Ogino T

Synthesis of deoxyribonucleic acid in human and hamster kidney cells infected with human adenovirus types 5 and 12

Abstract The incorporation of 3H-labeled thymidine (3H-dT) into deoxyribonucleic acid (DNA) has been studied in human embryonic kidney and hamster kidney cells infected with human adenovirus types 5 and 12 (Ad5 and Ad12). The Ad5-infected human and hamster kidney cultures incorporated about 5–10 times as much 3H-dT into DNA as did uninfected cultures. The Ad12-infected human and hamster kidney cultures incorporated about 4–6 times and 3–5 times as much 3H-dT into DNA as did uninfected cultures. DNA-DNA hybridization experiments using a membrane filter technique were performed to elucidate the kind of DNA that was pulse-labeled with 3H-dT. In human and hamster kidney cells infected with Ad5, there was a progressive increase in the incorporation of 3H-dT into viral DNA. Incorporation of 3H-dT into cellular DNA was not immediately shut off. An increased incorporation during the early stage of infection (16–22 hours post infection) was followed by a decreased incorporation at later stages. The shutoff of incorporation into cellular DNA was found to occur gradually in hamster kidney cultures and rather rapidly in human kidney cultures. In human embryonic kidney cells infected with Ad12, incorporation of 3H-dT into viral DNA increased progressively until about 32 hours PI. Incorporation of 3H-dT into cellular DNA was stimulated somewhat at the early stage of infection, and was not shut off either during the period of viral DNA synthesis or for at least 20 hours after virus maturation had begun. In abortive infection of hamster kidney cells with Ad12, incorporation of 3H-dT into cellular DNA was stimulated. A small amount of incorporation of 3H-dT into viral DNA was detected at an early stage of infection. From these results, it can be concluded that in productive infection of human and hamster kidney cells by Ad5 or of human kidney cells by Ad12, cellular DNA synthesis is not immediately shut off. In abortive infection of hamster kidney cells by Ad12, cellular DNA synthesis is stimulated, whereas viral DNA is synthesized in small amounts at an early stage of infection.

Estimation of relative molecular length of DNA by electrophoresis in agarose gel

Abstract Using T 2 phage DNAs of various lengths, it was found that the mobility of DNA on electrophoresis in agarose gel was inversely related to its sedimentation rate in sucrose density gradient, which has been commonly used to assess the relative molecular length of DNA. Thus electrophoresis in agarose gel can conveniently be used to estimate the relative molecular length of DNA. The advantage of the agarose gel electrophoresis method over the sucrose density gradient method is discussed.

Induction of neutralizing antibody against varicella-zoster virus (VZV) by VZV gp3 and cross-reactivity between VZV gp3 and herpes simplex viruses gB

Glycoprotein gp3 (64K) is one of the major proteins specified by varicella-zoster virus (VZV). This glycoprotein was purified on an immunoadsorbent consisting of monoclonal antibody (clone 8) against gp3 linked to protein A-Sepharose. Rabbits were then immunized with the purified antigen to obtain monospecific antisera against gp3. The monospecific antisera and monoclonal antibody immunoprecipitated polypeptides with the same molecular weights of approximately 64,000 (64K), 106K, and 116K from a lysate of labeled cells infected with VZV. The monoclonal antibodies against gp3 did not have neutralizing activity against VZV, but anti-gp3 monospecific sera neutralized VZV infectivity. The antigenic relation of VZV to herpes simplex virus (HSV) was investigated by the immunofluorescent test, immunoprecipitation followed by analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the neutralizing antibody test with monoclonal antibodies and monospecific antisera. In the indirect immunofluorescent test, the cytoplasm of cells infected with HSV-type 1 or HSV-type 2 was stained with anti-gp3 monospecific antiserum but not with anti-gp3 monoclonal antibodies. This serum also precipitated the polypeptides of HSV-type 1 and HSV-type 2 with molecular weight of approximately 120,000, possibly corresponding to gB of HSV-1 or HSV-2, and this immunoprecipitation was blocked by anti-gB monoclonal antibody. However, anti-gp3 monospecific antisera did not neutralize either HSV-type 1 or HSV-type 2 infectivity. These results suggest that gp3 induces neutralizing antibody against VZV and that it also has a cross-reacting antigenic determinant with gB of HSV.

