Properties of deoxythymidine kinase partially purified from noninfected and virus-infected mouse fibroblast cells

Kit, Saul; Dubbs, D. R.

doi:10.1016/0042-6822(65)90021-8

Cited by 100 publications

(45 citation statements)

References 20 publications

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“…Many herpesviruses and poxviruses possess the gent encoding this enzyme (Kit & Dubbs, 1963;Dubbs & Kit, I964;Hackstadt & Mallavia, 1978). It may not be necessary for virus replication in cell culture but it has a high affinity for nucleoside analogues (Kit & Dubbs, 1965) and is a potential target for anti-viral drugs. The presence of a TK gene in EBV was first demonstrated by Hampar et al (1971).…”

Section: Introductionmentioning

confidence: 99%

Functional analysis of C-terminal deletion mutants of Epstein--Barr virus thymidine kinase

Hsu

Liu

Chang

et al. 1996

Journal of General Virology

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Thymidine kinase fiR) activity was detected following expression of the TK gene of Epstein-Barr virus (EBV) using the pET expression plasmid and E. coli BL21(DE3)pLysS. To study the amino acid residues required at the C terminus of the EBV TK protein for enzymatic activity, a series of C-terminal deletion mutants was generated by direct truncation, linker insertion or PCR mutagenesis to create stop codons at particular sites. Deletion of nine residues from the C terminus caused a 35% reduction in TK activity, while a ten-residue deletion completely abolished the activity. A single point mutation at residue Cys570, corresponding to Cys336 of herpes simplex virus TK, did not alter the TK activity. Single amino acid changes within the last seven to ten residues also did not affect activity. The results indicate that maintenance of the conformation of the C terminus is important for enzyme activity.

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Section: Introductionmentioning

confidence: 99%

Functional analysis of C-terminal deletion mutants of Epstein--Barr virus thymidine kinase

Hsu

Liu

Chang

et al. 1996

Journal of General Virology

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“…Identification of these proteins and their functions has helped delineate the biology of herpesvirus replication and provided new targets for development of antiviral drugs. Among the enzymes known to be encoded by herpesviruses are DNA polymerase (39), DNase (2), dUTPase (38), helicase/primase (10,50), maturational protease (31,47), phosphotransferase (20), protein kinase (11,16), ribonucleotide reductase (1), terminase (41,45), thymidine kinase (25), ubiquitin ligase (7,9), and uracil deglycosidase (8). Most recently added to this list is a ubiquitin-specific cysteine protease (USP), also called deubiquitinating enzyme (DUB), discovered in human fibroblasts infected with herpes simplex virus type 1 (HSV) (23).…”

mentioning

confidence: 99%

High-Molecular-Weight Protein (pUL48) of Human Cytomegalovirus Is a Competent Deubiquitinating Protease: Mutant Viruses Altered in Its Active-Site Cysteine or Histidine Are Viable

