Lack of correlation between thymidine kinase activity and the antiviral or antiproliferative response to interferon

Murine L cells lacking thymidine kinase activity (Ltk-) do not respond to the antiviral or antiproliferative activity of interferon, whereas human tk- cells show a normal response. Furthermore, a clone of tk+ cells derived from Ltk- cells by DNA-mediated transfer of the tk herpes gene does not respond to interferon. Cells of this clone and Ltk- cells do not produce interferon when infected with Newcastle disease virus or treated with poly(I) X poly(C) and DEAE-dextran. Possible reasons for this inability to produce interferon and the lack of response to interferon are discussed.     

Effect of an amber mutation in the herpes simplex virus thymidine kinase gene on polypeptide synthesis and stability

KG111 is a mutant of herpes simplex virus (HSV), strain KOS, that exhibits temperature-dependent drug resistance. For example, it is almost as resistant as a thymidine kinase (tk)-deficient virus at 39 degrees, but is relatively sensitive to acyclovir at 34 degrees, Using marker transfer techniques, we have mapped the mutation conferring temperature-dependent drug resistance in KG111 to the 5' portion of the tk gene. Sequencing of this region revealed an amber mutation at codon 44, which lies between the first and second methionine codons of the tk polypeptide. This mutation is identical to that found in TK4, an HSV mutant derived from Cl 101 (L. Haarr et al., 1985, J. Virol. 56, 512-519). Analyses of immunoprecipitated tk proteins from KG111- and TK4-infected cells showed that KG111 and TK4 do not synthesize full-length tk polypeptides, but instead produce a truncated form of the protein. Small amounts of a similar truncated tk polypeptide are also produced in wild-type-infected cells and are thought to arise from initiation at a downstream AUG. The relative amounts and size of the mutant tk proteins compared with those of the wild-type are consistent with the amber mutation eliminating translation of full-length polypeptide and causing a four- to fivefold increase in the utilization of downstream AUG codons for initiation. The truncated polypeptides specified by KG111 and TK4 are less stable than the full-length polypeptide at 39 degrees, which may contribute to the conditional drug-resistant phenotype. On the other hand, the truncated polypeptides normally expressed by wild-type virus at low levels and the more highly expressed truncated tk polypeptides from a deletion mutant are relatively stable at 39 degrees. These results suggest that stability of the truncated tk polypeptide is influenced by the amount of tk present.     

Synthesis of vaccinia virus thymidine kinase in microinjected Xenopus oocytes

Messenger RNA was extracted from vaccinia virus (VV) -infected thymidine kinase (tk)-deficient cells early in infection and introduced into the cytoplasm of Xenopus laevis oocytes by microinjection. Extracts prepared from these oocytes after 24 hr incubation exhibited a thymidine kinase activity that was more than 250-fold greater than the endogenous oocyte level. Messenger RNA extracted late during VV infection, or from cells infected with a tk^? mutant of VV, failed to direct the increase in tk activity. The enzyme synthesized in oocytes appeared to be identical to authentic VV tk from infected human tk^? cells by the criteria of thermal inactivation kinetics, sedimentation velocity, and electrophoretic mobility under nondenaturing conditions. Moreover, tk synthesis was specifically arrested if, prior to microinjection, the early VV mRNA was hybridized to a recombinant plasmid that contained the VV tk gene. However, attempts to detect the synthesis of VV tk in Xenopus oocytes which had been injected intranuclearly with VV DNA fragments containing the tk gene were uniformly unsuccessful.     

