FREE SHIPPING over $40
OVER 10000 FIVE-STAR REVIEWS
5% OFF ALL SUBSCRIPTIONS
100% HAPPINESS GUARANTEE
Analyze Diet
0
Journal of virology1980; 34(2); 474-483; doi: 10.1128/JVI.34.2.474-483.1980

Biochemical characterization of equine herpesvirus type 3-induced deoxythymidine kinase purified from lytically infected horse embryo dermal fibroblasts.

Abstract: Infection of horse KyED cells with equine herpesvirus type 3 (EHV-3) resulted in a sevenfold increase in cytosol deoxythymidine kinase (dTK) activity. The EHV-3 dTK was purified from KyED cytosol dTK by affinity chromatography on deoxythymidine-Sepharose and characterized with respect to its electrophoretic mobility, molecular weight, substrate specificity, phosphate donor specificity, and immunological specificity. The purified EHV-3 dTK migrated in polyacrylamide gels with an Rf of 0.30 and sedimented in glycerol gradients with an S value of 5.13, corresponding to a molecular weight of 83,000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis yielded a single band with a molecular weight of 38,000 to 40,000. Antiserum prepared against the EHV-3 dTK induced in KyED cells neutralized the EHV-3-induced enzyme activity but not the dTK purified from uninfected cells. EHV-3 dTK was less sensitive to feedback inhibition to dTTP and had a lower Ki for the antiviral compound 1-beta-D-arabinofuranyosylthymine and a lower Km for the substrate deoxythymidine. These results indicate that infection of cells with EHV-3 results in the induction of a new virus-coded dTK activity which meets the criteria of Jensen for an evolutionary primitive enzyme.
Get Full Text
Publication Date: 1980-05-01 PubMed ID: 7373717PubMed Central: PMC288726DOI: 10.1128/JVI.34.2.474-483.1980Google Scholar: Lookup
The Equine Research Bank provides access to a large database of publicly available scientific literature. Inclusion in the Research Bank does not imply endorsement of study methods or findings by Mad Barn.
LinkedInCopy
  • Journal Article
  • Research Support
  • U.S. Gov't
  • P.H.S.

Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

This research investigates how infection by equine herpesvirus type 3 (EHV-3) in horse cells leads to a significant increase in a particular enzyme activity, resulting in the production of a new virus-coded enzyme. This enzyme exhibits unique characteristics which may convey evolutionary advantages to the virus.

About the Research

The primary focus of this research was on a specific enzyme known as deoxythymidine kinase (dTK) that is produced in horse cells upon infection by Equine Herpesvirus 3 (EHV-3).

  • The scientists monitored the activity of this enzyme in cells infected with EHV-3 and found that its activity increased sevenfold.
  • The enzyme was then purified from the cells and subjected to detailed analysis to understand its characteristics that could potentially add an evolutionary advantage to the virus.

Procedure

The researchers used sophisticated analysis methods to study the properties of the EHV-3 dTK enzyme.

  • For the purification of the EHV-3 dTK from the cells, affinity chromatography was used. This involves a biochemical technique to separate and purify a specific molecule, the dTK, from a mix of molecules, in this case, the cytosol from infected horse cells.
  • After the enzyme was purified, its molecular structure, weight, substrate specificity, immunological specificity and other properties were examined.
  • Electrophoretic mobility study, conducted using polyacrylamide gels, and sedimentation analysis in glycerol gradients were used to determine the approximate molecular weight of the enzyme.

Results

The results of the study bring forward various unique characteristics of the EHV-3 dTK enzyme.

  • It was found that the molecular weight of the enzyme was between 38,000 and 40,000, which was determined by Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE).
  • The antiserum prepared against EHV-3 dTK was able to neutralize the dTK induced enzyme activity, but not the dTK from uninfected cells. This indicates that this particular dTK is a result of EHV-3 infection.
  • The enzyme was also found to be less susceptible to feedback inhibition, had a lower Ki for the antiviral compound, and a lower Km for the substrate deoxythymidine, indicating its evolutionary advantage.

Conclusion

The results of this research indicate that EHV-3 infection results in a new virus-coded dTK activity which displays characteristics of an evolutionary primitive enzyme. Understanding this enzyme can help reveal further insights about the virus, its infection process, and potential ways to counter it.