Wang

Loveland

Kattenhorn

et al. 2006

J Virol

118

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We show here that the high-molecular-weight protein (HMWP or pUL48; 253 kDa) of human cytomegalovirus (HCMV) is a functionally competent deubiquitinating protease (DUB). By using a suicide substrate probe specific for ubiquitin-binding cysteine proteases (DUB probe) to screen lysates of HCMV-infected cells, we found just one infected-cell-specific DUB. Characteristics of this protein, including its large size, expression at late times of infection, presence in extracellular virus particles, and reactivity with an antiserum to the HMWP, identified it as the HMWP. This was confirmed by constructing mutant viruses with substitutions in two of the putative active-site residues, Cys24Ile and His162Ala. HMWP with these mutations either failed to bind the DUB probe (C24I) or had significantly reduced reactivity with it (H162A). More compellingly, the deubiquitinating activity detected in wild-type virus particles was completely abolished in both the C24I and H162A mutants, thereby directly linking HMWP with deubiquitinating enzyme activity. Mutations in these active-site residues were not lethal to virus replication but slowed production of infectious virus relative to wild type and mutations of other conserved residues. Initial studies, by electron microscopy, of cells infected with the mutants revealed no obvious differences at late times of replication in the general appearance of the cells or in the distribution, relative numbers, or appearance of virus particles in the cytoplasm or nucleus.Herpesviruses contain a large double-stranded DNA genome that codes for many of the enzymes required to produce new infectious virus. Identification of these proteins and their functions has helped delineate the biology of herpesvirus replication and provided new targets for development of antiviral drugs. Among the enzymes known to be encoded by herpesviruses are DNA polymerase (39), DNase (2), dUTPase (38), helicase/primase (10, 50), maturational protease (31, 47), phosphotransferase (20), protein kinase (11, 16), ribonucleotide reductase (1), terminase (41, 45), thymidine kinase (25), ubiquitin ligase (7, 9), and uracil deglycosidase (8). Most recently added to this list is a ubiquitin-specific cysteine protease (USP), also called deubiquitinating enzyme (DUB), discovered in human fibroblasts infected with herpes simplex virus type 1 (HSV) (23). Increasing awareness of DUBs as major regulators in biological systems focuses attention on this viral enzyme for the information that it can provide about unknown steps in virus replication and potential sites for pharmacological intervention, as well as for insights that it may provide into more general aspects of this class of enzymes.The HSV DUB was identified by using a ubiquitin-derived active-site-directed probe (DUB probe) designed to selectively bind these enzymes by forming a covalent adduct with their active-site cysteine (5). Analysis of the isolated DUB (Ϸ57 kDa with probe bound) established that its sequence is the same as the amino end of virion protein 1/2 (VP1/2, 336 ...

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“…The herpesvirus TKs can be considered functionally equal to the cellular TK2 and dCK. By contrast, poxvirus and asfavirus TKs are very similar to mammalian TK1: they have a strict substrate specificity for dT [26,27]. It has been reported that viral TKs are responsible for virus-efficient replication in some specific cell type, virus virulence, pathogenesis and reactivation from latency [20,28].…”

Section: Introductionmentioning

confidence: 99%

Rana grylio virus thymidine kinase gene: an early gene of iridovirus encoding for a cytoplasmic protein

et al. 2009

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The presence of thymidine kinase (TK) is a feature of many large DNA viruses. Here, a TK gene homologue was cloned and characterized from Rana grylio virus (RGV), a member of family Iridoviridae. RGV TK encodes a protein of 195 aa with a predicted molecular mass of 22.1 kDa. Homologues of the protein were present in all the currently sequenced iridoviruses, and phylogenetic analysis showed that it was much close to cellular TK type 2 (TK2), deoxycytidine kinase (dCK) and deoxyguanosine kinase (dGK). Subsequently, Western blotting revealed TK expression increased with time from 6 h postinfection in RGV-infected cells. Using drug inhibition analysis by protein synthesis inhibitor (cycloheximide) and DNA replication inhibitor (cytosine arabinofuranoside), RGV TK was classified as the early expression gene during in vitro infection. Subcellular localization by TK-GFP fusion protein expression and immunofluorescence staining showed RGV TK was an exclusively cytoplasmic protein in fish cells. Collectively, current data indicate that RGV TK was an early gene of iridovirus which encoded a cytoplasmic protein in fish cells.

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Properties of deoxythymidine kinase partially purified from noninfected and virus-infected mouse fibroblast cells

Cited by 100 publications

References 20 publications

Functional analysis of C-terminal deletion mutants of Epstein--Barr virus thymidine kinase

Functional analysis of C-terminal deletion mutants of Epstein--Barr virus thymidine kinase

High-Molecular-Weight Protein (pUL48) of Human Cytomegalovirus Is a Competent Deubiquitinating Protease: Mutant Viruses Altered in Its Active-Site Cysteine or Histidine Are Viable

Rana grylio virus thymidine kinase gene: an early gene of iridovirus encoding for a cytoplasmic protein

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