Purification of Avian Myeloblastosis Virus DNA Polymerase by Affinity Chromatography on Polycytidylate-Agarose

Polycytidylic acid [poly(rC)] covalently linked to cyanogen bromide-activated agarose is an effective affinity matrix for the RNA-dependent DNA polymerase from avian myeloblastosis virus. Poly(rC)-agarose is capable of binding large quantities of avian myeloblastosis DNA polymerase, which is then eluted by using a linear KCl gradient of increasing concentration. The DNA polymerase isolated from crude, detergent-disrupted virions by a single pass through columns of poly(rC)-agarose appears nearly homogeneous (approximately 90% pure) as determined by sodium dodecyl sulfate-polyacrylamide disc gel electrophoresis. Complete recovery of input enzymatic activity was obtained. Results suggest that polyribonucleotide columns may provide a high-yield, rapid method for the purification of oncornaviral DNA polymerase.     

Outer Membrane of Avian Myeloblastosis Virus

Guinea pigs immunized intracerebrally with avian myeloblastosis virus (AMV) produced antiserum which reacted with intact virus particles in complement fixation. The antigen in question appeared to be located on the surface of the virion and could be distinguished from the type-specific virus envelope and the group-specific internal antigens of chicken leukosis-sarcoma viruses (ChiLSV). The material could be isolated by sequential treatments of AMV with bromelin, Tween 20, and freeze-thawing, and could be purified by differential centrifugation. Electron microscopy analysis indicated the presence of a component resembling the outer membrane of the particle. The antigenic determinant was designated virus membrane antigen (Vm). Further analyses revealed the presence of protein, lipid, and carbohydrate in a material having a molecular weight of about 6,000 as determined by sodium dodecyl sulfate gel electrophoresis. Serological studies suggested that the outer membranes of AMV and other ChiLSV are represented mainly by host cellular material.     

Effect of Chemically Modified 70S RNA from Avian Myeloblastosis Virus (AMV) upon the Activity of AMV DNA Polymerase

Adenine residues of 70S avian myeloblastosis virus (AMV) RNA are modified when reacted with chloroacetaldehyde. This modification introduces characteristic fluorescent epsilon-adenosine (epsilonA) probes which were used to monitor the reaction. Under suitable conditions, modified 70S(epsilonA) RNA was maintained intact and was inactive as a template for the AMV DNA polymerase. Furthermore, it inhibited the reaction catalyzed by AMV polymerase when 70S RNA was used as template-primer and had no effect on the two tested bacterial polymerases. Protection against the 70S (epsilonA) RNA inhibition was observed when 70S RNA was primed with oligo(dT) indicating preference of the polymerase for the oligo(dT) primed regions.     

Extent of Double Strandedness in Avian Myeloblastosis Virus RNA

The extent of double strandedness of avian myeloblastosis virus 70S RNA has been determined from fluorescence measurements of the intercalation of ethidium bromide. We have shown that 50% of the nucleotides of 70S RNA in solution are in a stable helical configuration. This value does not include small helical regions that are too unstable to permit intercalation of the dye. The avian myeloblastosis virus RNA as it exists within the virion has the same degree of helicity as the free 70S RNA. Heating the free 70S RNA to 55 or 70 C, followed by cooling, does not measurably change the degree of helicity; the subunits therefore have as much helicity as the parent molecule.     

Chromatographic Analyses of Isoaccepting tRNAs from Avian Myeloblastosis Virus

Avian myeloblastosis virus (AMV) 4S RNA was tested for amino acid acceptor activity for 18 of the 20 amino acids. A nonrandom distribution of viral tRNAs was found compared with tRNA from normal liver or from AMV-infected leukemic myeloblasts, confirming previous reports. Methionine and proline tRNAs were considerably enriched, whereas glutamic acid, glutamine, serine, tyrosine, and valine tRNAs were markedly depleted in AMV relative to homologous cellular tRNAs. The seven AMV tRNAs with the greatest amino acid acceptance capacities, which were in order methionine, proline, lysine, arginine, histidine, isoleucine, and threonine tRNAs, were compared with homologous tRNAs from leukemic myeloblasts and liver by reversed-phase 5 chromatography. Of the 25 isoaccepting chromatographic fractions identified, no tRNA species unique to AMV was detected. Only methionyl-tRNA showed a substantial quantitative variation in isoaccepting species compared with the host cell. Thus, viral selectivity for amino acid-specific tRNAs is not, generally, paralleled by selectivity for individual isoaccepting tRNA species. Qualitative differences in arginyl- and histidyl-tRNA isoaccepting species were discovered in virus and leukemic myeloblasts compared with liver. This indicates the existence of structural differences in these tRNA species which could be related to virus replication or expression.     

