D. R. Dubbs

Recombination between temperature-sensitive mutants of simian virus 40

Abstract Seven temperature-sensitive (ts) SV40 mutants have been isolated and characterized. All of the mutants were defective in a late function. Four of the mutants were assigned to complementation group B, one to a new group designated C and two could not be assigned to a complementation group. Recombination occurred between mutants in the B and C complementation groups and between mutants in the B group. The recombination frequency (RF) between tsB302 and tsB306 was about 2.0 × 10−4 when virus was used for infection, but was 10-fold higher when infectious ts SV40 DNAs were used. Treatment of doubly infected cells with 1-β- d -arabinofuranosylcytosine (ara-C) to interrupt DNA synthesis increased the RF 3- to 14-fold. Ultraviolet irradiation of viral inocula to 1–5% survival resulted in a 25- to 40-fold increase in RF. However, only a 2-fold increase in RF was obtained when uv-irradiated ts SV40 DNAs were used for infection. Ultraviolet irradiation of host CV-1 cells or pretreatment of host CV-1 cells with nonirradiated or uv-irradiated CV-1 DNA prior to infection failed to increase the RF between tsB302 and tsB306.

Regulation of herpesvirus thymidine kinase activity in LM(TK−) cells transformed by ultraviolet light-irradiated herpes simplex virus

Abstract Thymidine kinase (TK) activity of herpes simplex virus-transformed cells [LM(TK − )/HSV-1 and LM(TK-)/HSV-2] was stimulated 6 to 9 hr after the cells were infected with TK-negative mutants of either type 1 or type 2 herpes simplex virus (HSV-1 TK-, HSV-2 TK − ) or with TK-negative mutants of marmoset herpesvirus (MarHV TK − ) and pseudorabies virus (PRV TK − ) Inhibitors of DNA synthesis did not prevent this stimulation, but inhibitors of RNA and protein synthesis did. In contrast, equine herpesvirus (EHV-1) and TK-negative mutants of vaccinia virus did not enhance the TK activity of the transformed cells, showing that stimulation was not a consequence merely of nonspecific virus infection. HSV-1 132015, a mutant deficient in inducing TK activity in productively infected LM(TK − ) cells at 37°, stimulated HSV-transformed cell TK at this temperature, suggesting that the gene which controls TK turn-on probably functions normally in mutant 132015. These experiments suggest that enhancement by TK-negative herpesviruses can occur regardless of whether the superinfecting herpesvirus produces an inactive TK polypeptide or none at all. Autoradiographic and enzyme studies on a subline of LM(TK-)/HSV-1 cells grown in nonselective medium without HAT (hypoxanthine, aminopterin, and thymidine) demonstrated that essentially all of the cells contained significant TK activity. The TK activity of this subline and of clonal sublines isolated in nonselective medium or in counterselective medium with bromodeoxyuridine (BrUdR) was enhanced by HSV-1 TK − infection, supporting the suggestion by Davidson and co-workers that the HSV TK gene was retained in most transformed cells grown in nonselective medium. Immunological studies, using type-specific immunoglobulin (IgG) fractions prepared from sera of rabbits that had been immunized with HSV-1 TK or HSV-2 TK, demonstrated that the enhanced TK activity of HSV-1-transformed cells was inhibited by anti-HSV-1 IgG and that of HSV-2-transformed cells was inhibited by anti-HSV-2 IgG, regardless of whether the transformed cells were superinfected with HSV-1 TK − , HSV-2 TK − or MarHV TK − . Neither antiserum inhibited the TK activity induced by wild-type MarHV in LM(TK − ) cells, indicating that the TK activity stimulated by TK-negative herpesvirus infections of HSV-transformed cells had the antigenic specificity of the resident virus TK.

Thymidine Kinases Induced by Avian and Human Herpesviruses

Disc PAGE, isoelectric focusing, and glycerol gradient centrifugation experiments were carried out to characterize thymidine (dT) kinase isozymes induced by herpesvirus of turkeys and infectious laryngotracheitis virus in the cytosol fraction of infected chick cells. The avian herpesvirus dT kinases differed from chick cytosol dT kinase in electrophoretic mobility, isoelectric point and phosphate donor specificity. The avian herpesvirus dT kinases resembled chick mitochondrial dT kinase in electrophoretic mobilityy and isoelectric point, but exhibited larger sedimentation coefficients. The avian herpesvirus enzymes closely resembled dT kinases induced by human herpes simplex viruses types 1 and 2.