Immunochemical characterization of pyrimidine kinase induced by varicella-zoster virus

Thymidine kinase (TK) induced by varicella-zoster virus (VZV) was precipitated with ammonium sulphate and purified by Sephadex G-150, QAE-Sephadex and Blue Sepharose column chromatographies. The purified TK fraction also contained deoxycytidine kinase (dCK) activity and a 35000 mol. wt. (35K) polypeptide as a major component. The TK and dCK activities were both neutralized by anti-VZV serum. Antiserum to an extract of cells infected with a bromodeoxyuridine (BUdR)-resistant mutant virus contained no antibody to the viral TK and dCK activities or to the 35K polypeptide. Antiserum to the purified viral TK fraction was prepared and absorbed with a lysate of BUdR-resistant mutant virus-infected cells. The resulting absorbed antiserum (anti-vTK serum) neutralized the viral activities of both TK and dCK, and specifically immunoprecipitated a 35K polypeptide from the lysate of parental virus-infected cells, but did not immunoprecipitate any detectable polypeptide from cells infected with BUdR-resistant mutant virus. Anti-vTK serum stained mainly the nuclei of cells infected with the parental virus strain, but did not stain those infected with BUdR-resistant mutant virus by an indirect fluorescent antibody test. These results suggest that the 35K polypeptide produced in VZV-infected cells is responsible for the viral TK and dCK activities, and that the TK and dCK are mainly localized in the nuclei of infected cells.

Biochemical Transformation of Mouse Cells by Varicella-Zoster Virus

Mouse L cells lacking the enzyme thymidine kinase (Ltk-) were infected with varicella-zoster virus (VZV). Even though virus did not replicate in Ltk- cells, the presence of virus antigen could be observed by use of an anti-complement immunofluorescent technique at 4 h post-infection and the VZV-specific thymidine kinase could be detected in VZV-infected Ltk- cells. Ltk-cells were converted to a tk+ phenotype (Ltk+) by infection with cell-associated VZV. Clones possessing the ability to grow in selective medium were isolated and cultured successfully for more than 20 passages. One of the clones grew very slowly, but other clones showed almost the same growth rate as that of the parental Ltk- cells. The chromosome analyses of Ltk- cells and transformed cells revealed that the isolated clones were of mouse origin. VZV-specific antigen could be detected in the nuclei of Ltk+ cell clones by an immunofluorescent test, while tk activity was greatly enhanced in extracts prepared from transformed cells and its activity was neutralized by hyperimmune serum against VZV.

Expression of Varicella-Zoster Virus-related Antigens in Biochemically Transformed Cells

L(O)cl 3 and L(K)cl 1 cells, biochemically transformed by varicella-zoster virus (VZV), were labelled with L-[35S]methionine and [32P]orthophosphate. Cell extracts were immunoprecipitated with anti-VZV monkey serum and analysed by SDS-polyacrylamide gel electrophoresis. Four polypeptides of apparent mol. wt. 135000 (135K), 48K, 44K and 35K were detected in the L-[35S]methionine-labelled extracts, and, of these, the 35K band showed marked intensity. However, this band was not detected in extracts from cells infected with a VZV tk- strain (Kanno strain). Also, the 35K polypeptide showed very low intensity when immunoprecipitated from extracts of transformed cells grown in non-selective (NS) medium, i.e. cells that had a very low thymidine kinase (tk) activity. In the case of [32P]orthophosphate-labelled cells, polypeptides of apparent mol. wt. 180K, 81K, 48K, 44K and 37K were obtained. In both instances, the 44K polypeptide was not immunoprecipitated from L(K)cl 1 cell extracts. From our data it is postulated that the expression of the 35K polypeptide is correlated with the VZV-specific tk activity of the cells.

Induction of cellular DNA synthesis by a temperature-sensitive mutant of herpes simplex virus type 2☆

Abstract A temperature-sensitive mutant (ts 4) of herpes simplex virus type 2 (HSV-2), having the ability to transform hamster embryo (HaE) cells at the nonpermissive temperature of 38.5°, has been investigated in several aspects. It is defective in thymidine (TdR) kinase induction at both the permissive (34°) and the nonpermissive temperatures and defective in viral DNA synthesis at the nonpermissive temperature. However, stimulation of chromosomal DNA synthesis was detected at 16–28 hr after infection at the nonpermissive temperature in HaE cells arrested with low serum concentration. DNA synthesis was estimated by the incorporation of [ 3 H]TdR or [ 3 H]CdR (deoxycytidine) into DNA, and differentiation of cellular from viral DNA was performed by buoyant density gradient centrifugation in CsCl or by DNA-DNA hybridization. By autoradiography with [ 3 H]TdR, it was found that the number of cells with grains in the nuclei increased in infected cultures at 16–28 hr after infection. Virus exposed to heat or uv light lost the ability to induce cellular DNA synthesis, indicating that active virus is responsible for stimulation of cellular DNA synthesis.