Student Theses and Dissertations

Date of Award

1970

Document Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

Abstract

Adenovirus type 2 productively infects human cells and hamster cells. Adenovirus type 12 productively infects human cells but abortively infects hamster cells. The metabolism of the DNA of these adenoviruses was determined in both human cells and hamster cells. The adenovirus DNA, both the parental DNA and the newly synthesized DNA, is found as three species within the cells. The species which sedimented rapidly is mainly cellular DNA which contains some integrated viral DNA. The species which cosediments with DNA extracted from the mature virions is homogeneously distributed. This species represents the intact viral genome. The species of DNA which sediments slowly is homogeneously distributed hybridizes only to viral DNA and represents segments of the adenovirus genome about one-fourth the size of the intact genome. The amount of each of these three species of DNA varies during the growth cycle of the adenovirus. There is a pattern of metabolism; the intact viral DNA is cleaved to the slow sedimenting DNA, the slow sedimenting DNA appears as a precursor which is integrated into the cellular DNA. Data from experiments with chemical inhibitors of macromolecular syntheses suggest that the slow sedimenting DNA is formed by a presynthesized endonuclease.

Extracts of cells infected with adenovirus contain the endonuclease activity. The endonuclease appears in the cells as both an early enzyme and a late enzyme. The endonuclease which appears early after infection correlates directly with the multiplicity of infection but is not effected by the inactivation of the viral genome. The endonuclease which appears late after infection is not effected by the multiplicity of infection if more than one infectious particle per cell is used. The endonuclease which appears late is not found in cells infected by virus which contain inactivated genomes. These results suggest that the endonuclease which appears early is carried into the cell as a portion of the virion and the endonuclease which appears late is newly synthesized as a virus-coded protein. The synthesis of endonuclease corresponds with the synthesis of structural proteins. Purified adenovirions have endonuclease activity when they are incubated with DNA in vitro. The endonuclease is inactivated by group-specific antiserum. This also suggests that the endonuclease is a virus-coded protein. The endonuclease made only double-strand scissions at specific sites in the adenovirus DNA.

Capsid subunits purified from KB cells infected with adenovirus type 2 were assayed for endonuclease activity. Only the penton, composed of a penton base and a fiber, cleaves the DNA substrate. The fiber does not compete with the penton for the substrate. The endonuclease was specifically inactivated by treating the penton with trypsin and group-specific antiserum. These results indicate that only the five, presumably identical, polypeptides of the penton base are the endonuclease. Thus the penton base is a structural subunit of the adenovirion and an endonuclease.

The penton endonuclease cleaves native DNA about 20 times faster than denatured DNA. Glucosylated DNA, double-stranded RNA, and single-stranded RNA are not cleaved. The site of attack on the DNA molecule apparently is a guanine-cytosine rich region. The endonuclease is inhibited by polydeoxyguanylic acid - polydeoxycytidylic acid but not by alternating copolymer of deoxyadenylic acid - deoxythymidylic acid.

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