Thymidine kinase activity of biochemically transformed mouse cells after superinfection by thymidine kinase-negative, temperature-sensitive, herpes simplex virus mutants☆

Abstract Previous studies have shown that thymidine kinase (TK)-negative mutants isolated from HSV-1 clone 101, HSV-2 (333), marmoset herpesvirus, and pseudorabies virus enhanced the resident TK activity of biochemically transformed mouse fibroblast [LM(TK − )/HSV-1 Cl 7] cells. The present study shows that 10 additional TK-negative HSV-1 mutants isolated from HSV-1 strains HF and KOS also stimulated the resident TK activity of LM(TK − )/HSV-1 Cl 7 cells. To identify possible HSV-1 gene(s) regulating the formation of the resident HSV-1 TK, the LM(TK − )/HSV-1 Cl 7 cells were superinfected at permissive (34.5°) and nonpermissive (39°) temperatures with TK-negative mutants that were also temperature-sensitive (ts) for virus replication. TK-negative, HSV-1 ts mutants from 12 complementation groups (tsA1, B2, C4, E6, F18, G3, J12, K13, M19, N20, O22, and ts-24) were studied. All of these TK-negative HSV-1 ts mutants enhanced the resident TK activity of LM(TK − )/HSV-1 Cl 7 cells at the permissive temperature, and all mutants, except HSV-1 ts B2, did so when infections were carried out at the nonpermissive temperature. The resident TK activity of LM(TK − )/HSV-1 Cl 7 cells was either unaffected, or it was depressed after infection at 39° by mutant ts B2 at multiplicities of infection of 3–25 PFU/cell. The experiments indicate that the product of HSV-1 gene B is one of the regulatory proteins required for TK enhancement in biochemically transformed cells.

Characterization of nucleoside phosphotransferase and thymidine kinase activities of chick embryo cells and of chick-mouse somatic cell hybrids

Abstract Experiments were carried out to characterize the thymidine (dT) phosphorylating activities of chick embryo, chick erythrocytes, and of chick mouse somatic cell hybrids derived from fused chick erythrocytes and dT kinase-deficient LM(TK) mouse cells. Disc PAGE, isoelectric focusing, and glycerol gradient centrifugation analyses revealed that chick embryo cells contained four distinctive dT phosphorylating activities, two dT kinases and two nucleoside phosphotransferases. Thymidine kinase F. found principally in the cytosol, was also detected in mitochondrial and nuclear extracts, but was very low or absent from chick erythrocytes. Thymidine kinase A corresponds to the mitochondrial-specific isozyme found in bromodeoxyuridine-resistant mammalian cells. Nucleoside phosphotransferase activities were very active in chick embryo cytosol and were detected in embryo mitochondria! and nuclear extracts and cytosol and nuclear extracts of chick erythrocytes. Most of the chick embryo nucleoside phosphotransferase activity could be removed by purification of cytosol dT kinase F. Chick-mouse somatic cell hybrids exhibited chick dT kinase F, but neither chick dT kinase A. chick nucleoside phosphotransferase, nor mouse cytosol dT kinase activities. The results indicate (1) the genetic determinant for chick cytosol dT kinase F is on a different chromosome from the determinants for the chick nucleoside phosphotransferases and mitochondrial dT kinase A, and/or (2) only the chick cytosol dT kinase F, but neither the chick nucleoside phosphotransferases nor dT kinase A, was reactivated in the hybrids.

Biochemical transformation of mouse cells by a purified fragment of marmoset herpesvirus DNA.

Although the size of marmoset herpesvirus (MarHV) DNA, estimated by velocity sedimentation in sucrose gradients, was similar to that of herpes simplex virus type 1 (HSV-1) DNA, the restriction endonuclease sites of MarHV and HSV-1 DNAs were quite different. A specific BamHI restriction fragment (6.2 x 10(6) daltons) of MarHV DNA biochemically transformed LM(TK-) mouse fibroblasts to the thymidine kinase(TK)-positive phenotype. Rabbit antisera, prepared against MarHV TK, inhibited MarHV-induced TK, but not HSV-1, HSV-2, or cellular TKs. Disc PAGE analyses and enzyme neutralization experiments with the anti-MarHV TK sera demonstrated that the TK expressed in MarHV transformants was MarHV-specific.

Biochemical and serological properties of the thymidine-phosphorylating enzymes induced by herpes simplex virus mutants temperature-dependent for enzyme formation

