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.     

Effects of 5-bromodeoxyuridine on metamorphosis and on cell cycle kinetics of ganglion cells in Drosophila melanogaster

In order to determine suitable experimental conditions for estimating the accurate spontaneous frequency of sister chromatid exchanges (SCEs) in vivo in somatic cells of Drosophila melanogaster, the effects of bromodeoxyuridine (BUdR) on metamorphosis as well as on cell cycle kinetics were examined. The rate of growth of third-instar larvae, fed on BUdR-containing synthetic medium, markedly delayed with increasing concentrations of BUdR, but this toxic effect of BUdR was not observed below 150 μg/ml.Furthermore, the rate of eclosion drastically decreased by the incorporation of BUdR: it was reduced to about one-half of that in the control when the larvae were exposed to 100 (μg/ml. On the other hand, little difference in the rate of pupation was found within the range of 0–800 μg/ml BUdR. These results indicate that the developmental stage from pupa to adult is the most sensitive phase to BUdR.To test the effect of BUdR on cell cycle, metaphase cells were classified as having undergone each replication cycle in the presence of different BUdR concentrations according to the pattern of differential staining of sister chromatids, and the proportion of each replication cycle cells examined. No inhibition of cellular kinetics was observed at BUdR concentrations below 200 μg/ml.On the basis of these results, 100 μg/ml was chosen as suitable BUdR concentration for the analysis of cell cycle kinetics and according to the distribution of replication cycle metaphase cells as a function of time after the initiation of BUdR treatment, the cell cycle duration of the third-instar larval ganglion cells was roughly estimated to be about 7–8 h, at least under our experimental conditions.     

Effects of a bacteriocin from Mycobacterium smegmatis on BALB/3T3 and simian virus 40-transformed BALB/c mouse cells

The effects of a bacteriocin from Mycobacterium smegmatis ATCC 14468 on simian virus 40-transformed BALB/c mouse cells (mKS-A TU-7 cells) and nontransformed BALB/3T3 cells originating from the BALB/c mouse strain were studied. The percentage of nigrosin-unstained (viable) cells in the bacteriocin-treated mKS-A TU-7 cells decreased time-dependently with an increase in the bacteriocin activity. There was a time-dependent decrease in the bacteriocin activity after treatment with the cell membrane preparation from mKS-A TU-7 cells. There was no apparent effect of the bacteriocin on the viability of nontransformed BALB/3T3 cells. Wheat germ agglutinin blocked the toxic effect of bacteriocin on mKS-A TU-7 cells. These results indicate that the higher sensitivity and binding capacity of the tumor cells to the bacteriocin is probably due to the presence of a large amount of N-acetyl-glucosamine or closely related sugar residues with a high affinity for bacteriocin, as compared with normal cells. The bacteriocin produced morphological alterations and inhibition of synthesis of ribonucleic acid, deoxyribonucleic acid and protein in the transformed but not in the nontransformed cells.     

Effect of L-phenylalanine on I-methyladenine production and spontaneous oocyte maturation in starfish

5-Bromodeoxyuridine (BUdR)-resistant cells were obtained from N-methyl-N′-nitro-N-nitrosoguanidine (NTG)-treated soybean protoplasts and cultured in liquid nutrient medium containing BUdR (20 μg/ml) and uridine (100 μg/ml). Addition of uridine to the medium improved growth of the BUdR-resistant cells. The growth of BUdR-resistant cells was partly inhibited when hypoxanthine, aminopterine, glycine and thymidine were added to the medium. Both BUdR-resistant and BUdR-sensitive cells exhibited thymidine kinase activity. CsCl density gradient analyses showed that the DNA of BUdR-resistant cells, which were cultured in the presence of BUdR, had a buoyant density of 1.703 g/ml, while the DNA of the parental soybean cells grown without BUdR had a buoyant density of 1.692 g/ml. Uptake of ³H-thymidine or ¹⁴C-BUdR by the cells occurred in both BUdR-resistant and BUdR-sensitive cells. CsCl density gradient patterns of labelled DNA also demonstrated that ¹⁴C-BUdR and ³H-thymidine were incorporated into the DNA of BUdR-resistant cells, as well as into that of BUdR-sensitive cells.     

