Walter Doerfler

Nonproductive infection of baby hamster kidney cells (BHK21) with adenovirus type 12

Abstract Adenovirus type 12 (Ad 12) infects baby hamster kidney cells (BHK21), but does not multiply in these cells. During a period of 114 hours after infection, replication of Ad 12 DNA cannot be detected by equilibrium sedimentation in CsCl density gradients, by zone sedimentation in sucrose density gradients or by DNA-DNA hybridization on membrane filters. The latter technique is the most sensitive one and allows detection of Ad 12 DNA when it constitutes ≥ 0.1% of the total intracellular DNA. Cellular DNA synthesis continues for at least 114 hours after infection. Starting between 24 and 50 hours after infection with Ad 12 at high multiplicity, cellular DNA fragments of 5–10 × 10 6 daltons in molecular weight are observed. They are conceivably due to an endonuclease cleaving double-stranded DNA. Adenovirus type 2 (Ad 2) multiplies in BHK21 cells, and replication of Ad 2 DNA begins between 14 and 16 hours after infection. Cellular DNA synthesis is turned off approximately 17 hours after infection. When BHK21 cells are doubly infected with Ad 2 and Ad 12 simultaneously, only Ad 2 DNA synthesis can be detected.

Denaturation pattern of the DNA of adenovirus type 2 as determined by electron microscopy.

Abstract The DNA of human adenovirus type 2 has been found in electron microscope studies of DNA-protein monolayers to be a linear molecule which is 10.9 μ long. When partially denatured by heating at low salt concentrations and in the presence of formaldehyde to various temperatures in the range between 52 and 58 °C, denaturation sites can be recognized in the electron microscope, and a map of these regions can be constructed. These sites presumably correspond to segments in the DNA molecule which are rich in adenine thymine base pairs. In the range of 10 to 25% denaturation (55 to 57 °C), a unique melting pattern is found in adenovirus type 2 DNA with three major and one minor denaturation sites. On a unit length scale of the molecule, these sites are located between 0 and 0.15, 0.5 and 0.6, and 0.8 and 1.0; and at 0.4. These findings argue strongly against the possibility that the gene sequence in adenovirus type 2 DNA is circularly permuted. Fragments of adenovirus type 2 DNA generated by shear breakage of the molecule can be separated in Hg(II)-Cs 2 SO 4 density gradients. This finding supports the interpretation that adenine-thymine-rich clusters are distributed unequally in adenovirus type 2 DNA.

Adenovirus endonuclease: Association with the penton of adenovirus type 2

Abstract Previous results have defined an endonuclease in cells infected with adenovirus type 2 or 12, and this endonuclease is associated with purified adenovirions. When the viral capsid subunits are purified from KB cells infected with adenovirus type 2 and are assayed for endonuclease activity, only the penton is active. Neither the hexon nor the fiber of the penton competes with the penton endonuclease. Selective digestion of the penton base by trypsin destroys the endonuclease activity. In partially purified preparations of penton, the amount of endonuclease activity correlates with the amount of penton base antigen and not with the amount of total protein. The endonuclease is inhibited by antiserum prepared against pentons. These results suggest that the endonuclease resides in the peptides of the penton base. The penton endonuclease is specific for DNA. The endonuclease hydrolyzes native DNA at a rate 20 times greater than denatured DNA. Both strands of the DNA are cleaved. The preferred site for hydrolysis apparently is a region rich in guanine and cytosine bases. These data indicate that the penton base of adenovirus type 2 functions as both an endonuclease and a structural subunit.

Incomplete particles of adenovirus. I. Characteristics of the DNA associated with incomplete adenovirions of types 2 and 12.

Abstract When KB or HeLa cells are infected with either human adenovirus type 2 or 12, in addition to the complete infectious adenovirions, five discrete types of incomplete particles are produced in large quantity. These particles can be purified by equilibrium centrifugation in CsCl density gradients. Each type of incomplete adenovirion has a unique buoyant density in CsCl, and contains a specific-sized fragment of adenovirus DNA. The incomplete adenovirions have a very low specific infectivity as compared to that of the mature virion. Although other explanations cannot be excluded, this infectivity is likely to be due to a low level of contamination with complete virions.

