Magdalena Piña

Biochemical studies on adenovirus multiplication: IV. Isolation, purification, and chemical analysis of adenovirus

Abstract A method is described for the isolation and purification of type 2 adenovirus from infected suspension cultures of KB cells. Yields of 5–15 mg of purified virus were obtained from 6 to 9 × 10 8 cells. The procedure involved extraction with Tris buffer, treatment with genetron, centrifugation upon a layer of RbCl solution, and two RbCl-density gradient centrifugations. The virus was found to contain 13% DNA and 87% protein. Base analysis of viral DNA gave molar percentages of 22, 21, 27, and 29 for adenine, thymine, guanine, and cytosine, respectively. These ratios are strikingly different from that of host cell DNA in which adenine and thymine predominate. Evidence for the purity of the virus and for the double-stranded nature of viral DNA is discussed. Type 4 adenovirus prepared by the above procedure had a similar chemical composition and DNA base ratios as type 2 adenovirus.

Biochemical studies on adenovirus multiplication: XIV. Macromolecule and enzyme synthesis in cells replicating oncogenic and nononcogenic human adenovirus

Abstract Exponentially growing KB cells infected with nononcogenic Ad 2 and oncogenic Ad 12 and 31 stopped dividing but continued to synthesize and accumulate protein, DNA, and RNA. The protein and DNA content per infected cell was twice that of uninfected cells at 36 hours after infection and the RNA content increased by 30–80% until 20 hours after infection and then sharply decreased to the initial levels. The rate of synthesis of protein, DNA, and RNA per cell increased initially and then decreased in cells infected with Ad 2, 12, and 31 as determined by pulse labeling with radioactive valine, thymidine, and uridine. Stimulation and inhibition occurred later after infection with oncogenic adenoviruses. Infected cells incorporated two to three times more valine, thymidine, and uridine into the acid-soluble fraction than did uninfected cells. The species of DNA synthesized at different times after infection were identified by density gradient centrifugation and by DNA-DNA hybridization. Host cell DNA synthesis was blocked completely at 24 to 36 hours after infection with Ad 2, 7, 12, 18, and 31. DNA polymerase activity did not increase after infection of exponentially growing KB cells with Ad 2, 12, or 31. Thymidine kinase activity was increased 2- to 3-fold in cells infected with Ad 12 and 31, but not in cells infected with Ad 2. Stimulation of enzyme activity in adenovirus-infected cells may depend upon the base level of enzyme activity in the uninfected host cell and the growth rate of the virus.

A simple purification procedure for adenovirus type 12 T and tumor antigens and some of their properties

Abstract Human adenovirus (Ad) type 12 T and tumor antigens are present predominantly in the isolated nuclear fraction of Ad12-infected KB cells and Ad12-transformed hamster embryo cells, respectively. Hydroxylapatite chromatography of nuclear extracts provide 200- to 400-fold purification of T and tumor antigens. Sucrose gradient centrifugation and polyacrylamide gel electrophoresis demonstrated three and four antigenic components in purified T and tumor antigen preparations, respectively; it is not known whether these represent different forms of the same protein or different viral-coded proteins. Purified preparations of T and tumor antigen (1) do not stimulate DNA synthesis in contact-inhibited mouse 3T3 cells, (2) do not possess thymidine kinase, DNA polymerase, RNA polymerase, RNase, or DNase activities, and (3) do not alter thymidine kinase, DNA polymerase, and “DNA synthesizing enzyme” system activities in cell-free extracts or RNA polymerase activities in isolated nuclei.