Failure to Demonstrate the Interaction of Subunits of Thymidine Kinase in Cells Simultaneously Infected with Herpes Virus and a Kinaseless Mutant

Buchan, A.; Luff, Sandra; Wallis, C.M.

doi:10.1099/0022-1317-9-3-239

Cited by 17 publications

(4 citation statements)

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“…The inability to detect complementation (table III) suggested that subunits were not present and thus indicated that all mutants were defective in the same gene for TK. These results agree with those of Buchan et al [19] who failed to show serologically an interaction of subunits of TK.…”

Section: Discussionsupporting

confidence: 83%

Thymidine Kinase Activity of Herpes Simplex Virus Temperature-Sensitive Mutants

Aron¹,

Schaffer²,

Courtney³

et al. 1973

Intervirology

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Eighteen temperature-sensitive (ts) mutants of herpes simplex virus type 1 were studied with regard to their ability to synthesize virus-induced thymidine kinase (TK) at permissive (34°) and nonpermissive (39°) temperatures. Five mutants were TK^– at both temperatures (TK^– mutants), 10 were TK⁺ at both temperatures (TK⁺ mutants) and 3 showed reduced TK activity at 39° but not at 34° (TK^± mutants). Two of the 3 TK^± mutants were ts in the production of TK at 39° while the third mutant synthesized a ts TK at both temperatures. Polyacrylamide gel electrophoresis of extracts of both uninfected and TK-mutant-infected cells exhibited one major and one minor peak of TK activity, whereas extracts of wild-type (WT) virus-infected cells revealed at least one additional major peak of TK activity. Gel patterns of TK^± mutants were similar to those of the WT virus in 34 and 39° extracts; however, a reduction in TK activity of the viral peaks was observed in 39° extracts.

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Section: Discussionsupporting

confidence: 83%

Thymidine Kinase Activity of Herpes Simplex Virus Temperature-Sensitive Mutants

Aron¹,

Schaffer²,

Courtney³

et al. 1973

Intervirology

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“…The observation that the peptide of about 40,000 daltons is missing in the TK-deficient extracts and present in revertants which were selected for return of TK activity confirms the immunologic data of others (6,8) that the peptide in question is the viral TK peptide. Except for the missing 40,000 dalton protein and the presence of the peptide fragments noted above, no other differences in the electropherograms of mutant, wild-type, and revertant extracts were observed.…”

Section: Methodssupporting

confidence: 60%

“…(4). Immunological evidence strongly supports the conclusion that the peptide missing in B2006 is the HSV thymidine kinase (6,8). Fig.…”

supporting

confidence: 61%

Possible peptide chain termination mutants in thymide kinase gene of a mammalian virus, herpes simplex virus.

Summers¹,

Wagner²,

Summers³

1975

Proc. Natl. Acad. Sci. U.S.A.