Cite This Article

APA
McGowan JJ, Allen GP, Barnett JM, Gentry GA. (1980). Biochemical characterization of equine herpesvirus type 3-induced deoxythymidine kinase purified from lytically infected horse embryo dermal fibroblasts. J Virol, 34(2), 474-483. https://doi.org/10.1128/JVI.34.2.474-483.1980

Publication

Journal of virology
ISSN: 0022-538X
NlmUniqueID: 0113724
Country: United States
Language: English
Volume: 34
Issue: 2
Pages: 474-483

Researcher Affiliations

McGowan, J J
    Allen, G P
      Barnett, J M
        Gentry, G A

          MeSH Terms

          • Animals
          • Cations, Divalent / pharmacology
          • Cell Line
          • Enzyme Induction
          • Fibroblasts
          • Herpesviridae / enzymology
          • Herpesviridae / growth & development
          • Horses
          • Kinetics
          • Molecular Weight
          • Nucleotides / metabolism
          • Substrate Specificity
          • Thymidine Kinase / isolation & purification
          • Thymidine Kinase / metabolism

          References

          This article includes 35 references
          1. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4.. Nature 1970 Aug 15;227(5259):680-5.
            pubmed: 5432063doi: 10.1038/227680a0google scholar: lookup
          2. Munyon W, Kraiselburd E, Davis D, Mann J. Transfer of thymidine kinase to thymidine kinaseless L cells by infection with ultraviolet-irradiated herpes simplex virus.. J Virol 1971 Jun;7(6):813-20.
            pubmed: 4327589doi: 10.1128/JVI.7.6.813-820.1971google scholar: lookup
          3. Honess RW, Watson DH. Herpes simplex virus-specific polypeptides studied by polyacrylamide gel electrophoresis of immune precipitates.. J Gen Virol 1974 Feb;22(2):171-85.
            pubmed: 4362401doi: 10.1099/0022-1317-22-2-171google scholar: lookup
          4. Kemp MC, Perdue ML, Rogers HW, O'Callaghan DJ, Randall CC. Structural polypeptides of the hamster strain of equine herpes virus type 1: products associated with purification.. Virology 1974 Oct;61(2):361-75.
            pubmed: 4418750doi: 10.1016/0042-6822(74)90274-8google scholar: lookup
          5. Thouless ME, Wildy P. Deoxypyrimidine kinases of herpes simplex viruses types 1 and 2: comparison of serological and structural properties.. J Gen Virol 1975 Feb;26(2):159-70.
            pubmed: 163287doi: 10.1099/0022-1317-26-2-159google scholar: lookup
          6. Kit S, Leung WC, Jorgensen GN, Trkula D, Dubbs DR. Thymidine kinase isozymes of normal and virus-infected cells.. Cold Spring Harb Symp Quant Biol 1975;39 Pt 2:703-15.
            pubmed: 169026doi: 10.1101/sqb.1974.039.01.084google scholar: lookup
          7. Leung WC, Dubbs DR, Trkula D, Kit S. Mitochondrial and herpesvirus-specific deoxypyrimidine kinases.. J Virol 1975 Sep;16(3):486-97.
            pubmed: 169387doi: 10.1128/JVI.16.3.486-497.1975google scholar: lookup
          8. Dobersen MJ, Greer S. An assay for pyrimidine deoxyribonucleoside kinase using gamma-32P-labeled ATP.. Anal Biochem 1975 Aug;67(2):602-10.
            pubmed: 169711doi: 10.1016/0003-2697(75)90335-8google scholar: lookup
          9. Kraiselburd E, Gage LP, Weissbach A. Presence of a herpes simplex virus DNA fragment in an L cell clone obtained after infection with irradiated herpes simplex virus I.. J Mol Biol 1975 Oct 5;97(4):533-42.
            pubmed: 171420doi: 10.1016/s0022-2836(75)80057-xgoogle scholar: lookup
          10. Kit S, Leung WC, Jorgensen GN, Trkula D, Dubbs DR. Viral-induced thymidine kinase isozymes.. Prog Med Virol 1975;21:13-34.
            pubmed: 174152
          11. Lee LS, Cheng YC. Human deoxythymidine kinase. I. Purification and general properties of the cytoplasmic and mitochondrial isozymes derived from blast cells of acute myelocytic leukemia.. J Biol Chem 1976 May 10;251(9):2600-4.
