Deoxyribonucleic Acid Polymerase Associated with Avian Tumor Viruses: Secondary Structure of the Deoxyribonucleic Acid Product

The products of the deoxyribonucleic acid (DNA) polymerase associated with Rous sarcoma virus and avian myeloblastosis virus were characterized by correlative analyses with equilibrium centrifugation and stepwise elution from hydroxyapatite. The initial enzymatic product consists of nascent DNA chains which are hydrogen-bonded to 70S viral ribonucleic acid (RNA), whereas the final enzymatic product is double-stranded DNA. Appreciable amounts of free single-stranded DNA were not detected at any point during the course of the enzymatic reaction, but the data in this regard are not decisive. The time course of synthesis of DNA:RNA hybrids and double-stranded DNA has been analyzed. It is concluded that the synthesis of double-stranded DNA is a sequel to and is probably dependent upon the synthesis of DNA:RNA hybrid.     

Separation of DNA Sequences Complementary to the RNA of Avian Myeloblastosis Virus from Chicken DNA by Alkaline Cesium Chloride Density Sedimentation

Density gradient sedimentation in alkaline cesium chloride of DNA from normal chicken embryos or leukemic myeloblasts fragmented to a size of 13S revealed that the DNA sequences complementary to 70S avian myeloblastosis virus RNA sedimented in the high guanine plus cytosine region ahead of the main peak of cellular DNA. When the DNA was fragmented into pieces of 6.6S there was a broader distribution of the DNA sequences complementary to the viral RNA. This technique could be employed as a step towards the isolation of DNA copies of the entire viral RNA genome from the mass of host cellular DNA.     

Ribonucleotide Sequence Homology Among Avian Oncornaviruses

RNA sequence relatedness among avian RNA tumor virus genomes was analyzed by inhibition of DNA-RNA hybrid formation between ³H-labeled 35S viral RNA and an excess of leukemic or normal chicken cell DNA with increasing concentrations of unlabeled 35S viral RNA. The avian viruses tested were Rous associated virus (RAV)-0, avian myeloblastosis virus (AMV), RAV-60, RAV-61, and B-77 sarcoma virus. Hybridization of ³H-labeled 35S AMV RNA with DNA from normal chicken cells was inhibited by unlabeled 35S RAV-0 RNA as efficiently (100%) as by unlabeled AMV RNA. Hybridization between ³H-labeled 35S AMV RNA and DNA from leukemic chicken myeloblasts induced by AMV was suppressed 100 and 68% by unlabeled 35S RNA from AMV and RAV-0, respectively. Hybridization between ³H-labeled RAV-0 and leukemic chicken myeloblast DNA was inhibited 100 and 67% by unlabeled 35S RNA from RAV-0 and AMV, respectively. It appears therefore that the AMV and RAV-0 genomes are 67 to 70% homologous and that AMV hybridizes to RAV-0 like sequences in normal chicken DNA. Hybridization between AMV RNA and leukemic chicken DNA was inhibited 40% by RNA from RAV-60 or RAV-61 and 50% by B-77 RNA. Hybridization between RAV-0 RNA and leukemic chicken DNA was inhibited 80% by RAV-60 or RAV-61 and 70% by B-77 RNA. Hybridization between ³H-labeled 35S RNA from RAV-60 or RAV-61 and leukemic chicken myeloblast DNA was reduced equally by RNA from RAV-60, RAV-61, AMV or RAV-0; this suggests that RNA from RAV-60 and RAV-61 hybridizes with virus-specific sequences in leukemic DNA which are shared by AMV, RAV-0, RAV-60, and RAV-61 RNAs. Hybridization between ³H-labeled 35S RNA from RAV-61 and normal pheasant DNA was inhibited 100% by homologous viral RNA, 22 to 26% by RNA from AMV or RAV-0, and 30 to 33% by RNA from RAV-60 or B-77. Nearly complete inhibition of hybridization between RAV-0 RNA and leukemic chicken DNA by a mixture of AMV and B-77 35S RNAs indicates that the RNA sequences shared by B-77 virus and RAV-0 are different from the sequences shared by AMV and RAV-0. It appears that different avian RNA tumor virus genomes have from 50 to 80% homology in nucleotide sequences and that the degree of hybridization between normal chicken cell DNA and a given viral RNA can be predicted from the homology that exists between the viral RNA tested and RAV-0 RNA.     