HSV_1tk/GCV基因治疗系统的旁观者效应观察

探讨逆转录病毒 (retrovirus ,RV )载体介导的单纯疱疹病毒胸苷激酶 (HSV1 tk )基因转染 ,联合抗病毒药物核苷类似物羟甲基无环鸟苷 (ganciclovir,GCV )对人卵巢上皮癌细胞系TYK细胞杀伤过程中所产生的旁观者效应。采用脂质体介导法将PLNTK5质粒转入包装细胞PA317后 ,以滴度最高的PA317病毒上清液感染TYK细胞 ,遗传霉素G418筛选后 ,得到带有HSV1 tk基因的TYK细胞。将细胞按不同比例混合培养后 ,给予 10 μg/mlGCV ,4d后用MTT法计算细胞存活率 ,观察旁观者效应。PLNTK5质粒成功转入PA317细胞 ,病毒滴度最高者为 6× 10 5cfu/ml。用病毒上清液感染TYK细胞 ,成功地得到了表达HSV1 tk的卵巢癌细胞株TYK/tk。混合培养结果显示 ,TYK/tk细胞占混合细胞 10 %时 ,低浓度的GCV就可使一半以上的细胞杀死 ,证明了HSV1 tk/GCV治疗方法存在着旁观者效应。逆转录病毒可介导HSV1 tk基因转入TYK细胞并获稳定表达 ,HSV tk/GCV系统存在旁观者效应。     

Synthesis of herpes simplex virus DNA in preparations of chromatin from infected cell nuclei.

Chromatin prepared from cells infected with Herpes simplex virus type 1 or type 2 can synthesize both virus and cell DNA in vitro. The rate of synthesis is comparable to that of isolated whole nuclei. Incorporation is limited, and both cell and virus DNA synthesis are sensitive to the presence of virus-specific antiserum and phosphonoacetate. In chromatin from cells infected with a phosphonoacetate resistant virus mutant, both types of DNA synthesis are resistant to the presence of the inhibitor.     

Application of antisera to interferon in studying oncogenic viruses.

Susceptibility of BALB/c mice to infection with Moloney sarcoma virus (MSV) and to Herpes simplex virus type 2 (HSV-2) was considerably increased by administration of sheep anti-mouse interferon (anti-IF) serum. The regression of the MSV-induced tumors was inhibited when the weanling (three-to-four-week-old) mice were injected with the anti-IF serum. Using the anti-IF serum it has been found that the antagonism between HSV-2 and Rauscher leukemia virus in the mouse is not mediated by interferon. It is suggested that interferon is an important factor controlling growth of virus and/or virus induced tumor cells in the mouse before it develops a strong immunological response.     

Low levels of herpes simplex virus thymidine- thymidylate kinase are not limiting for sensitivity to certain antiviral drugs or for latency in a mouse model

Herpes simplex virus mutant KG111 contains a nonsense mutation at codon 44 of the viral thymidine kinase (tk) gene and produces low amounts of a truncated tk polypeptide. We tested mutant KG111 and related viruses that specify varying amounts of similar truncated tk polypeptides for their sensitivities to antiviral nucleoside analogs at different temperatures using plaque reduction assays. The results of these assays showed that the nonsense mutation confers high resistance to bromovinyldeoxyuridine (BVdU) at any temperature and temperature-dependent resistance to acyclovir (ACV), buciclovir (BCV), ganciclovir (DHPG), and fluoroiodoarabinouracil (FIAU). Above relatively low threshold levels of tk that varied depending on the drug tested, viruses exhibited full sensitivity to ACV, BCV, DHPG, and FIAU at 34 degrees. Below these threshold levels, however, decreases in drug sensitivity were linear with decreases in tk levels, forming the basis of a pharmacological assay for tk gene expression. Studies of thymidine (TdR) anabolism in infected 143 tk-cells showed that when high TdR concentrations were added to the medium, KG111 directed thymidine monophosphate (TMP) formation at rates consonant with the amount of tk polypeptide produced by the mutant. When low concentrations to TdR were added to the medium, however, KG111 directed TMP formation at a rate similar to that directed by wild-type virus, indicating that the truncation of the tk polypeptide had little or no effect on tk activity at 34 degrees. Subsequent anabolism to thymidine diphosphate and thymidine triphosphate was reduced in KG111-infected cells, indicating a defect in TMP kinase activity that explains this mutant's resistance to BVdU. Despite the low levels of tk and TMP kinase activity expressed by KG111, this mutant established reactivatable latent infections as efficiently as wild-type virus in a mouse model.     

