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Bacteriophage P4's superinfection immunity mechanism is unique among those of other known bacteriophages in several respects: (i) the P4 immunity factor is not a protein but a short, stable RNA (CI RNA); (ii) in the prophage the expression of the replication operon is prevented by premature transcription termination rather than by repression of transcription initiation; (iii) transcription termination is controlled via RNA-RNA interactions between the CI RNA and two complementary target sequences on the nascent transcript; and (iv) the CI RNA is produced by processing of the same transcript it controls. It was thought that several host-encoded factors may participate in the molecular events required for P4 immunity expression, i.e., RNA processing, RNA-RNA interactions, and transcription termination. To identify such factors we searched for Escherichia coli mutations that affect P4 lysogenization. One such mutation, bfl-1, severely reduced P4's lysogenization frequency and delayed both the disappearance of the long transcripts that cover the entire replication operon and the appearance of the CI RNA. By physical mapping and genetic analysis we show that bfl-1 is allelic to pnp, which codes for polynucleotide phosphorylase, a 3'-to-5' exonucleolytic enzyme. A previously isolated pnp null mutant (pnp-7) exhibited a phenotype similar to that of bfl-1. These results indicate that the polynucleotide phosphorylase of E. coli is involved with the maturation pathway of bacteriophage P4's RNA immunity factor.

Adenovirus-associated virus (AAV)-specific RNA present in the cytoplasm of cells coinfected with a helper adenovirus was analyzed by sucrose gradient sedimentation and gel electrophoresis. In aqueous conditions both gels or gradients revealed three AAV RNA components corresponding to 30S, 27S, and 20S and having apparent molecular weights of 2.6 x 10(6), 1.75 x 10(6) to 1.8 x 10(6), and 0.9 x 10(6) to 1.0 x 10(6), respectively. In nonaqueous, denaturing solvents only the 20S AAV RNA species was observed. For this reason, and because they would be apparently significantly larger than a single AAV DNA strand, both the 30S and 27S species are believed to result from conformational or aggregation effects in the aqueous nondenaturing systems. It is concluded that only a single RNA molecule having a molecular weight of approximately 0.9 x 10(6) to 1.0 x 10(6) is synthesized by AAV.

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.

Electrophoretic analysis of KB cells coinfected with adenovirus-associated virus (AAV) type 2, a defective parvovirus, and adenovirus type 5 (as helper) have revealed the synthesis in vivo of at least five AAV-specific polypeptides. The three largest polypeptides, with molecular weights of 90,700, 71,600, and 60,000 comigrated in polyacrylamide gels with the three AAV structural polypeptides. The remaining two polypeptides had molecular weights of 24,900 and 15,800. The concentrations of the AAV-induced polypeptides relative to one another remained approximately constant during the infectious cycle, and the structural components were present in proportions similar to those found in purified virions. As determined by pulse-chase experiments, all polypeptides were generated at the level of protein synthesis and not by posttranslational proteolytic processing. Although inhibitors of proteolytic enzymes failed to influence the pattern of AAV-induced polypeptides, and amino acid analog, L-canavanine, blocked the appearances of both the major structural polypeptide (60,000 daltons) and the larger nonstructural polypeptide (24,900 daltons). Taken in conjunction with pulse-chase data, this result supports a model whereby the major virion polypeptide is produced by proteolytic cleavage of the nascent polypeptide chain.

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.

Simian sarcoma-associated virus type 1 propagated in human rhabdomyosarcoma cells exhibited characteristics typical of oncornaviruses but seemed to have several aberrant properties. It had a buoyant density of 1.14 g/cm3, had RNA-dependent DNA polymerase activity, seemed to be labile to high salt concentrations, and contained little 50 to 60S RNA but relatively large amounts of human ribosomal RNA. In addition to 50 to 60S RNA, purified virions contained smaller RNA molecules with sedimentation coefficients of 28 to 30S, 18 TO 20S, and 4 to 10S. Unlike the 50 to 60S RNA species, the smaller virion-associated RNAs lacked polyadenylic acid, and the 28 to 30S RNA had an average base composition similar to that of human ribosomal RNA. Upon heat denaturation, the native 50 to 60S RNA genome yielded polyadenylic acid-containing 28 to 30S subunits that degraded in to 18 to 20S molecules upon further heat treatment. The 50 to 60S viral RNA had a guanine plus cytosine content of 56%.

Efficient potentiation of adenovirus-associated viruses (AAV) replication occurs in coinfections with either of two DNA-minus temperature-sensitive mutants of adenovirus type 5 (Ad5), ts125 and ts149. The helper activity of these mutants does not result from leakiness. At the nonpermissive termperature (39.5 C) there was little or no detectable adenovirus DNA synthesis, and only a relatively low level of adenovirus transcription was observed. However, the synthesis of AAV DNA and RNA and the yield of infectious AAV were comparable in amounts to those found when wild-type Ad5 was the helper. Furthermore, an apparent lag in the initiation of AAV transcription after the onset of AAV DNA synthesis was seen in coinfections with both wild type or ts125. These findings strongly suggest that the adenovirus factor(s) required for AAV multiplication is produced early in the adenovirus DNA replication, this requirement does not include all factors directly needed for adenovirus DNA synthesis.