Properties of visna virus particles harvested at short time intervals: RNA content, infectivity, and ultrastructure

The major RNA component of Visna virus harvested at short intervals of time (5 min) is not the 60 to 70S RNA but a molecule of higher electrophoretic mobility. This RNA has been isolated and characterized. Its sedimentation coefficient is identical to that of 30 to 40S RNA subunits obtained by heat denaturation of the 60 to 70S RNA. In 1.8% acrylamide gels without agarose the electrophoretic mobility of 30 to 40S RNA subunits present in rapidly harvested virus is slightly lower than that of the subunits obtained by denaturation of the 60 to 70S RNA; after heat denaturation the mobilities are identical. These free RNA subunits present in early virus particles assemble into a 60 to 70S RNA complex as shown by following the RNA content of early virus incubated at 37 C for various lengths of time. The rate of this maturation process is slow. There is no difference between the infectivity of immature and mature virus particles. Both particles have a dense core when examined in sections of virus pellets.     

RNA-Dependent DNA Polymerase Activity of RNA Tumor Viruses II. Directing Influence of RNA in the Reaction

The role of ribonucleic acid (RNA) in deoxyribonucleic acid (DNA) synthesis with the purified DNA polymerase from the avian myeloblastosis virus has been studied. The polymerase catalyzes the synthesis of DNA in the presence of four deoxynucleoside triphosphates, Mg(2+), and a variety of RNA templates including those isolated from avian myeloblastosis, Rous sarcoma, and Rauscher leukemia viruses; phages f2, MS2, and Qbeta; and synthetic homopolymers such as polyadenylate.polyuridylic acid. The enzyme does not initiate the synthesis of new chains but incorporates deoxynucleotides at 3' hydroxyl ends of primer strands. The product is an RNA.DNA hybrid in which the two polynucleotide components are covalently linked. Free DNA has not been detected among the products formed with the purified enzyme in vitro. The DNA synthesized with avian myeloblastosis virus RNA after alkaline hydrolysis has a sedimentation coefficient of 6 to 7S.     

Small RNAs of Rous Sarcoma Virus: Characterization by Two-Dimensional Polyacrylamide Gel Electrophoresis and Fingerprint Analysis

Approximately 15 to 20 different species of small (4 to 7S) RNAs have been purified by two-dimensional polyacrylamide gel electrophoresis of RNA isolated from virions of Schmidt-Ruppin D strain of Rous sarcoma virus. Each species of small RNA has been isolated free of 70S RNA; nine of them, including 5S and 7S RNAs, were also found associated with the 70S genomic RNA. Most of the 4S RNAs are present at an average of less than one copy per virion. The 4S RNAs have T1 RNase (EC 2.7.7.26) fingerprints, which are very similar to those of tRNAs. One of the smallest 4S RNAs, which can act as a primer for initiation of RNA-directed DNA synthesis, is associated with the 70S RNA in 1 to 2 copies per complex, whereas an additional 6 to 8 copies of this molecule are free.     

Covalent linkage between ribonucleic Acid primer and deoxyribonucleic Acid product of the avian myeloblastosis virus deoxyribonucleic Acid polymerase

Initiation of deoxyribonucleic acid (DNA) synthesis by the avian myeloblastosis virus DNA polymerase was previously suggested to involve a ribonucleic acid (RNA) primer, the initial product being a DNA molecule joined by a phosphodiester bond to the RNA primer. The existence and nature of such an RNA-DNA joint was investigated by assaying for transfer of a ³²P atom from an α-³²P-deoxyribonucleotide to a 2′(3′)-ribonucleotide after alkaline hydrolysis of the polymerase product. Such a transfer was observed, but only from α-³²P-deoxyadenosine triphosphate and only to 2′(3′)-adenosine monophosphate. This same transfer was observed in both the endogenous DNA polymerase reaction of purified virions and the reconstructed reaction of purified DNA polymerase plus purified 60 to 70S viral RNA. These results indicate a high level of specificity for the initiation process and support the idea of a low-molecular-weight initiator RNA as part of the 60 to 70S RNA complex.     

Acquisition of new DNA sequences after infection of chicken cells with avian myeloblastosis virus

DNA-RNA hybridization studies between 70S RNA from avian myeloblastosis virus (AMV) and an excess of DNA from (i) AMV-induced leukemic chicken myeloblasts or (ii) a mixture of normal and of congenitally infected K-137 chicken embryos producing avian leukosis viruses revealed the presence of fast- and slow-hybridizing virus-specific DNA sequences. However, the leukemic cells contained twice the level of AMV-specific DNA sequences observed in normal chicken embryonic cells. The fast-reacting sequences were two to three times more numerous in leukemic DNA than in DNA from the mixed embryos. The slow-reacting sequences had a reiteration frequency of approximately 9 and 6, in the two respective systems. Both the fast- and the slow-reacting DNA sequences in leukemic cells exhibited a higher T_m (2 C) than the respective DNA sequences in normal cells. In normal and leukemic cells the slow hybrid sequences appeared to have a T_m which was 2 C higher than that of the fast hybrid sequences. Individual non-virus-producing chicken embryos, either group-specific antigen positive or negative, contained 40 to 100 copies of the fast sequences and 2 to 6 copies of the slowly hybridizing sequences per cell genome. Normal rat cells did not contain DNA that hybridized with AMV RNA, whereas non-virus-producing rat cells transformed by B-77 avian sarcoma virus contained only the slowly reacting sequences. The results demonstrate that leukemic cells transformed by AMV contain new AMV-specific DNA sequences which were not present before infection.     