Effects of controlled exposure of L cells to bromodeoxyuridine: II. Turnover rates and activity profiles during cell cycle of bromodeoxyuridine-sensitive and -resistant enzymes

Fluorodeoxyuridine (FUdR)-synchronized mouse L cells were allowed to incorporate 5-bromodeoxyuridine (BUdR) at restricted intervals in the S phase and the effects of the selective incorporation of BUdR in DNA on the activities of seven randomly chosen enzymes (five dehydrogenases and two phosphatases) were analysed. Reductions to 56.9 and 83.3 % of the control levels were noted for glucose-6-phosphate dehydrogenase (G6PD) and alcohol dehydrogenase (ADH) activities respectively, when cells were exposed to BUdR during the 1st h of S. Acid phosphatase (AcP) activity was reduced to 81.9% of the control level following exposure to the analogue during the 3rd h of S. Exposure of cells to BUdR for the entire S period failed to increase the magnitude of the reductions in activity for any of these three enzymes. Alternately, when cells were allowed to synthesize DNA in the presence of thymidine for the 1st h of S and the remainder in the presence of BUdR, the activities of G6PD and ADH were comparable to those found in untreated cells. Exposure of cells to thymidine for the 3rd h of S, combined with exposure to BUdR for the preceding and subsequent hours of S, provided complete protection against the BUdR-mediated reduction in AcP activity. The activities of lactate dehydrogenase (LDH), 6-phosphogluconate dehydrogenase (6pGD), isocitrate dehydrogenase (IDH) and alkaline phosphatase (A1P) were found to be insensitive to treatment with BUdR, even when the period of analogue exposure encompassed the entire S period.Additional investigations carried out with G6PD for characterization of the nature of the BUdR effects suggest that the BUdR-mediated reductions in enzyme activities are not caused by the increased rates of degradation of the enzymes, formation of enzyme inhibitors or by the disproportionate replication of A-T base pairs during BUdR treatment. The alterations of enzyme activities appear to result from decreased rates of synthesis of enzymes in BUdR-treated cells. The results of the present study clearly suggest that pulse labelling of cells with BUdR at various intervals of the S phase may provide a useful approach for determining temporal localization of replication time of DNA segments that are critical for the synthesis or regulation of specific gene products.     

Origin of Thymidine Kinase in Adenovirus-Infected Human Cell Lines

Human adenovirus type 5 enhances the thymidine kinase activity of KB cells but does not induce the enzyme in kinase-deficient HeLa (BU25) cells. Vaccinia induces thymidine kinase activity in both KB and HeLa (BU25) cells. Human adenovirus types 2, 4, 7, and 12 also fail to induce the enzyme in HeLa (BU25) cells. Vaccinia replicates equally well in the presence or absence of HATG (hypoxanthine-aminopterin-thymidine-glycine) in KB and HeLa (BU25) cells. Adenovirus type 5 replicates in KB and in HeLa (BU25) cells in the absence of HATG, and adenovirus type 5 replicates in kinase-positive KB cells in the presence of HATG. However, replication of adenovirus type 5 is grossly inhibited in HeLa (BU25) cells in the presence of HATG. These results suggest that human adenoviruses do not code for a new virus-specific thymidine kinase.     

Characterization of pyrimidine deoxyribonucleoside kinase (thymidine kinase) and thymidylate kinase as a multifunctional enzyme in cells transformed by herpes simplex virus type 1 and in cells infected with mutant strains of herpes simplex virus.

Pyrimidine deoxyribonucleoside kinase (thymidine kinase [TK]) was purified from two herpes simplex virus type 1 (HVS-1)-transformed TK-deficient mouse (LMTK-) cell lines and from LMTK- cells infected with HSV-1 mutant viruses coding for variant TK enzymes. These preparations exhibited normal or variant virus-induced thymidylate kinase activities correlating with their relative TK activities. Neither virus-induced activity was detected in LMTK- cells infected with an HSV-1 TK-deficient mutant. These results suggest that HSV-1 thymidylate kinase activity and TK activity are mediated by the same protein.     