            pubmed: 1063125
          12. Cheng YC, Ostrander M. Deoxythymidine kinase induced in HeLa TK- cells by herpes simplex virus type I and type II. II. Purification and characterization.. J Biol Chem 1976 May 10;251(9):2605-10.
            pubmed: 177418
          13. Kowal EP, Markus G. Affinity chromatography of thymidine kinase from a rat colon adenocarcinoma.. Prep Biochem 1976;6(5):369-85.
            pubmed: 972854doi: 10.1080/00327487608061625google scholar: lookup
          14. Jensen RA. Enzyme recruitment in evolution of new function.. Annu Rev Microbiol 1976;30:409-25.
            pubmed: 791073doi: 10.1146/annurev.mi.30.100176.002205google scholar: lookup
          15. Cheng YC. Deoxythymidine kinase induced in the HELA TK- cells by herpes simplex virus type I and type II. Substrate specificity and kinetic behavior.. Biochim Biophys Acta 1976 Dec 8;452(2):370-81.
            pubmed: 188465doi: 10.1016/0005-2744(76)90186-8google scholar: lookup
          16. Závada V, Erban V, Rezácová D, Vonka V. Thymidine-kinase in cytomegalovirus infected cells.. Arch Virol 1976;52(4):333-9.
            pubmed: 13768doi: 10.1007/BF01315622google scholar: lookup
          17. Walboomers JM, van Ketel R, Becker-Bloemkolk MJ. A sensitive method for the detection of herpes simplex virus type 2 specific thymidine kinase.. Intervirology 1977;8(3):186-92.
            pubmed: 192698doi: 10.1159/000148894google scholar: lookup
          18. Allen GP, Bryans JT. Replication of equine herpesvirus type 3: kinetics of infectious particle formation and virus nucleic acid synthesis.. J Gen Virol 1977 Mar;34(3):421-30.
            pubmed: 558280doi: 10.1099/0022-1317-34-3-421google scholar: lookup
          19. Kit S, Jorgensen GN, Liav A, Zaslavsky V. Purification of vaccinia virus-induced thymidine kinase activity from [35S]methionine-labeled cells.. Virology 1977 Apr;77(2):661-76.
            pubmed: 193257doi: 10.1016/0042-6822(77)90490-1google scholar: lookup
          20. Aswell JF, Allen GP, Jamieson AT, Campbell DE, Gentry GA. Antiviral activity of arabinosylthymine in herpesviral replication: mechanism of action in vivo and in vitro.. Antimicrob Agents Chemother 1977 Aug;12(2):243-54.
            pubmed: 197886doi: 10.1128/AAC.12.2.243google scholar: lookup
          21. Dobersen MJ, Greer S. Herpes simplex virus type 2 induced pyrimidine nucleoside kinase: enzymatic basis for the selective antiherpetic effect of 5-halogenated analogues of deoxycytidine.. Biochemistry 1978 Mar 7;17(5):920-8.
            pubmed: 204334doi: 10.1021/bi00598a028google scholar: lookup
          22. Pellicer A, Wigler M, Axel R, Silverstein S. The transfer and stable integration of the HSV thymidine kinase gene into mouse cells.. Cell 1978 May;14(1):133-41.
            pubmed: 208776doi: 10.1016/0092-8674(78)90308-2google scholar: lookup
          23. Matsukage A, Ono K, Ohashi A, Takahashi T, Nakayama C, Saneyoshi M. Inhibitory effect of 1-beta-D-arabinofuranosylthymine 5'-triphosphate and 1-beta-D-arabinofuranosylcytosine 5'-triphosphate on DNA polymerases from murine cells and oncornavirus.. Cancer Res 1978 Sep;38(9):3076-9.
            pubmed: 79444
          24. Cheng YC. A rational approach to the development of antiviral chemotherapy: alternative substrates of herpes simplex virus Type 1 (HSV-1) and Type 2 (HSV-2) thymidine kinase (TK).. Ann N Y Acad Sci 1977 Mar 4;284:594-8.
            pubmed: 212988doi: 10.1111/j.1749-6632.1977.tb21992.xgoogle scholar: lookup
          25. Fyfe JA, Keller PM, Furman PA, Miller RL, Elion GB. Thymidine kinase from herpes simplex virus phosphorylates the new antiviral compound, 9-(2-hydroxyethoxymethyl)guanine.. J Biol Chem 1978 Dec 25;253(24):8721-7.