Comparison of an Avian Osteopetrosis Virus with an Avian Lymphomatosis Virus by RNA-DNA Hybridization

Myeloblastosis-associated virus (MAV)-2(0), a virus which was derived from avian myeloblastosis virus and induced a high incidence of osteopetrosis, was compared with avian lymphomatosis virus 5938, a recent field isolate which induced a high incidence of lymphomatosis. The following information was obtained. (i) MAV-2(0) induced osteopetrosis, nephroblastoma, and a very low incidence of hepatocellular carcinoma. No difference was seen in the oncogenic spectrum of end point and plaque-purified MAV-2(0). (ii) ¹²⁵I-labeled RNA sequences from MAV-2(0) formed hybrids with DNA extracted from osteopetrotic bone at a rate suggesting five proviral copies per haploid cell genome. The extent of hybridization of MAV-2(0) RNA with DNA from osteopetrotic tissue was more extensive (87%) than was observed in reactions with DNA from uninfected chicken embryos (52%). (iii) Competition of unlabeled viral RNA in hybridization reactions between the radioactive RNA from the two viruses and their respective proviral sequences present in tumor tissues showed that 15 to 20% of the viral sequences detected in these reactions were unshared. In contrast, no differences were detected in competition analyses of RNA sequences from the two viruses detected in DNA of normal chicken cells. (iv) MAV-2(0) 35S RNA was indistinguishable in size from avian lymphomatosis virus 5938 35S RNA by polyacrylamide gel electrophoresis.     

In vitro association of unique species of cellular 4S RNA with 35S RNA of RNA tumor viruses

Unique 4S RNA species from AKR mouse embryo cells hybridize with AKR murine leukemia virus and avian myeloblastosis virus 35S RNAs $\text{in vitro}$. Analyses by reversed-phase column chromatography indicate that the major 4S species that hybridize with the two viral RNAs are probably the same. A 4S RNA species with similar chromatographic properties is a major component of the AKR viral 4S RNA which associates with the viral 70S RNA $\text{in vivo}$.     

Association of 4S Ribonucleic Acid with Oncornavirus Ribonucleic Acids

Oncornavirus 60 to 70S ribonucleic acids (RNA), such as those from avian myeloblastosis virus, Schmidt-Ruppin virus, or mouse sarcoma-mouse leukemia viruses, isolated by conventional techniques, contain 4S transferlike RNA molecules that are released upon dissociation of the 60 to 70S RNA with heat. The 4S RNA represents 2.5 to 3.0% of the RNA in the 65S aggregate or 4 to 5 molecules per molecule of 35S RNA formed.     

Template recognition and chain elongation in DNA synthesis in vitro.

DNA polymerase from Escherichia coli (Pol I) and from avian myeloblastosis virus (AMV polymerase) were compared for the manner in which they catalyze the polymerization of deoxynucleotides upon a variety of synthetic and natural templates. It was found that the rates of nucleotide incorporation with different natural RNAs were similar. Both polymerases have an associated RNA endonuclease which hydrolyses RNA templates containing double-stranded regions. This activity depends on the presence of the complementary deoxynucleoside triphosphates, and/or polymerization. Both enzymes copy natural DNA, which has been sonicated and treated with E. coli exonuclease III, at the same rate. However, avian myeloblastosis virus DNA polymerase, which has no associated DNA exonuclease activity, is unable to copy double-stranded DNA and copies DNAase-treated DNA only 10% as well as Pol I. Pol I copied all the homopolymers investigated at a greater rate than AMV polymerase with the exception of poly(C) · oligo(dG). However, the initial rate of chain elongation, as measured by gel electrophoresis, was the same for the two polymerases, approximately 300 nucleotides incorporated per minute. Template saturation experiments show a stoichiometric relationship between template and enzyme at optimal rates of nucleotide incorporation which suggests that all enzyme molecules are potential catalysts. Enzyme saturation experiments indicate that not all enzyme molecules are “effectively” bound to a template. Fewer AMV polymerase than Pol I molecules are functionally bound to a particular template. From these data, it is concluded that the two polymerases elongate DNA chains in a similar way and that the manner in which the polymerases bind to a particular template accounts for the discrepancies found in their turnover numbers.     

