Biochemical and immunological characterization of deoxythymidine kinase of thymidine kinaseless HeLa cells biochemically transformed by herpes simplex virus type (original) (raw)
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Infection and Immunity
Protection against Plasmodium falciparum can be induced by vaccination in animal models with merozoite surface protein 1 (MSP1), which makes this protein an attractive vaccine candidate for malaria. In an attempt to produce a product that is easily scaleable and inexpensive, we expressed the C-terminal 42 kDa of MSP1 (MSP1 42) in Escherichia coli, refolded the protein to its native form from insoluble inclusion bodies, and tested its ability to elicit antibodies with in vitro and in vivo activities. Biochemical, biophysical, and immunological characterization confirmed that refolded E. coli MSP1 42 was homogeneous and highly immunogenic. In a formulation suitable for human use, rabbit antibodies were raised against refolded E. coli MSP1 42 and tested in vitro in a P. falciparum growth invasion assay. The antibodies inhibited the growth of parasites expressing either homologous or heterologous forms of P. falciparum MSP1 42. However, the inhibitory activity was primarily a consequence of antibodies directed against the C-terminal 19 kDa of MSP1 (MSP1 19). Vaccination of nonhuman primates with E. coli MSP1 42 in Freund's adjuvant protected six of seven Aotus monkeys from virulent infection with P. falciparum. The protection correlated with antibody-dependent mechanisms. Thus, this new construct, E. coli MSP1 42 , is a viable candidate for human vaccine trials.
Antimicrobial Agents and Chemotherapy, 1987
The thymidine analog 5'-ethynylthymidine was a potent inhibitor of herpes simplex virus type 1 (strain K(S)-induced thymidine kinase with a Ki value of 0.09 ,uM. 5'-Ethynylthymidine was less inhibitory against herpes simplex virus type 2 (strain 333)-induced thymidine kinase with a Ki of 0.38 ,uM and showed no inhibition against human cytosolic thymidine kinase under the conditions tested. The compound was effective against the altered thymidine kinase induced by acyclovir-and bromovinyldeoxyuridine-resistant virus variants. At 100 FM 5'-ethynylthymidine, the cellular pool size of dTTP in herpes simplex virus type 1-infected cells was 5% that of infected cells receiving no drug treatment, while there was no significant effect on the pool sizes of dATP, dGTP, and dCTP. There was a positive correlation between dTTP pools and the intracellular thymidine kinase activity of herpes simplex virus type 1-infected cells. When tested alone, 5'-ethynylthymidine exhibited no antiviral activity, but it antagonized the antiviral efficacy of five compounds which require viral thymidine kinase for their action. Herpes simplex viruses type 1 (HSV-1) and type 2 (HSV-2) and varicella-zoster virus induce unique virusspecified thymidine kinases (dThd kinase) in infected cells (2, 8, 18, 20). In the past, major efforts were made to develop antiviral compounds that would serve as "selective alternative substrates" (3) for virus-specified dThd kinase. While the activity of viral dThd kinase does not appear to be critical for virus replication in cell culture systems (19), studies have suggested that it is important for virus pathogenicity and reactivation of latent virus from neural cells (9, 14, 30). In this report, we describe the effects of 5'ethynylthymidine (5'-Et-dThd) (Fig. 1) as a selective inhibitor for HSV dThd kinase and its impact on deoxynucleotide metabolism in virus-infected cells. MATERIALS AND METHODS Materials. All chemicals used were reagent grade or better. 3H-labeled deoxynucleoside 5'-triphosphates (dNTPs) were purchased from ICN Radiochemicals, Irvine, Calif. dThd, dNTPs, and calf thymus DNA were purchased from Sigma Chemical Co., St. Louis, Mo. DNA polymerase I (endonuclease-free) and hydroxyurea were purchased from Boehringer Mannheim Biochemicals, Indianapolis, Ind. RPMI 1640 medium, fetal bovine serum, and kanamycin were purchased from Hazleton Research Products, Inc., Denver, Pa. 5'-Et-dThd was synthesized by published procedures (29). Acyclovir (ACV) was a gift from Burroughs Wellcome Co., Research Triangle Park, N.C., and 9-(1,3dihydroxy-2-propoxy-methyl)guanine (DHPG) was from Syntex Co. 5'-Amino-dThd (5'-NH2-dThd) was provided by William Prusoff, Yale University, New Haven, Conn. Fluoroiodo-arabinosyl-cytosine (FIAC) and (E)-5-(2bromovinyl)-2'-deoxyuridine (BVDU) were gifts from J. J. Fox (Sloan-Kettering Cancer Institute) and G. D. Searle & Co., respectively.
