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

Hsu, Ting-Yu; Liu, Min-Wei; Chang, Ya-Ru; Pai, Chien-Ying; Yang, Czau‐Siung; Chen, Jen‐Yang

doi:10.1099/0022-1317-77-8-1893

Cited by 6 publications

(8 citation statements)

References 38 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In this sense, the reduced activity of the Δ8 mutant relative to the other C-terminal deletion mutants (Table 3) is unanticipated. However, we note that similar effects have been reported by others who carried out C-terminal deletion studies, including the IN269 mutation for the integrase of HIV [104], the Δ8 and Δ9 mutants of the thymidine kinase of Epstein-Barr virus [105], and the D5 and D10 mutants of the plant vacullar H (+)-pyrophosphatase [106]. Differential proteolysis may also explain why the Δ8 construct does not follow the same trend as the other mutants.…”

Section: Discussionsupporting

confidence: 83%

Carboxyl-Terminal Truncations Alter the Activity of the Human α-Galactosidase A

et al. 2015

View full text Add to dashboard Cite

Fabry disease is an X-linked inborn error of glycolipid metabolism caused by deficiency of the human lysosomal enzyme, α-galactosidase A (αGal), leading to strokes, myocardial infarctions, and terminal renal failure, often leading to death in the fourth or fifth decade of life. The enzyme is responsible for the hydrolysis of terminal α-galactoside linkages in various glycolipids. Enzyme replacement therapy (ERT) has been approved for the treatment of Fabry disease, but adverse reactions, including immune reactions, make it desirable to generate improved methods for ERT. One approach to circumvent these adverse reactions is the development of derivatives of the enzyme with more activity per mg. It was previously reported that carboxyl-terminal deletions of 2 to 10 amino acids led to increased activity of about 2 to 6-fold. However, this data was qualitative or semi-quantitative and relied on comparison of the amounts of mRNA present in Northern blots with αGal enzyme activity using a transient expression system in COS-1 cells. Here we follow up on this report by constructing and purifying mutant enzymes with deletions of 2, 4, 6, 8, and 10 C-terminal amino acids (Δ2, Δ4, Δ6, Δ8, Δ10) for unambiguous quantitative enzyme assays. The results reported here show that the k cat/K m approximately doubles with deletions of 2, 4, 6 and 10 amino acids (0.8 to 1.7-fold effect) while a deletion of 8 amino acids decreases the k cat/K m (7.2-fold effect). These results indicate that the mutated enzymes with increased activity constructed here would be expected to have a greater therapeutic effect on a per mg basis, and could therefore reduce the likelihood of adverse infusion related reactions in Fabry patients receiving ERT treatment. These results also illustrate the principle that in vitro mutagenesis can be used to generate αGal derivatives with improved enzyme activity.

show abstract

Section: Discussionsupporting

confidence: 83%

Carboxyl-Terminal Truncations Alter the Activity of the Human α-Galactosidase A

et al. 2015

View full text Add to dashboard Cite

show abstract

“…This hypothesis is very unlikely. Indeed, several studies of herpesviruses and poxviruses have demonstrated previously that the C-terminal region of TK is essential for its activity (23). For example, it has been demonstrated previously that the last 10 residues of the 607-aa-long Epstein-Barr virus TK are essential for its activity (23).…”

Section: Discussionmentioning

confidence: 99%

“…Indeed, several studies of herpesviruses and poxviruses have demonstrated previously that the C-terminal region of TK is essential for its activity (23). For example, it has been demonstrated previously that the last 10 residues of the 607-aa-long Epstein-Barr virus TK are essential for its activity (23). In comparison to EpsteinBarr virus TK, KHV TK is rather small, consisting of only 217 aa, among which only the first 185 residues are expressed by the FL BAC-excised strain.…”

Section: Discussionmentioning

confidence: 99%

Cloning of the Koi Herpesvirus Genome as an Infectious Bacterial Artificial Chromosome Demonstrates That Disruption of the Thymidine Kinase Locus Induces Partial Attenuation in Cyprinus carpio koi

