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Satellite phage P4 directs the capsid proteins of its helper phage, P2, to form a head which is only one-third the size of the normal P2 head. The P2 head contains a genome of molecular weight 22 x 10(6), while the small P4 head contains a genome with a molecular weight of only 7 x 10(6). We have used in vitro DNA packaging to test whether P2 and P4 phage head sizes are determined by DNA size. The small DNA of satellite phage P4 added to a P2-infected cell extract was packaged primarily into particles containing three copies of the P4 genome. This process occurred with approximately the same efficiency as P2 DNA packaging in the same cell extract. In contrast, the large DNA of P2 was packaged 300-fold less efficiently than the small DNA of P4 in an extract derived from P4-infected, P2-lysogenic cells. These results suggest that DNA size is not sufficient to determine head size. The results are compatible with DNA packaging via the filling of preformed empty capsids.

Escherichia coli C strains containing different deoxyribonucleic acid (DNA) synthesis mutations have been tested for their support of the DNA synthesis of bacteriophage P2 and its satellite phage P4. Bacteriophage P2 requires functional dnaB, dnaE, and dnaG E. coli gene products for DNA synthesis, whereas it does not require the products of the dnaA, dnaC, or dnaH genes. In contrast, the satellite virus P4 requires functional dnaE and dnaH gene products for DNA synthesis and does not need the products of the dnaA, dnaB, dnaC, and dnaG genes. Thus the P2 and P4 genomes are replicated differently, even though they are packaged in heads made of the same protein.

The cellular sites of integration of the avian myeloblastosis-associated virus type 2 (MAV-2) DNA have been examined by Southern blot analysis of cellular DNA from infected cloned and uncloned chicken embryonic fibroblasts. Provirus-cell juncture fragments were not detected in restriction enzyme digests of DNA from MAV-2-infected uncloned cells. However, each MAV-2-infected cell clone examined produced a unique set of junctive bands. Thse findings indicate that multiple sites of integration exists for MAV-2 proviruses in cellular DNA.

The adsorption of adenovirus-associated virus (AAV) type 2 by KB cells and the subsequent penetration of the AAV genome to the cell nucleus was measured with and without helper adenovirus type 2 (Ad2). It was found that the helper virus did not enhance either process. On the other hand, a synthesis of AAV deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) was not detected by nucleic acid hybridization after KB cells were infected with AAV type 2 alone, whereas both AAV DNA and RNA synthesis were readily detected when cells were additionally infected with Ad2 or herpes simplex virus type 1, a partial helper of AAV replication. AAV RNA synthesis was initially observed 10 to 11 hr after simultaneous infection with Ad2, but the interval between AAV infection and AAV transcription could be reduced to 4 to 5 hr when cells were first infected with Ad2 for 10 hr. It was estimated that AAV DNA synthesis accounted for 3% of the total DNA in cells after a simultaneous infection with Ad2. These findings, together with the previous observation that adenovirus provides a helper function(s) after AAV uncoating, suggest that AAV are defective only subsequent to the uncoating process, and that helper viruses may provide a factor(s) needed for initiating synthesis of AAV DNA, RNA, or both.

Nucleic acid hybridization procedures were used to measure the extent of transcription of adenovirus-associated virus (AAV) deoxyribonucleic acid (DNA) in KB cells in the presence of either adenovirus or herpes simplex virus as the helper. Annealing of AAV ribonucleic acid to AAV DNA was monitored by a hybridization inhibition assay on nitrocellulose filters or by hydroxyapatite chromatography. These experiments confirmed the previous observation that, in the presence of either type of helper virus, only one strand of AAV DNA (the thymidine-rich or "minus" strand) is transcribed in vivo. However, it was found that only 70 to 80% of this strand appears to be transcribed in vivo. Furthermore, studies with minus strands employing hydroxyapatite chromatography and nuclease S(1), which specifically degrades single-stranded DNA, indicated that up to 20% of the minus strand is self-complementary. It seems likely that these self-complementary sequences account for the bulk of that portion of the minus strand (20 to 30%) which is not transcribed in vivo.

The two complementary strand species of 5-bromodeoxyuridine-substituted, adenovirus-associated virus type 2 (AAV-2) deoxyribonucleic acid were preparatively separated in CsCl density gradients and further purified by sedimentation through 5 to 20% sucrose. The base composition of each strand species was determined, and it was found that the species banding at a greater density in CsCl (heavy strands) had an expected higher thymidine content (26.5%) than that 21.7%) of the less dense species (light strands). Furthermore, the base composition of in vivo-synthesized, AAV-specific ribonucleic acid was similar to that of light-strand deoxyribonucleic acid, and this ribonucleic acid apparently hybridized only with heavy strands. These observations indicate that the heavy-strand species alone serves as the transcriptional template in vivo. This study represents the first instance in which the base composition and specificity of in vivo transcription have been determined for each of the complementary strands of an animal virus deoxyribonucleic acid.