesion. Unlike other human herpesvirus TKs, KSHV TKs and related rhadinovirus TKs are constitutively tyrosine phosphorylated; a KSHV TK mutant that was hypophosphorylated failed to detach and grow in suspension. Loss of adhesion may enhance terminal differentiation, viral replication, and egress at the cellular level and at the organism level may facilitate detachment and distant migration of KSHV-replicating cells within body fluids-promoting oropharyngeal transmission and perhaps contributing to the multifocal lesions that characterize KS.

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“…EBV encodes a TK that shows sequence and functional homology with HSV-1 TK (22,24,26,27,53). The EBV TK is larger than the HSV-1 TK and encodes a 243-amino-acid N terminus whose function is unknown (22,26).…”

mentioning

confidence: 99%

“…The EBV TK is larger than the HSV-1 TK and encodes a 243-amino-acid N terminus whose function is unknown (22,26). The EBV protein, like its HSV-1 homologue, but unlike the homologues in HSV-2 and varicella-zoster virus, has both TK and thymidylate kinase activity (6,19).…”

mentioning

confidence: 99%

Induction of Epstein-Barr Virus Kinases To Sensitize Tumor Cells to Nucleoside Analogues

Moore¹,

Cannon²,

Tanhehco³

et al. 2001

Antimicrob Agents Chemother

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The presence of Epstein-Barr virus (EBV) in the tumor cells of some EBV-associated malignancies may facilitate selective killing of these tumor cells. We show that treatment of an EBV ؉ Burkitt's lymphoma cell line with 5-azacytidine led to a dose-dependent induction of EBV lytic antigen expression, including expression of the viral thymidine kinase (TK) and phosphotransferase (PT). Azacytidine treatment for 24 h modestly sensitized the cell line to all nucleosides tested. To better characterize EBV TK with regard to various nucleoside analogues, we expressed EBV TK in stable cell clones. Two EBV TK-expressing clones were moderately sensitive to high doses of acyclovir and penciclovir (PCV) (62.5 to 500 M) and to lower doses of ganciclovir (GCV) and bromovinyldeoxyuridine (BVdU) (10 to 100 M) compared to a control clone and were shown to phosphorylate GCV. Similar experiments in a transient overexpression system showed more killing of cells transfected with the EBV TK expression vector than of cells transfected with the control mutant vector (50 M GCV for 4 days). A putative PT was also studied in the transient transfection system and appeared similar to the TK in phosphorylating GCV and conferring sensitivity to GCV, but not in BVdU-or PCVmediated cell killing. Induction of EBV kinases in combination with agents such as GCV merits further evaluation as an alternative strategy to gene therapy for selective killing of EBV-infected cells.

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The Epstein Barr Virus Genome Encodes Deoxythymidine Kinase Activity in a Nested Internal Open Reading Frame

Cited by 7 publications

References 9 publications

Human Herpesvirus 8 Open Reading Frame 21 Is a Thymidine and Thymidylate Kinase of Narrow Substrate Specificity That Efficiently Phosphorylates Zidovudine but Not Ganciclovir

Human Herpesvirus 8 Open Reading Frame 21 Is a Thymidine and Thymidylate Kinase of Narrow Substrate Specificity That Efficiently Phosphorylates Zidovudine but Not Ganciclovir

Functional Divergence of Kaposi's Sarcoma-Associated Herpesvirus and Related Gamma-2 Herpesvirus Thymidine Kinases: Novel Cytoplasmic Phosphoproteins That Alter Cellular Morphology and Disrupt Adhesion

Induction of Epstein-Barr Virus Kinases To Sensitize Tumor Cells to Nucleoside Analogues

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