Adeno-associated satellite virus type 4, obtained by repeated undiluted passage, failed to produce distinct bands at the expected density of 1.43 g/cm(3) after density gradient centrifugation in CsCl. This phenomenon occurred regardless of the hemagglutinating activity of the starting material. Sharp bands were found at a density of 1.34 to 1.35 g/cm(3). These bands contained adenovirions and numerous satellite particles. These latter particles could be distinguished by electron microscopy from standard dense satellite particles by their flattened profiles and deep penetration of negative stains. Dense bands of satellite virus at 1.43 g/cm(3) were constantly observed when the inoculum was comprised of highly diluted seed virus. Light satellite particles had a particle to HA ratio comparable with dense particles, but possessed low infectivity. Measurements of contour lengths of extracted deoxyribonucleic acid (DNA) indicate that light particles contain only a small amount of DNA, possibly less than 0.5 x 10(6) daltons, compared to 1.4 x 10(6) for the complete satellite DNA molecule.

Three major structural proteins were found in adenovirus-associated virus (AAV) type 3H virions which were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular weights of the polypeptides were determined to be approximately 66,000 (VP1), 80,000 (VP2), and 92,000 (VP3). The component having a molecular weight of 66,000 comprised about 80% of the total virion protein in the major AAV-3H particle, and the other two components comprised about 10% each. Proteins of the same molecular weight were found in the minor dense AAV-3H virion, but the 80,000- and 92,000-molecular-weight components were present at about one-half the concentration. The AAV-3H virion contains about 72 molecules of VP1 and 8 and 7 molecules of VP2 and VP3, respectively.

Three clones of morphologically altered cells (L(-)MC29) of singular properties were isolated from MC29 (subgroup A) leukosis virus-infected chick embryo cells. Supernatant fluids from cultures of the cloned cells produced no transforming or interfering activity on chick embryo cells susceptible to known avian leukosis-sarcoma viruses. No virus associated with the cells was demonstrable by fluorescent-antibody staining or by electron microscopy. All L(-)MC29 clone cells were activated, however, by four strains of Rous-associated viruses (RAV) representative of A, B, C, and D subgroup avian leukosis viruses and by two strains of MC29 virus. Virus L(-)MC29 cells activated by superinfection with RAV-1 and RAV-2 was characterized by helper-dependent and helper-independent properties. These findings suggest that the strain MC29 leukosis virus, or a component thereof, possesses properties of defectiveness similar to those of the Bryan high-titer Rous sarcoma virus.

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.

A method for preparing large membrane fragments and cell ghosts was developed for uninfected and Rous sarcoma virus-transformed chicken embryo fibroblasts in culture. Membrane proteins were analyzed by electrophoresis in acrylamide gels containing sodium dodecyl sulfate. A major amino-acid-containing component of uninfected cell membranes was greatly diminished in amount or absent in membranes of virus-transformed cells. This component, called MP-1, had an electrophoretic mobility in sodium dodecyl sulfate-containing gels similar to that of a protein of a mol wt of 1.42 x 10(5). MP-1 was not altered by changes in cell growth rate or in cells infected with the nontransforming virus RAV-1.

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.

We have analyzed ribonucleic acid (RNA) synthesized in KB cells co-infected with adenovirus-associated virus (AAV) type 2 and either adenovirus type 2 (Ad2) or herpes simplex virus type 1 (HSV-1). With either type of helper virus, synthesis of AAV RNA was readily detected by deoxyribonucleic acid (DNA)-RNA hybridization. As is the case for AAV RNA synthesized with helper Ad2, the AAV RNA synthesized with HSV-1 as helper annealed only to the thymidine-rich (minus) AAV DNA strand. In addition, AAV RNA synthesized with either type of helper (i) contained similar nucleotide sequences as determined by hybridization inhibition tests and (ii) had a mean molecular weight of approximately 7.5 x 10(5) based on sedimentation in dimethylsulfoxide-sucrose gradients. These experiments suggest that the restricted helper function of HSV-1 is not due to abnormal transcription of the AAV genome. Since the mean molecular weight of AAV RNA is equivalent to 40 to 50% of the AAV genome, as few as two or three AAV RNA species may be transcribed in vivo. In contrast to adenovirus RNA, cleavage of AAV RNA after transcription was not observed.

The preparation of antisera to the three purified sodium dodecyl sulfate (SDS)-treated polypeptide components (VP1, VP2, VP3) of adenovirus-associated virus (AAV) type 3H is described. In immunofluorescence tests (FA), these antisera stained heat-stable antigens with distinct morphologies in cells co-infected with either adenovirus or herpes simplex virus. Kinetic studies of antigen formation showed that VP1 antiserum first stained the cytoplasm (14 hr) and later (by 18 hr) stained both cytoplasmic and intranuclear areas. VP2 antiserum stained only discrete intranuclear areas, and VP3 antiserum stained nearly the entire nucleus. All three VP antigens appeared at about the 14th hr postinfection, about 2 hr prior to the appearance of whole virion antigen. The VP antisera cross-reacted in FA with AAV types 1 and 2 (all at one-eighth of the homologous titer), but did not react with other parvoviruses, i.e., rat virus, hemadsorbing enteric virus of calves, minute virus of mice, or H-1 virus. These non-neutralizing antisera reacted specifically with SDS-treated AAV virion antigens in complement fixation and immunodiffusion tests, and antiserum prepared against SDS-treated helper adenovirus structural polypeptides reacted with adenovirus polypeptide antigens. All antisera to SDS-treated polypeptides were specific for new antigens revealed on the dissociated peptides and did not react with whole virions, whereas whole-virion antisera did not cross-react with the polypeptide antigens. These findings suggest that antigens unique to the polypeptides of AAV are revealed by SDS treatment and that these antigens can be detected in cells prior to the folding of the polypeptides into the molecular configuration they possess as virion subunits. These results also indicate that at least one AAV polypeptide component is synthesized in the cell cytoplasm.