Infection and immunity, 1980
An optimized assay for herpes simplex virus type 1- and 2-induced deoxythymidine kinase (dTk) is described which used [125I]iododeoxyuridine (IUdr) as a substrate. Values for Km and Vsat were determined for both viral and cellular dTK, using either deoxythymidine or IUdR as a substrate. A comparison between the two substrates revealed that higher reaction velocities and lower Km values were obtained with IUdR. A standard assay was designed which uses 10(-7) M IUdR as a substrate. This assay can detect herpes simplex-induced dTK from as few as two infected cells and is several orders of magnitude more sensitive than conventional dTK assays which use 10(-5) M dT as a substrate. An easily detectable blocking activity, which was shown to be mainly confined to the immunoglobulin G antibody class, was found in most human sera which were positive for complement-fixing antibody against herpes simplex virus.
Infection and immunity, 1977
Mouse L cells lacking thymidine kinase (Ltk-) that had been transformed to the thymidine kinase-positive (tk+) phenotype by herpes simplex virus type 1 (HSV-1) were cultured in medium containing tritiated thymidine. Six clonal lines of cells surviving this treatment were found to have the following properties: (i) the cells were tk- and had spontaneous back-reversion frequencies to the tk+ phenotype of 10(-5) or less, (ii) the cells contained HSV antigens, although in lesser amounts than in the parental transformed cells, and (iii) the cells were retransformable to the tk+ phenotype by HSV-1 at frequencies of about 1 to 13% of the frequency of the primary transformation of LtK- cells. HSV-1 plaqued as efficiently on monolayers of these cells and replicated in them to the same extent as it did in Ltk- cells. These results indicate that HSV-1-transformed L cells surviving selection with tritiated thymidine are unlike the parental Ltk- cells in that they are damaged in such a way that ...
Journal of General Virology, 1982
We have defined the minimal size and physical map locations in the genomes of both herpes simplex virus types 1 and 2 (HSV-1 and HSV-2) for DNA sequences capable of conferring stable biochemical transformation under thymidine kinase (TK) selection. The experiments involved transfection of Ltk-cells with either isolated virus DNA fragments or cloned pBR322 plasmids containing the 3.5 kilobase (kb) BamHI-O fragment from HSV-I(MP) or the 5.6 kb SalI-G fragment from HSV-2(333). Mapping of restriction enzyme sites within these cloned DNAs, followed by assays for colony formation in HAT medium after transfection with cleaved DNA, localized the biologically active TK-transforming sequences to lie between coordinates 0.300 and 0.313 in HSV-1 and between 0.303 and 0.315 in HSV-2. Experiments with a series of cloned plasmids containing deletions of the BamHI-O fragment towards either the 3'-or 5'-ends of the TK gene indicated that the sequences required for stable HSV-1 TK transformation lay within a 1600 base pair (bp) region at 0.303 to 0.313 map units. An internal deletion mutant plasmid, selected by a novel bacterial transfection assay for the absence of the KpnI site at 0.308, also failed to rescue Ltk-cells. With the exception of cleavage at the StuI site at 0-303 in HSV-2, which reduced activity only eightfold, all cleavages that affected TK transformation reduced the efficiency at least 50-fold. A direct comparison of the HSV-1 and HSV-2 minimal transforming regions with the nucleotide sequence of the structural HSV-1 TK gene indicates that the HSV-2 StuI site lies 30 bp beyond the poly(A) addition site at the 3'-end of TK mRNA. On the other hand, cleavage at the Sinai site in HSV-1 TK, located 80 bp in front of the poly(A) addition point, abolishes colony formation. Comparison of the putative 5'-end of the HSV-2 TK gene defined by transfection assays, with a 250 bp non-transcribed region at the front of the HSV-1 TK gene, suggests that the promoter regions contain a much higher frequency of conserved cleavage sites than do the coding portions of the two genes. Direct nucleotide sequencing of the 5'-flanking sequences for HSV-2 TK confirmed that large portions of the two promoters possess greater than 95 % sequence homology. At least 140 bp, but no more than 200 bp, of this 5'-promoter region are essential for efficient transfer and expression of the viral TK gene. Combining the results from HSV-1 and HSV-2, we conclude that a contiguous sequence of 1480 to 1540 bp is necessary to achieve at least 10 % of the maximum transformation efficiency.
Journal of clinical microbiology, 1982
We describe two methods for typing of herpes simplex virus (HSV). One procedure is based on the finding that the multiplication of HSV type 1 strains in primary rabbit kidney cells is inhibited by 2 x 10(-5) M iododeoxyuridine, whereas growth of HSV type 2 strains is considerably less affected. Forty-nine different HSV isolates were typed according to this method. For all isolates except two the results were found to be in agreement with results obtained by another typing procedure, the counterimmunoelectroosmophoretic method (S. Jeansson, Appl. Microbiol. 24:96-100, 1972). One HSV type 1 isolate behaved as a type 2 strain and was found to be a deoxythymidine kinase-negative mutant strain. The other deviant strain exhibited an intermediate iododeoxyuridine sensitivity, thus being impossible to type with this method. Another, faster typing procedure which is based on the immunological difference between HSV type 1 and 2 deoxythymidine kinase is also presented. This assay, in combinat...