An endonuclease in cells infected with adenovirus and associated with adenovirions

Abstract Extracts prepared from confluent cultures of baby hamster kidney cells (BHK), human embryonic lung cells, or KB cells infected with purified adenovirus type 2 or 12 contain high endonuclease activity and little or no cellular exonuclease activity. In infected cells, the intact adenovirus DNA is cleaved to 18 S DNA, which sediments homogeneously. Sedimentation in alkaline sucrose density gradients shows that the fragments of viral DNA are free of single-strand scissions. The endonuclease extracted from cells infected with adenovirus cleaves the viral DNA in vitro. Extracts of BHK cells infected with adenovirus type 2 or 12 show marked differences in endonuclease activity at various times after infection. In extracts of BHK cells productively infected with adenovirus type 2, an “early” endonuclease activity is detected between 2 and 8 hr after infection. This “early” activity nearly disappears, and at 12 hr after infection “late” endonuclease activity appears and rapidly increases to about 20 times the level of the “early” endonuclease. In BHK cells abortively infected with adenovirus. type 12, only the “early” endonuclease activity is found. When the genome of the infecting adenovirion is inactivated, the “late” endonuclease is not synthesized. These data are consistent with the hypothesis that the “early” endonuclease activity is due to the structural proteins introduced into the cell with the parental virions, while the “late” endonuclease is newly synthesized. When purified adenovirions are incubated with extracted adenovirus DNA, the adenovirus DNA is hydrolyzed to 18 S fragments which are free of single-strand scissions. These data suggest that the endonuclease activity found in cells infected with adenovirions is derived from the infecting virions.

Inhibition of adenovirus replication by 5,6-Dichloro-1-,β-d-ribofuranosylbenzimidazole

Abstract The halogenated benzimidazole derivative 5,6-dichloro-1-β- d -ribofuranosylbenzimidazole (DRB) inhibits reversibly the replication of human adenovirus type 2 (Ad2) and its DNA in human KB cells. Viral DNA replication is almost completely blocked when the drug is added earlier than 4 hr postinfection in concentrations between 50 and 150 μM. Replication of viral DNA in all four size-classes (>100 S, 50–90 S, 34 S, and 95%, as demonstrated by DNA-RNA filter hybridization. Thus, the block in viral DNA replication is best explained by the inhibition of the synthesis of early virus-specific RNA.

Intracellular forms of Adenovirus DNA in Productively Infected Cells: Evidence for Integration of the Viral Genome

In order to understand the mechanism of replication of adenovirus DNA in productively infected human cells, the intracellular forms of newly synthesized DNA have been studied. KB cells growing in monolayers were inoculated with CsCl-purified adenovirus type 2 (Ad2) at a multiplicity of 100 PFU/cell. At various times after infection, the cells were labeled with 3H-uridine or 3H-thymidine. In some experiments the cells were prelabeled with 14C-thymidine. The intracellular DNA was extracted after lysis of the cells with SDS or with alkali and was analyzed in dye-buoyant density gradients or by zonal centrifugation in neutral or alkaline sucrose gradients. The results of the experiments can be summarized as follows: 1) There is no evidence that parental or newly synthesized Ad 2 DNA becomes supercoiled. 2) A virus-specific DNA-RNA complex can be isolated in dye-buoyant density gradients. This complex is probably involved in transcription. 3) In CsCl density gradients viral DNA of high buoyant density is observed which is a precursor to virion DNA as judged from pulse-chase experiments. These molecules are in part single-stranded. 4) In alkaline sucrose gradients viral DNA is detected which sediments at a rate of 50–90 S. The evidence suggests that this DNA may represent an integrated form of the viral genome.