Interspersion of sequences in avian myeloblastosis virus rna that rapidly hybridize with leukemic chicken cell DNA.

Liquid hybridization of progressively smaller fragments (35S, 27S, 15.5S, 12.5S, and 8S) of poly(A)-selected avian myeloblastosis virus RNA with excess DNA from leukemic chicken myeloblasts revealed that all sizes of RNA contained sequences complementary to both slowly and rapidly hybridizing cellular DNA sequences. Apparently, the RNA sequences which hybridize rapidly with excesses of cellular DNA are not restricted to any one region of the avian myeloblastosis virus 35S RNA. Instead, they appear to be randomly distributed over the entire 35S avian myeloblastosis virus RNA molecule with some positioned within 200 nucleotides of the poly(A) tract at the 3' end of the RNA.     

The presence of polyriboadenylic acid sequences in calf lens messenger RNA

The presence of poly(rA) sequences in lens RNA has been demonstrated by the isolation of RNase A and T₁-resistant fragments of approximately 50 nucleotide residues. These poly(rA)-rich sequences, obtained from lenses incubated for six hours in organ culture with [³H]adenosine, are located at the 3′ termini of mRNA as determined by 3′ exoribonuclease digestion. Limited digestion of the [³H]adenosine-labeled mRNA with the enzyme led to the abolition of binding to poly(rU)-filters and a concomitant loss of template activity with avian myeloblastosis virus RNA-dependent DNA polymerase. Furthermore, after incubation of lenses in organ culture with 3′-deoxyadenosine, the isolated polysomal RNA was unable to function as a template in an avian myeloblastosis virus RNA-dependent DNA polymerase-catalyzed reaction system.     

Transcription of 70S RNA by DNA polymerases from mammalian RNA viruses.

DNA polymerases purified by the same procedure from four mammalian RNA viruses, simian sarcoma virus type 1, gibbon ape lymphoma virus, Mason-Pfizer monkey virus, and Rauscher murine leukemia virus are capable of transcribing heteropolymeric regions of viral 70S RNA without any other primer. In this reconstituted system the enzymes from simian sarcoma virus type 1, Mason-Pfizer monkey virus, and Rauscher murine leukemia virus transcribe viral 70S RNA almost as efficiently as the DNA polymerase from the avian myeloblastosis virus, but gibbon ape lymphoma virus DNA polymerase is approximately three-to fivefold less efficient. Although there is a substantial difference among the sizes of these DNA polymerases (160,000 daltons for the avian myeloblastosis virus enzyme, 110,000 daltons for the Mason-Pfizer monkey virus enzyme, and 70,000 daltons for the mammalian type C viral polymerases), the ability to transcribe viral 70S RNA is a characteristic common to these enzymes.     

Lysine tRNA''s associated with avian myeloblastosis virus 70S RNA.

The lysine tRNA released from the 70S RNA of avian myeloblastosis virus was separated by reversed-phase chromatography. All of the AAG-coding lysine tRNA's were present in the 70S-associated fraction; however, the AAA-coding lysine tRNA could not be detected. Chromatography of the lysine tRNA released at various temperatures did not show any preferential release of one AAG-coding species over another.     

Inhibition of reverse transcriptase activity of RNA-tumor viruses by fagaronine+.

Fagaronine, a benzophenanthridine alkaloid from roots of $\text{Fagara zanthoxyloides}$ (Rutaceae), has been reported to possess anti-leukemic activity. It inhibited RNA-directed DNA polymerase activity from avian myeloblastosis virus, Rauscher leukemia virus and simian sarcoma virus. With poly rA·oligo dT, the alkaloid concentration for 50% inhibition of the enzyme activity from these viruses was in the range of 6–12 μg (15 – 31 nmoles) per ml of reaction mixture. The enzyme reaction was also inhibited with activated DNA and 70S RNA as templates; however, with poly rC·oligo dG no inhibition of enzyme activity was obtained. These results suggest that fagaronine inhibits enzyme activity by interaction with the A:T templateprimer.     

Subunits of oncornavirus high-molecular-weight RNA. I. Stepwise conversion of 60 S AMV (avian myeloblastosis virus) RNA to subunits

The effect of formamide on the dissociation of aggregate structure of high-molecular-weight RNA of avian myeloblastosis virus, an oncornavirus, was studied. It has been found that the pretreatment with increasing formamide concentration leads to the stepwise conversion of 60 – 70 S RNA molecule to 50 – 54 S and 30 – 40 S components; the 50 – 54 S intermediate is then further converted to 30 – 40 S subunits and smaller heterogenous RNAs. It is suggested that the subunits forming the aggregate RNA molecule of oncornaviruses are held together by not equally stable double stranded regions.