Inhibition of Herpes Simplex Virus Type 2 Replication by Thymidine

Replication of herpes simplex virus type 2 (HSV-2) was impeded in KB cells which were blocked in their capacity to synthesize DNA by 2 mM thymidine (TdR). The degree of inhibition was dependent upon the concentration of TdR. In marked contrast, HSV-1 is able to replicate under these conditions. The failure of HSV-2 to replicate is probably due to the inhibition of viral DNA synthesis; there was a marked reduction in the rate of DNA synthesis as well as the total amount of HSV-2 DNA made in the presence of 2 mM TdR. We postulated that the effect of TdR on viral replication occurs at the level of ribonucleotide reductase in a manner similar to KB cells. However, unlike KB cells, an altered ribonucleotide reductase activity, highly resistant to thymidine triphosphate inhibition, was found in extracts of HSV-2-infected KB cells. This activity was present in HSV-2-infected cells incubated in the presence or absence of TdR. Ribonucleotide reductase activity in extracts of HSV-1-infected KB cells showed a similar resistance to thymidine triphosphate inhibition. These results suggest that the effect of TdR on HSV-2 replication occurs at a stage of DNA synthesis other than reduction of cytidine nucleotides to deoxycytidine nucleotides.     

Virus Type-Specific Thymidine Kinase in Cells Biochemically Transformed by Herpes Simplex Virus Types 1 and 2

Transformation of mouse cells (Ltk(-)) and human cells (HeLa Bu) from a thymidine kinase (TK)-minus to a TK(+) phenotype (herpes simplex virus [HSV]-transformed cells) has been induced by infection with ultraviolet-irradiated HSV type 2 (HSV-2), as well as by HSV type 1 (HSV-1). Medium containing methotrexate, thymidine, adenine, guanosine, and glycine was used to select for cells able to utilize exogenous thymidine. We have determined the kinetics of thermal inactivation of TK from cells lytically infected with HSV-1 or HSV-2 and from HSV-1- and HSV-2-transformed cells. Three hours of incubation at 41 C produces a 20-fold decrease in the TK activity of cell extracts from HSV-2-transformed cells and Ltk(-) cells lytically infected with HSV-2. The same conditions produce only a twofold decrease in the TK activities from HSV-1-transformed cells and cells lytically infected with HSV-1. This finding supports the hypothesis that an HSV structural gene coding for TK has been incorporated in the HSV-transformed cells.     

The amplified expression of factors regulating myogenesis in L6 myoblasts

A strategy for increasing the expression of the factors regulating myogenesis was developed based upon the observation that increased amounts of regulatory factors could overcome the inhibition of differentiation produced by 5-bromodeoxyuridine (BUdR). L6 rat myoblasts were subjected to multiple cycles of cloning in progressively increasing concentrations of BUdR. The first clones to differentiate were picked and replated for the next cycle of selection. After 28 cycles in BUdR, cells were isolated that could differentiate in the presence of 8 microM BUdR. Cell hybrids between myoblasts subjected to 21 cycles of selection (BU21 cells) and differentiation-defective myoblasts exhibited a high probability of differentiation, consistent with the hypothesis that BU21 cells were overproducing factor(s) involved in the decision to differentiate. The selection of cells able to differentiate in the presence of BUdR may provide a general approach for increasing the expression of the regulatory molecules controlling terminal differentiation.     

Mutations in the M112/M113-Coding Region Facilitate Murine Cytomegalovirus Replication in Human Cells