Costes

Fournier

Michel

et al. 2008

J Virol

View full text Add to dashboard Cite

Koi herpesvirus (KHV) is the causative agent of a lethal disease in koi and common carp. In the present study, we describe the cloning of the KHV genome as a stable and infectious bacterial artificial chromosome (BAC) clone that can be used to produce KHV recombinant strains. This goal was achieved by the insertion of a loxP-flanked BAC cassette into the thymidine kinase (TK) locus. This insertion led to a BAC plasmid that was stably maintained in bacteria and was able to regenerate virions when permissive cells were transfected with the plasmid. Reconstituted virions free of the BAC cassette but carrying a disrupted TK locus (the FL BAC-excised strain) were produced by the transfection of Cre recombinase-expressing cells with the BAC. Similarly, virions with a wild-type revertant TK sequence (the FL BAC revertant strain) were produced by the cotransfection of cells with the BAC and a DNA fragment encoding the wild-type TK sequence. Reconstituted recombinant viruses were compared to the wild-type parental virus in vitro and in vivo. The FL BAC revertant strain and the FL BAC-excised strain replicated comparably to the parental FL strain. The FL BAC revertant strain induced KHV infection in koi carp that was indistinguishable from that induced by the parental strain, while the FL BAC-excised strain exhibited a partially attenuated phenotype. Finally, the usefulness of the KHV BAC for recombination studies was demonstrated by the production of an ORF16-deleted strain by using prokaryotic recombination technology. The availability of the KHV BAC is an important advance that will allow the study of viral genes involved in KHV pathogenesis, as well as the production of attenuated recombinant candidate vaccines.

show abstract

“…The recombinant plasmids obtained were used to transform the Escherichia coli strain BL21(DE3)pLysS [29]. One colony was …”

Section: Expression Of the Tk Proteinmentioning

confidence: 99%

“…However, the structurefunction relationship of EBV TK has been addressed rarely. In our previous study, we have shown that the C-terminus of EBV TK is important for its activity [29]. In the present study, we sought to identify and characterize the nucleoside-binding sites of the enzyme.…”

Section: Introductionmentioning

confidence: 99%

Identification and characterization of the conserved nucleoside-binding sites in the Epstein-Barr virus thymidine kinase

Chen

Chang

et al. 2004

Biochemical Journal

View full text Add to dashboard Cite

Thymidine kinase (TK), encoded by EBV (Epstein-Barr virus), is an attractive target for antiviral therapy and provides a novel approach to the treatment of EBV-associated malignancies. Despite the extensive use of nucleoside analogues for the treatment of viral infections and cancer, the structure-function relationship of EBV TK has been addressed rarely. In the absence of any structural information, we sought to identify and elucidate the functional roles of amino acids in the nucleoside-binding site using site-directed mutagenesis. Through alignment with other human herpesviral TK protein sequences, we predicted that certain conserved regions comprise the nucleoside-binding site of EBV TK and, through site-directed mutagenesis, showed significant changes in activity and binding affinity for thymidine of site 3 (-DRH-) and 4 (-VFP-) mutants. For site 3, only mutants D392E (Asp 392 → Glu) and R393H retain activity, indicating that a negative charge is important for Asp 392 and a positive charge is required for Arg 393 . The increased binding affinities of these two mutants for 3 -deoxy-2 ,3 -didehydrothymidine suggest that the two residues are also important for substrate selection. Interestingly, the changed metal-ion usage pattern of D392E reveals that Asp 392 plays multiple roles in this region. His 394 cannot be compensated by other amino acids, also indicating a crucial role. In site 4, the F402Y mutant retains full activity; however, F402S retains only 60 % relative activity. Strikingly, when Phe 402 is substituted with serine residue, the original preferred pyrimidine substrates, such as 3 -azido-3 -deoxythymidine, iododeoxyuridine and β-L-5-iododioxolane uracil (L-form substrate), have decreased competitiveness with thymidine, suggesting that Phe 402 plays a crucial role in substrate specificity and that the aromatic ring is important for function.

show abstract

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

Cited by 6 publications

References 38 publications

Carboxyl-Terminal Truncations Alter the Activity of the Human α-Galactosidase A

Carboxyl-Terminal Truncations Alter the Activity of the Human α-Galactosidase A

Cloning of the Koi Herpesvirus Genome as an Infectious Bacterial Artificial Chromosome Demonstrates That Disruption of the Thymidine Kinase Locus Induces Partial Attenuation in Cyprinus carpio koi

Identification and characterization of the conserved nucleoside-binding sites in the Epstein-Barr virus thymidine kinase

Contact Info

Product

Resources

About