Purified recombinant protein encoded by the BXLF-I open reading frame of the Epstein-Barr virus genome has thymidine kinase activity. The substrate behaviors of various nucleosides toward this enzyme were tested. Halogenated deoxyuridines, zidovudine, and bromovinyldeoxyuridine are efficient substrates, while acyclovir and dihydroxypropylmethylguanine are relatively poor substrates for the Epstein-Barr virus thymidine kinase.Like most other herpesviruses, Epstein-Barr virus (EBV) encodes a virus-induced protein with thymidine kinase (TK) activity. This enzyme catalyzes the transfer of the gamma phosphoryl group of ATP to the 5' hydroxyl of a variety of deoxynucleosides to produce the corresponding nucleoside monophosphate. It is presumed that the further phosphorylations to the diphosphate and triphosphate forms are carried out by cellular kinases. In the case of herpes simplex virus type 1 (HSV-1) and HSV-2, the substrate specificity of the viral TK is much less restricted than that of the mammalian cell TK (12). Thus, in infected cells the viral enzyme initiates the conversion of nucleoside analogs to phosphorylated forms which either are inhibitors of the viral DNA polymerase or are relatively toxic when incorporated into the viral DNA (5). If the EBV TK has a similar broad substrate specificity, it may provide a route to the activation of nucleoside analogs for the production of useful antiviral nucleotides. To examine this possibility, we examined the use of various nucleoside analogs by EBV TK made in a bacterial expression system programed with the cloned EBV DNA sequence encoding this enzyme.Reagents. Nucleoside analogs, thymidine, ATP, morpholinoethanesulfonic acid (MES), and bovine serum albumin (BSA) were purchased from Sigma. [14C] mM spermidine-HCl (pH 7.5) and were lysed by the addition of 300 ,ug of lysozyme per ml. The lysed cells were sonicated briefly to reduce the viscosity before pelleting the cellular debris. The supernatant was loaded directly onto a DEAE Biogel column (4 by 30 cm), and the column was eluted with 1 liter of a linear gradient of salt (0 to 500 mM NaCl). Fractions (6 ml) were collected and assayed for TK activity (see below). Fractions containing TK activity were pooled and applied to a hydroxylapatite column (1 by 8 cm) equilibrated with 1 mM potassium phosphate buffer (pH 6.8). The column was washed in succession with one column volume of the equilibrating buffer, the equilibrating buffer containing 500 mM NaCl, and finally the equilibrating buffer. The column was eluted with a 100-ml linear gradient of 10 to 300 mM potassium phosphate (pH 6.8) (10). Fractions with TK activity were dialyzed against 0.2 mM thymidine-50% glycerol-50 mM Tris (pH 6.8) and were stored at -20°C. The enzyme was judged to be about 30% pure on the basis of the staining intensity of the TK polypeptide on polyacrylamide gels.Standard TK assay. Enzyme activity was determined by the differential binding of phosphorylated versus unphosphorylated nucleoside to positively charged DEAE paper (26). All assay...

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Angular distributions have be'en measured for the reactions Sr(' 0, ' C) Zr and Zr(' 0, ' C) Mo at 80 MeU, and 'Sr('~C, ' Be) Zr and Zr("C, ' Be) Mo at 60 MeV. The data were analyzed with full recoil, coupled-channel Born approximation calculations, in which both direct (one-step) and sequential (two-step) reaction routes were considered. Detailed shell model wave functions were used to corstruct all form. factors. The calculated sequential contributions to the cross sections were found to be significantly larger than those associated with direct transfer, and inclusion of the two-step routes substantially improved agreement with experiment. The calculated angular distribution shapes were found to depend on both the intermediate Q value of the sequential process, and also on the microscopic configurations involved in the transfer. In addition, a previously unreported configuration dependence of the recoil corrections was noted in the calculations.NUCLEAR REACTIONS 88Sr(160 160) 88Sr(16 0 14C) 90Z r(160, 16 0), OZr (16 p 14C), 0 MeV. 88Sr (12C 12C 12C) 88Sr (12C 10ge) 9 OZ r (12C 12C) 904 r(12C 10ge) @ 6P MeV; enriched targets, measured 0'(&). Finite range DWBA and CCBA analyses of direct and sequential transfer, compared with data. .

The iodoacetate-dependent and iodoacetamide-dependent inhibition of cytoplasmic malate dehydrogenase (s-MDH) has been examined. We have confirmed previous reports that iodoacetate inhibits this dimeric enzyme by modifying a single active site methionine per s-MDH subunit. Time courses for the inactivation of the solution-state enzyme with both reagents indicate each s-MDH subunit is modified with equal rapidity in the absence of substrate or cofactor. However, the subunits react with distinctly different rates in the presence of cofactor or cofactor/substrate combinations, indicating some conformational asymmetry between subunits occurs when these ligands are bound. This is consistent with solution-state s-MDH behaving as a cooperative enzyme. Apo and holo crystalline s-MDH are also inhibited by iodoacetic acid. However, subunits of the crystalline enzyme are inhibited with different rates in the presence or absence of active site ligands. This suggests subunit conformations of the dimeric enzyme are not identical in crystalline s-MDH preparations regardless of ligand binding. Furthermore, by the criterion of inhibition rate constants, subunit conformations of the crystalline enzyme are not rigid but are perturbed by ligand binding. Comparisons of inactivation time courses for solution- and crystalline-state s-MDH suggest crystalline s-MDH exhibits at least some of the subunit asymmetry associated with the solution-state enzyme.

Human immunodeficiency virus (HIV) resistance to the nonnucleoside reverse transcriptase inhibitors emerges very rapidly under selection in culture and in patients. In contrast, zidovudine (3-azido-3-deoxythymidine [AZT])-resistant HIV generally emerges in patients only after more-prolonged therapy. Although HIV can be cultured from many patients shortly after the initiation of AZT treatment, characterization of the virus that is cultured generally indicates that it is sensitive to AZT. To initiate an evaluation of the mechanisms contributing to early HIV breakthrough in the presence of AZT and other nucleoside analogs, we have utilized replication-defective HIV encoding reporter genes. These recombinant HIV allow a quantitative analysis of a single cycle of infection. Results with these defective HIV indicate that early infection in the presence of AZT often results from the infection of a cell which is refractory to the antiretroviral effects of AZT. Characterization of a cell line derived from one such cell has demonstrated decreased accumulation of AZT triphosphate, increased phosphorylation of thymidine to thymidine triphosphate, and increased levels of thymidine kinase activity. In addition, AZT inhibition of replication-competent HIV infection is also significantly impaired in this cell line. Attempts to detect and characterize the mechanisms responsible for early viral infection after initiation of AZT therapy may result in the development of new strategies for prolonged suppression of viral infection prior to the emergence of drug-resistant virus.