Cytomegaloviruses, representatives of the Betaherpesvirinae, cause opportunistic infections in immunocompromised hosts. They infect various cells and tissues in their natural host but are highly species specific. For instance, human cytomegalovirus (HCMV) does not replicate in mouse cells, and human cells are not permissive for murine cytomegalovirus (MCMV) infection. However, the underlying molecular mechanisms are so far poorly understood. In the present study we isolated and characterized a spontaneously occurring MCMV mutant that has gained the capacity to replicate rapidly and to high titers in human cells. Compared to the parental wild-type (wt) virus, this mutant formed larger nuclear replication compartments and replicated viral DNA more efficiently. It also disrupted promyelocytic leukemia (PML) protein nuclear domains with greater efficiency but caused less apoptosis than did wt MCMV. Sequence analysis of the mutant virus genome revealed mutations in the M112/M113-coding region. This region is homologous to the HCMV UL112-113 region and encodes the viral early 1 (E1) proteins, which are known to play an important role in viral DNA replication. By introducing the M112/M113 mutations into wt MCMV, we demonstrated that they are sufficient to facilitate MCMV replication in human cells and are, at least in part, responsible for the efficient replication capability of the spontaneously adapted virus. However, additional mutations probably contribute as well. These results reveal a previously unrecognized role of the viral E1 proteins in regulating viral replication in different cells and provide new insights into the mechanisms of the species specificity of cytomegaloviruses.Cytomegaloviruses (CMVs) are prototypes of the β subfamily of the Herpesviridae. Representatives of this subfamily have been identified in various animal species, and these viruses cause similar symptoms in their respective hosts (36). HCMV is an opportunistic pathogen that causes generally mild infections in people with a fully functional immune system. However, this virus is also responsible for serious medical problems, particularly in newborns and immunocompromised patients (39).Since their first isolation in cell culture, CMVs have been recognized as highly species specific (57). They replicate only in cells of their own or a closely related species. For instance, simian CMV can replicate in human fibroblasts (32), and HCMV can replicate in chimpanzee skin fibroblasts (41). Similarly, murine cytomegalovirus (MCMV) productively infects rat cells (7, 46), but a rat cytomegalovirus did not replicate in murine fibroblasts (7). However, cells of other more distantly related species are usually nonpermissive to infection. Several studies have shown that CMVs can enter cells of other species and express a subset of viral genes (19, 20, 29, 32). This finding has led to the conclusion that the restriction to CMV replication in nonpermissive cells is associated with a postpenetration block to viral gene expression and DNA replication but not due to a failure to enter the cell (36).Recently, we picked up on this topic and tried to gain new insights into the molecular mechanisms underlying the species specificity of CMVs. We showed that CMVs of mice and rats induce apoptosis when they infect human fibroblasts or retinal epithelial cells (26). The induction of apoptosis prevented a sustained replication of these viruses in human cells and reduced progeny production to insignificant levels. When apoptosis was inhibited by the overexpression of Bcl-2 or a functionally similar protein, MCMV was able to replicate to substantial titers in human cells. These results indicated that the induction of apoptosis is an important limitation to cytomegalovirus cross-species infections (26). However, the fact that MCMV replication in human cells in the presence of apoptosis inhibition was somewhat delayed and less efficient than that in murine cells indicated that other limiting factors likely exist. Another study suggested that MCMV can replicate to low levels in human cells with the help of HCMV immediate-early 1 (IE1) and HCMV tegument proteins (50).In the present study, we describe the isolation and characterization of a mutant MCMV that has spontaneously acquired the ability to replicate rapidly and to high titers in human retinal pigment epithelial (RPE-1) cells. We show that this virus induces less apoptosis and replicates its DNA faster than the parental wild-type (wt) MCMV. Moreover, the mutant virus disrupts intranuclear sites of intrinsic antiviral defense more efficiently than the wt virus. Sequence analysis of the human cell-adapted MCMV strain revealed several alterations, including mutations in the M112/M113-coding region. By targeted mutagenesis we showed that mutations in M112/M113 are sufficient to facilitate MCMV replication in human cells. However, additional mutations most likely contribute to the remarkably efficient replication of the adapted strain.     

Interaction of herpes simplex virus type 2 with a rat glioma cell line

The interaction between herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) and two neural cell lines, mouse neuroblastoma (N1E-115) and rat glioma (C6-BU-1), was investigated. N1E-115 cells were permissive to both types of HSV. In C6-BU-1 cells, on the other hand, all the HSV-1 strains tested so far showed persistent infection, and the infectious virus of HSV-2 strains disappeared spontaneously. The HSV-2-infected C6-BU-1 cells were positive for HSV-2-specific DNA sequences, virus-specific RNA, HSV-2-specific antigens and thymidine kinase activity, when no infectious virus was detected. The HSV-2 was reactivated from those C6-BU-1 cells by superinfection with murine cytomegalovirus (MCMV), but not with UV-irradiated MCMV or human cytomegalovirus. The reactivated HSV-2 was identical to the parental virus, when examined by restriction endonuclease cleavage analysis.     