Abstract Evidence is presented in support of the hypothesis that: (i) an enzyme induced by wild-type herpes simplex virus type 1 (HSV-1) is a deoxypyrimidine kinase with a single active site for the phosphorylation of both thymidine (TdR) and deoxycytidine (CdR); (ii) enzymes induced by HSV-1 mutants that are temperature dependent for enzyme induction are altered with respect to nucleoside acceptor specificity and antigenicity; and (iii) altered polypeptides are synthesized in mutant virus-infected cells at both the permissive (31°) and restrictive (37.5°) temperatures. HSV-1 mutants B2010 and B2015 induce less TdR-phosphorylating activities at 31° than wild-type HSV-1 and very low but detectable enzyme activities at 37.5°. The TdR-phosphorylating activities induced by the mutant viruses at 31° have the same electrophoretic mobilities as that of the wild-type HSV-1-induced enzyme but, unlike the wild-type enzyme, the mutant enzymes lack CdR-phosphorylating activity. Immunoglobulin G (IgG) prepared from rabbits immunized with cytosol extracts from rabbit kidney cells infected with wild-type HSV-1 inhibit the TdR-phosphorylating activities induced by wild-type and mutant viruses, as well as the CdR-phosphorylating activity induced by wild-type HSV-1. Extracts purified from cells infected at 31° with wild-type and mutant HSV-1 blocked the neutralizing activity of anti-HSV-1 IgG for the CdR- as well as the TdR-phosphorylating activities of wild-type HSV-1 enzyme, despite the lack of CdR-phosphorylating activity of the mutant enzyme. However, the mutant virus-infected cell extracts were less effective than wild-type extracts in serum blocking activity. Extracts from cells infected with mutant B2015 at 37.5° did not exhibit serum blocking power.

Integration site(s) of herpes simplex virus type 1 thymidine kinase gene and regional assignment of the gene for aminoacylase-1 in human chromosomes

To investigate the chromosomal sites of integration of the herpes simplex virus type 1 (HSV-1) thymidine kinase (TK) gene in HSV-1-transformed human HeLa(BU25)/KOS 8-1 cells, the biochemically transformed cells were fused with TK-negative mouse LM(TK-) cells, and human-mouse somatic cell hybrid lines (LH81) were isolated using a HATG-ouabain selection system. The presence of HSV-1 TK activity in the hybrid lines was verified by disc polyacrylamide gel electrophoresis (PAGE) and by enzyme neutralization with type-specific rabbit anti-HSV-1 TK immunoglobulin. Karyotype analyses of several somatic cell hybrid clones using G-banding, Hoechst 33258 staining, and combined G-banding and Hoechst staining demonstrated that they retained only a few human chromosomes. A marker chromosome, M7, consisting of a chromosome 17 translocated to the short arm of 3, occurred in 25 of the 28 metaphases examined. Also chromosomes 8 and X were found in a minority of metaphases. Isozyme analyses showed that all 19 hybrid clones analyzed expressed human aminoacylase-1 (ACY1) and esterase D (ESD), markers for 3 and 13, respectively. Back-selection of somatic cell hybrid clones with 5-bromodeoxyuridine resulted in the isolation of several subclones lacking HSV-1 TK activity, human ACY1, human ESD, and the human chromosomes. These experiments suggest that the HSV-1 TK gene is associated with either M7 or a segment of 13, or both, in biochemically transformed HeLa(BU25)/KOS 8-1 cells. These experiments also permit localization of the ACY1 structural gene to the pter leads to p12 region of 3.

Gel Electrophoresis, Isoelectric Focusing, and Localization of Thymidine Kinase in Normal and Simian Virus 40-Infected Monkey Cells

Following SV40 infection of CV-1 monkey cells, cytosol thymidine (dT) kinase activity increased 4- to 10-fold. This enzyme was not significantly different from the cytosol dT kinase of uninfected cells with respect to mobility, isoelectric point, sedimentation coefficient, and phosphate donor specificity. Monkey mitochondria contain a distinctive dT kinase isozyme localized in the mitochondrial matrix. The distinctive mitochondrial isozyme had a higher electrophoretic mobility, lower isoelectric point, and smaller sedimentation coefficient than the cytosol dT kinase, and could utilize either ATP or UTP as phosphate donor; it did not increase after SV40 infection. Mitochondria also contained a cytosol-like dT kinase which was enhanced in infected cells. These findings suggest that SV40 infection derepresses the normal cytosol dT kinase of host cells.

Mapping thymidine kinase-deficient mutants of vaccinia virus by marker rescue with hybrid plasmid DNAs containing portions of the HindIII-J fragment of virus DNA

Five hybrid plasmids were constructed, each containing a portion of the vaccinia virus DNA HindIII-J fragment. These plasmid DNAs were used in marker rescue experiments to map the mutations in the thymidine kinase (TK) gene of three TK- vaccinia virus mutants. The TK gene of each of the three mutants was rescued by DNA from plasmid pPJ701, which contained about one-half of the HindIII-J fragment. Two mutants, 1004B and 1017-1, but not the third, 1016-1, were rescued by DNA of two plasmids, pPJ702 and pPJ703, which contained 16 and 18%, respectively, of one end of the J fragment. Mutant 1016-1 could be rescued by plasmid pPJ705 containing a 1.69-kb fragment of the HindIII-J fragment. The J fragment DNA in plasmid pPJ705 is located adjacent to that and separated by an EcoRI site from pPJ703 in the vaccinia virus genome. These results indicate that the mutation site in the TK gene of 1016-1 differs from that in 1004B or 1017-1 and suggests that the structural gene for the vaccinia virus TK lies near one end of the HindIII-J fragment and spans the EcoRI site.