            pubmed: 214430
          26. Allen GP, McGowan JJ, Bryans JT, Randall CC, Gentry GA. Induction of deoxythymidine kinase activity in several mammalian cell lines after infection with six different strains of equine herpesvirus type 1 (EHV-1).. Virology 1978 Oct 15;90(2):351-9.
            pubmed: 214953doi: 10.1016/0042-6822(78)90319-7google scholar: lookup
          27. Bacchetti S, Graham FL. Characterization of human TK- cell lines transformed to a TK+ phenotype by herpes simplex virus type 2 DNA.. J Gen Virol 1979 Jan;42(1):149-57.
            pubmed: 215706doi: 10.1099/0022-1317-42-1-149google scholar: lookup
          28. Allen GP, McGowan JJ, Randall CC, Mancini W, Cheng YC, Gentry GA. Purification and characterization of deoxythymidine kinase (dTK) induced in dTK- 3T3 mouse cells by equine herpesvirus type 1 (EHV-1).. Virology 1979 Jan 30;92(2):367-74.
            pubmed: 218350doi: 10.1016/0042-6822(79)90141-7google scholar: lookup
          29. Anderson CM, Zucker FH, Steitz TA. Space-filling models of kinase clefts and conformation changes.. Science 1979 Apr 27;204(4391):375-80.
            pubmed: 220706doi: 10.1126/science.220706google scholar: lookup
          30. Cohen SS. Comparative biochemistry and drug design for infectious disease.. Science 1979 Sep 7;205(4410):964-71.
            pubmed: 382357doi: 10.1126/science.382357google scholar: lookup
          31. McGowan JJ, Allen GP, Gentry GA. Purification and biochemical characterization of deoxythymidine kinase of deoxythymidine kinase-deficient mouse 3T3 cells biochemically transformed by equine herpesvirus type 1.. Infect Immun 1979 Aug;25(2):610-4.
            pubmed: 489123doi: 10.1128/iai.25.2.610-614.1979google scholar: lookup
          32. Barnett JM, McGowan JJ, Gentry GA. Arabinosylthymine: inhibitor of splenic lymphocyte macromolecular synthesis in vitro.. Infect Immun 1979 Oct;26(1):294-7.
            pubmed: 500206doi: 10.1128/iai.26.1.294-297.1979google scholar: lookup
          33. Cheng YC, Tsou TY, Hackstadt T, Mallavia LP. Induction of thymidine kinase and DNase in varicella-zoster virus-infected cells and kinetic properties of the virus-induced thymidine kinase.. J Virol 1979 Jul;31(1):172-7.
            pubmed: 228052doi: 10.1128/JVI.31.1.172-177.1979google scholar: lookup
          34. LOWRY OH, ROSEBROUGH NJ, FARR AL, RANDALL RJ. Protein measurement with the Folin phenol reagent.. J Biol Chem 1951 Nov;193(1):265-75.
            pubmed: 14907713
          35. MARTIN RG, AMES BN. A method for determining the sedimentation behavior of enzymes: application to protein mixtures.. J Biol Chem 1961 May;236:1372-9.
            pubmed: 13767412

          Citations

          This article has been cited 3 times.
          1. Ebeling A, Keil G, Nowak B, Fleckenstein B, Berthelot N, Sheldrick P. Genome structure and virion polypeptides of the primate herpesviruses Herpesvirus aotus types 1 and 3: comparison with human cytomegalovirus. J Virol 1983 Feb;45(2):715-26.
            doi: 10.1128/JVI.45.2.715-726.1983pubmed: 6300430google scholar: lookup
          2. McGowan JJ, Wagner RR. Inhibition of cellular DNA synthesis by vesicular stomatitis virus. J Virol 1981 Apr;38(1):356-67.
            doi: 10.1128/JVI.38.1.356-367.1981pubmed: 6165831google scholar: lookup
          3. de Turenne-Tessier M, Ooka T, de The G, Daillie J. Characterization of an Epstein-Barr virus-induced thymidine kinase. J Virol 1986 Mar;57(3):1105-12.
            doi: 10.1128/JVI.57.3.1105-1112.1986pubmed: 3005613google scholar: lookup
          Subscribe to our newsletter
          Join 99,824 horse owners like you who receive our email updates on horse health and nutrition.