Action of human leukocyte interferon on poliomyelitis virus reproduction in resistant MIO(r) cells

The effect of human leukocyte interferon on reproduction of poliomyelitis virus in MIO cells resistant to this virus (MIOr) and sensitive MIO cells was studied. Interferon was shown to exert a short-time protective effect in the sensitive cells and to induce virus reproduction in the resistant cells. It is suggested that poliomyelitis virus reproduction in the resistant cells is due to activation of lysosomal enzyme, cathepsin D, in this system.     

Thymidine kinase of herpes simplex virus type 1 strain KOS lacks mutator activity

The effect of thymidine kinase (TK) encoded by herpes simplex virus type 1(HSV-1) strain KOS in DNA replication fidelity was examined by two different mutagenesis assays. Mutagenesis assay of the LacZ reporter gene present in recombinant tkLTRZ1, which contained the integrated LacZ gene in the tk locus, revealed a less than 0.05% mutation frequency of the LacZ gene regardless of whether the viruses were propagated in TK-expressing cells or control cells, conflicting an earlier report that a HSV-1 TK(+) strain replicated a 0.5% mutation frequency of the LacZ gene (R. B. Pyles and R. L. Thompson, 1994, J. Virol. 68, 4514-4524). Furthermore, TK-proficient and -deficient recombinant viruses replicated with similar mutation frequencies (0.027 and 0.026%, respectively) of the LacZ gene, which was integrated in the polymerase locus. Results of SupF mutagenesis assay demonstrated that neither the spectra of mutation nor the mutation frequencies of SupF gene, which was integrated in the tk locus of recombinant, were significantly different (P > 0.05) in progeny viruses grown in TK-expressing cells and control cells. Therefore, both LacZ and SupF mutagenesis assays demonstrated that TK of the HSV-1 strain KOS did not have detectable mutator activity.     

Herpes simplex virus type 1 alpha gene containing plasmids can inhibit expression regulated from an alpha promoter in CV-1 but not HeLa cells

Transfection of plasmids containing the herpes simplex virus type 1 (HSV-1) alpha gene 27 has been observed to inhibit gene expression from a virus alpha promoter in monkey (CV-1) but not human (HeLa) cells in a transient gene expression system. DNA mediated gene transfer to CV-1 and HeLa cells of the bacterial gene for chloramphenicol acetyl transferase (CAT) linked to the promoter regulatory domain from the HSV-1 alpha 4 gene results in production of substantial levels of CAT enzyme. Cotransfection of equal mole amounts of an alpha 27 containing plasmid with an alpha 4-CAT construct to CV-1 cells results in a greater than 85% inhibition of CAT activity. No significant inhibition of CAT activity was observed when transfection was done in HeLa cells, with the same concentrations tested. Intact alpha 27 structural genes were necessary to achieve inhibition since subgenomic fragments and restriction enzyme digested alpha 27 genes were not effective inhibitors. Cotransfection of alpha 27 genes to CV-1 cells also prevented alpha 0 as well as alpha 4 from mediating their trans-stimulation of the HSV thymidine kinase (tk) regulated CAT gene, B-CAT. This suggests that the alpha 27 gene product may down-regulate gene expression from alpha promoters.     

Construction of human cell lines which contain and express the adenovirus DNA binding protein gene by cotransformation with the HSV-1 tk gene

We have introduced the DNA binding protein (DBP) gene of human adenovirus type 5 (Ad5) into high molecular weight DNA of permissive human cells by cotransformation of tk- cells with the cloned DBP and HSV-1 thymidine kinase genes. 110 tk+ cell lines were isolated after selection in HAT medium. The amount and arrangement of adenovirus sequences in the tk+ cell lines were analyzed by restriction endonuclease digestion and filter hybridization. Twelve of the 110 lines carry at least a segment of the DBP gene while only three of these contain the entire DBP gene at approximately one copy per cell. Cytoplasmic, polyadenylated DBP mRNA is made in all three cell lines though the amount is very low compared to that present in infected HeLa cells. The cell line U13-2 which contains approximately 1/30 the steady-state level of DBP mRNA found in infected HeLa cells produces a few percent of the amount of DBP made during the peak period of DBP synthesis in infected cells. The other two lines contain lower levels of DBP mRNA and do not synthesize detectable levels of the protein. When these DBP-tk+ cell lines are infected with adenovirus mutants containing temperature-sensitive (ts) mutations in the DBP gene, only U13-2 permits some viral DNA replication (and hence late gene expression) at the nonpermissive temperature, indicating that sufficient quantities of DBP from the integrated gene are produced to allow complementation of the ts mutation in this cell line. However, growth of these ts mutants (as measured by virus production) is only partially complemented in U13-2 at the nonpermissive temperature.     