The ribonucleotide reductase R1 homolog of murine cytomegalovirus is not a functional enzyme subunit but is required for pathogenesis

Ribonucleotide reductase (RNR) is the key enzyme in the biosynthesis of deoxyribonucleotides. Alpha- and gammaherpesviruses express a functional enzyme, since they code for both the R1 and the R2 subunits. By contrast, betaherpesviruses contain an open reading frame (ORF) with homology to R1, but an ORF for R2 is absent, suggesting that they do not express a functional RNR. The M45 protein of murine cytomegalovirus (MCMV) exhibits the sequence features of a class Ia RNR R1 subunit but lacks certain amino acid residues believed to be critical for enzymatic function. It starts to be expressed independently upon the onset of viral DNA synthesis at 12 h after infection and accumulates at later times in the cytoplasm of the infected cells. Moreover, it is associated with the virion particle. To investigate direct involvement of the virally encoded R1 subunit in ribonucleotide reduction, recombinant M45 was tested in enzyme activity assays together with cellular R1 and R2. The results indicate that M45 neither is a functional equivalent of an R1 subunit nor affects the activity or the allosteric control of the mouse enzyme. To replicate in quiescent cells, MCMV induces the expression and activity of the cellular RNR. Mutant viruses in which the M45 gene has been inactivated are avirulent in immunodeficient SCID mice and fail to replicate in their target organs. These results suggest that M45 has evolved a new function that is indispensable for virus replication and pathogenesis in vivo.     

A simple technique for in vivo observation of SCE in mouse ascites tumor and spermatogonial cells

A simplified technique to observe sister chromatid exchange (SCE) in ascites tumor cells and spermatogonial cells of the mouse was described. The technique consisted of a single intraperitoneal injection of 5-bromo-2′-deoxyuridine (BUdR) absorbed on activated charcoal that assured continuous release of BUdR throughout two rounds of the replication period. The minimum dose of BUdR to detect SCE was found to be 5 mg/body, the SCE frequency being 4.50 – 4.90/ cell in tumor cells and 1.30 – 1.88/cell in spermatogonia, respectively.     

Use of [125I]deoxycytidine to detect herpes simplex virus-specific thymidine kinase in tissues of latently infected guinea pigs

The footpad skin and the lumbosacral dorsal root ganglia were removed from inbred guinea pigs at different times after subcutaneous infection with herpes simplex virus type 2 (HSV-2) in both hind footpads. These tissues, shown by our previous study to harbor latent HSV, were dispersed into single cells. The presence of virus-specific thymidine kinase (TK) in these cells was assayed by the uptake and phosphorylation of [125I]deoxycytidine in culture. [125I]deoxycytidine was shown to be a specific substrate for the HSV-coded TK. The method could detect herpes TK activity in a culture of 10(6) cells with less than 0.1% of the cells being virally infected. The enzyme was readily detected in footpad cells of acutely (24 h) but not of latently (14 days to 1 year) infected guinea pigs. No herpes TK was found either in the sensory ganglionic cells of guinea pigs during the early and late phases of latent infection. It is concluded that HSV-2, while residing in the footpads and the lumbosacral ganglia of the guinea pig during latent infection, does not express any viral TK function.     

Live covisualization of competing adeno-associated virus and herpes simplex virus type 1 DNA replication: molecular mechanisms of interaction

We performed live cell visualization assays to directly assess the interaction between competing adeno-associated virus (AAV) and herpes simplex virus type 1 (HSV-1) DNA replication. Our studies reveal the formation of separate AAV and HSV-1 replication compartments and the inhibition of HSV-1 replication compartment formation in the presence of AAV. AAV Rep is recruited into AAV replication compartments but not into those of HSV-1, while the single-stranded DNA-binding protein HSV-1 ICP8 is recruited into both AAV and HSV-1 replication compartments, although with differential staining patterns. Slot blot analysis of coinfected cells revealed a dose-dependent inhibition of HSV-1 DNA replication by wild-type AAV but not by rep-negative recombinant AAV. Consistent with this, Western blot analysis indicated that wild-type AAV affects the levels of the HSV-1 immediate-early protein ICP4 and the early protein ICP8 only modestly but strongly inhibits the accumulation of the late proteins VP16 and gC. Furthermore, we demonstrate that the presence of Rep in the absence of AAV DNA replication is sufficient for the inhibition of HSV-1. In particular, Rep68/78 proteins severely inhibit the formation of mature HSV-1 replication compartments and lead to the accumulation of ICP8 at sites of cellular DNA synthesis, a phenomenon previously observed in the presence of viral polymerase inhibitors. Taken together, our results suggest that AAV and HSV-1 replicate in separate compartments and that AAV Rep inhibits HSV-1 at the level of DNA replication.