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Mutations in the viral gene coding for the thymidine kinase (ATP:thymidine 5'-phosphotransferase, EC 2.7.1.75) induced by herpes simplex virus have been obtained by selection of virus resistant to bromoodeoxyuridine when grown in thymidine-kinase-deficient LMTK-mouse cells. Proteins labeled after infection of Vero (monkey) cells with herpes simplex virus were analyzed by gel electrophoresis and one protein of about 40,000 daltons was consistently altered in a number of thymidine-kinase-deficient mutants. Many viral mutants lacked this peptide and one class of these mutants induced the synthesis of new shorter peptides. Revertant virus could be selected which simultaneously regained the ability to induce thymidine kinase activity, regained the intact thymidine kinase peptide, and lost the ability to synthesize the shorter peptide fragment. These mutants comprise a class of animal virus mutants which have the properties expected of peptide chain termination mutants.The use of mutants which are sensitive to translational suppression has greatly advanced the study of bacteria and bacterial viruses (1). With the exception of fungi (2, 3), such mutations have not been demonstrated in eukaryotic systems. Two approaches to obtain such mutants are immediately apparent. First is to obtain a cell which has a suppressor gene and then to select mutants which are suppressible. Second, one can obtain potentially suppressible mutations and then use these to search for suppressor mutations. It is this latter course which we have taken, and we report here on the first part of the problem, namely the isolation and characterization of mutants which have the properties expected of peptide chain termination, potentially suppressible mutations.We have studied mutants in the thymidine kinase (ATP: thymidine 5'-phosphotransferase, EC 2.7.1.75) (TK) gene of herpes simplex virus (HSV). This system was chosen for several reasons. The enzyme thymidine kinase is not a required viral function, so defective mutants can be grown even in the absence of suppressor activity. TK-deficient mutants can be selected on the basis of their resistance to the lethal effects of 5-bromo-deoxyuridine (BrdUrd) (4). In addition, a sensitive and simple enzyme assay is available (5). The recent identification of the TK polypeptide by gel electrophoresis of immunoprecipitated, labeled infected cell proteins was also highly encouraging (6). The results of our studies show that a single viral-induced polypeptide is affected by mutation to BrdUrd resistance with simultaneous loss of TK activity, that revertants to BrdUrd sensitivity regain the affected polypeptide and TK activity, and that, in some cases, a shorter peptide is observed which appears in the mutants and is absent in the revertants. The simplest interpretation of these results is in terms of classical peptide chain termination mutants. Selection of Revertants. In order to select revertants of HSV-TK which had regained the ability to induce TK, certain of the virus mutants were grown in LMTK-cells...

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“…HVH thymidine kinase was chosen because it is a CPE as described previously. nonvirion protein, an early enzyme in the HVH replicative cycle, and an enzyme which has had some of its biochemical ENZYME properties established (4)(5)(6)10). The rabbit model of HVH encephalitis was utilized to evaluate some of the quantitative An extract containing thymidine (TdR) kinase activity was and qualitative aspects of this enzyme in CSF of infected prepared using HVH-1 strain 0-1 116 which had been "pasrabbits.…”

mentioning

confidence: 99%

Thymidine Kinase Activity in Cerebrospinal Fluid of Rabbits with Herpesvirus hominis Encephalitis

et al. 1975

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Extractdivloid fibroblast (W1-38 cells) and the virus was identified as HVH type 1 (HVH-1) by D;. AndrB J. Nahmias of Emory The rabbit model of Herpesvirus horninis (HVH) encepha-University. litis was utilized to determine whether thymidine (TdR) kinase (EC. 2.7.1.2.1), an early enzyme in the HVH replicative cycle, CELL CULTUREis present in the cerebrospinal fluid (CSF) of rabbits with HVH encephalitis. The mean TdR kinase activity in CSF from W1-38 human diploid fibroblasts were maintained in Eagles infected rabbits was 2.30 f 1.69 pmol product/l20 min/100 pl minimum essential medium (Eagle's MEM) which contained CSF, while the mean TdR kinase activity of uninfected CSF 2% fetal calf serum, NaHC03 30 mEq/liter, penicillin 250 was 0.34 + 0.25. The enzyme appeared to be cell associated units/ml, streptomycin 200 pg/ml, polymyxin 12.5 units/ml, since the TdR kinase activity in the sedimented cell suspension and chlortetracycline 25 pg/ml.

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Failure to Demonstrate the Interaction of Subunits of Thymidine Kinase in Cells Simultaneously Infected with Herpes Virus and a Kinaseless Mutant

Cited by 17 publications

References 1 publication

Thymidine Kinase Activity of Herpes Simplex Virus Temperature-Sensitive Mutants

Thymidine Kinase Activity of Herpes Simplex Virus Temperature-Sensitive Mutants

Possible peptide chain termination mutants in thymide kinase gene of a mammalian virus, herpes simplex virus.

Thymidine Kinase Activity in Cerebrospinal Fluid of Rabbits with Herpesvirus hominis Encephalitis

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