Induction of Natural Killer Cells and Interferon during Mouse Hepatitis Virus Infection of Resistant and Susceptible Inbred Mouse Strains

Following infection by mouse hepatitis virus (JHM strain), an induction of natural killer (NK) cell activity was observed in C3H mice, which are considered to be sensitive to JHM virus infection. In contrast, mice of the resistant SJL strain did not show any increase of NK cell activity after JHM virus infection. However, infection of both SJL and C3H mice with mouse hepatitis virus type 3 (MHV3) resulted in an increase of NK level, comparable to that observed with the JHM virus infection in the C3H strain. No significant differences were observed in the NK cell activity of the peritoneal exudate or spleen cells of infected mice. Low levels of interferon were detected in serum or peritoneal exudate of C3H mice infected with JHM virus 18 or 24 hours before, but no detectable early interferon production was found. Also no interferon could be detected in the resistant SJL mice. After JHM virus infection, the number of peritoneal exudate cells (PEC) was increased significantly in C3H mice but not in SJL mice. Macrophages obtained from the C3H mice supported virus replication, whereas SJL macrophages did not. Our data suggest that NK cells do not play a role in the resistance of SJL mice against JHM virus infection but may participate in the defence mechanisms against this virus in C3H mice.     

Studies of L cells persistently infected with VSV: factors involved in the regulation of persistent infection.

Infection of interferon-treated L cells with VSV led frequently to the establishment of L cells persistently infected with VSV (LVSV cells). These cells were characterized by the following properties; (I) no supplement of antiviral factors such as anti-VSV antiserum, interferon, was required for their maintenance; (2) virus antigens were detected in about 5 to 30% of the cells by immunofluorescence staining; (3) the cells were not only resistant to superinfection by homologous virus, but also resistant to challenge by heterologous viruses such as Mengo virus; (4) the cells were destroyed by co-cultivation with heterologous cells susceptible to VSV infection; (5) the cells could be cured by serial cultivation in medium containing antiviral antibody, and the cured cells were as susceptible to VSV as normal L cells. It was shown that at least three factors (interferon, defective interfering [DI] particles and a selection of small-plaque temperature-sensitive [ts] mutants) took part in the maintenance of LVSV cells although it was difficult to evaluate exactly the relative importance of these factors. The effect of antiviral antibody, interferon and incubation temperature upon the maintenance of LVSV cells are discussed further.     

Regulation of the interferon system: evidence that Vero cells have a genetic defect in interferon production.

A clone of Vero cells was isolated and shown to be totally unable to synthesize interferon and insensitive to the toxic effect of poly(rI).poly(rC) treatment. Cells of this clone and mouse L cells were fused by treatment with polyethylene glycol or Sendai virus. Hybrid cell clones were isolated following selection in medium containing hypoxanthine, thymidine and ouabain. The hybrids were sensitive to the antiviral effect of poly(rI).poly(rC) and synthesized mouse, but not primate, interferon. It is proposed that in Vero cells, the gene for interferon synthesis is defective or absent.     

Dominant negative selection of vaccinia virus using a thymidine kinase/thymidylate kinase fusion gene and the prodrug azidothymidine

The Escherichia coli thymidine kinase/thymidylate kinase (tk/tmk) fusion gene encodes an enzyme that efficiently converts the prodrug 3'-azido-2',3'-dideoxythymidine (AZT) into its toxic triphosphate derivative, a substance which stops DNA chain elongation. Integration of this marker gene into vaccinia virus that normally is not inhibited by AZT allowed the establishment of a powerful selection procedure for recombinant viruses. In contrast to the conventional vaccinia thymidine kinase (tk) selection that is performed in tk-negative cell lines, AZT selection can be performed in normal (tk-positive) cell lines. The technique is especially useful for the generation of replication-deficient vaccinia viruses and may also be used for gene knock-out studies of essential vaccinia genes.     

胸腺嘧啶激酶/丙氧鸟苷体外杀伤携带胸腺嘧啶激酶基因的C6胶质瘤细胞时细胞凋亡的检测

目的检测胸腺嘧啶激酶基因转染C6胶质瘤细胞后用丙氧鸟苷处理时细胞凋亡的发生。方法用脂质体介导的基因转染将单纯疱疹病毒胸腺嘧啶激酶基因转入C6胶质瘤细胞中,用G418筛选,建立稳定表达胸腺嘧啶激酶的C6细胞株;加入丙氧鸟苷作用不同时间,用原位末端标记、透射电镜和DNA凝胶电泳法检测。结果用丙氧鸟苷作用72h发现有大量原位末端标记阳性的细胞,阳性信号呈块状或半月形位于核膜内侧;透射电镜发现肿瘤细胞核内染色质呈块状或帽状位于核膜内侧,非染色质部分呈空泡状,细胞质有的固缩,细胞器基本保持完整;有的则高度肿胀,细胞内亚单位遭到破坏;DNA凝胶电泳发现用丙氧鸟苷作用72h细胞基因组DNA呈梯状带。结论实验结果提示胸腺嘧啶激酶/丙氧鸟苷系统作用于表达胸腺嘧啶激酶的C6细胞时该细胞主要是以凋亡的方式死亡的。     

Restricted replication of ectromelia virus in cell culture correlates with mutations in virus-encoded host range gene.

Ectromelia virus (strain Moscow) was shown to replicate poorly or not at all in cell lines derived from the rabbit or hamster. The failure of ectromelia virus to replicate in cell lines derived from the hamster suggested that the virus lacked a functional CHO host range (hr) gene required for multiplication in these cells. A DNA fragment which hybridized to the CHO hr gene was cloned from the ectromelia virus genome and shown by sequence analysis to be deleted of 506 bp within the ectromelia virus CHO hr homologue. Two additional ectromelia viruses (Hampstead and Mill Hill strains) were also shown to lack an intact CHO hr gene. Insertion of the CHO gene from cowpox virus into the ectromelia virus genome extended the host range of ectromelia virus in tissue culture. These results demonstrate that an intact CHO hr gene is not required for maintenance of ectromelia virus in nature and provide a partial explanation for ectromelia virus' narrow host range, as opposed to the broad host range of cowpox virus, which has a functional CHO hr gene.     

Component(s) of Sendai virus that can induce interferon in mouse spleen cells.

To identify the active component of Sendai virus that induces interferon in mouse spleen cells, infectious and noninfectious viruses, envelope particles derived from them, and isolated hemagglutinin-neuraminidase (HN) glycoproteins were examined for interferon induction. The interaction between membranous structures containing Sendai virus HN glycoprotein and the receptors on the cell surface was shown to be sufficient for interferon induction in mouse spleen cells, suggesting that the actual inducer of interferon in mouse spleen cells is the HN glycoprotein of Sendai virus. When mouse spleen cells were stimulated in vitro with Sendai virus grown in eggs or LLC-MK2 cells or with membranous structures containing glycoproteins obtained from these viruses, interferon could be detected in the culture fluid. Furthermore, isolated HN glycoprotein per se could induce interferon in the cells. A linear correlation was found between the titer of interferon induced and the hemagglutinating activity of the membranous structure containing the HN glycoprotein. It was concluded from these findings that HN glycoprotein was the active component of Sendai virus responsible for interferon induction in mouse spleen cells and that viral RNA and F glycoprotein were not required. The results also showed that the interaction between HN glycoprotein and receptors on the cell surface triggered production of type I interferon (IFN-alpha and IFN-beta). Although when Sendai virus was incubated at 56 degrees C for 5 min it lost its hemolytic and hemagglutinating activities, it induced a considerable amount of interferon in the culture fluid of mouse spleen cells. The interferon-inducing ability of heat-inactivated virus could be absorbed with mouse spleen cells but not with sheep erythrocytes or mouse erythrocytes, indicating that the inactivated virus retained ability to bind to mouse lymphoid cells.