Enzymatic Radioiodination of the Envelope Proteins of Avian Myeloblastosis Virus

Purified avian myeloblastosis virus was iodinated by the lactoperoxidase method. Disruption of the labeled virions and chromatography of the viral proteins showed that radioiodine was associated only with the glycoproteins of the viral envelope. Reaction of the radiolabeled virus with antiviral antibody followed by mild detergent treatment and subsequent recovery of immune complexes showed this technique to be useful for the isolation of viral envelope antigens.     

VSV Pseudotype Particles with the Coat of Avian Myeloblastosis Virus

PSEUDOTYPES of vesicular stomatitis virus (VSV) with the coat of avian myeloblastosis (AMV) or murine leukaemia viruses—VSV(AMV) and VSV(MLV)—can be produced by growing VSV in chick cells preinfected with AMV or in mouse cells preinfected with MLV¹. The VSV particles carrying their own neutralization antigen and double-neutralizable particles may be inactivated with antiserum against VSV. The surviving pseudotypes possess neutralization, host-range and interference specificities corresponding to the tumour virus donating their coat. It has also been shown that a conditional lethal mutant of VSV in which a structural protein is affected is complemented under restrictive conditions with AMV. This mutant, ts-45, when complemented with AMV again predominantly produces the pseudotype VSV(AMV).     

Transient Inhibition of Avian Myeloblastosis Virus Reproduction by Amethopterin and Fluorodeoxyuridine

Avian myeloblastosis virus cannot initiate its reproduction in the presence of amethopterin or fluorodeoxyuridine. This inhibition is reversed by thymidine. Addition of either inhibitor after virus production has started does not inhibit further virus synthesis. In presence of either inhibitor, deoxyribonucleic acid synthesis is inhibited by over 90%, but ribonucleic acid synthesis is not affected. Cells resume their normal growth rate 24 hr after removal of either inhibitor.     

Transfer Ribonucleic Acid Synthetase Activity Associated with Avian Myeloblastosis Virus

Avian myeloblastosis virions purified by conventional techniques were shown to be associated with or to contain transfer ribonucleic acid synthetase activity. Arginine, tryptophan, cystine, and lysine synthetase activities were observed.     

Absence of Ribonucleic Acid Methylase in the Avian Myeloblastosis Virus Core

N(2)-guanine-ribonucleic acid-methyltransferase, which is associated with avian myeloblastosis virus, is not a component of the viral core.     

Integrated State of Oncornavirus DNA in Normal Chicken Cells and in Cells Transformed by Avian Myeloblastosis Virus

The covalent linkage of oncornavirus-specific DNA to chicken DNA was investigated in normal chicken embryo fibroblasts (CEF) and in virus-producing leukemic cells transformed by avian myeloblastosis virus (AMV). The virus-specific sequences present in cellular DNA fractionated by different methods were detected by DNA-RNA hybridization by using 70S AMV RNA as a probe. In CEF and in leukemic cells, the viral DNA appeared to be present only in the nucleus. After cesium chloride-ethidium bromide density equilibrium sedimentation, the viral DNA was present as linear, double-stranded molecules not separable from linear chicken DNA. After extraction by the Hirt procedure, the viral DNA precipitated with the high-molecular-weight DNA. After alkaline sucrose velocity sedimentation, the viral DNA cosedimented with the high-molecular-weight cellular DNA. The results indicate that in both types of cells studied, the oncornavirus-specific DNA sequences were linked by alkali stable bonds to nuclear cellular DNA of high molecular weight and did not appear to be present in free form of any size.     

Amino Acid Sequence Analysis of Reverse Transcriptase Subunits from Avian Myeloblastosis Virus

The NH₂-terminal amino acid sequences of the α and β chains of avian myeloblastosis αβ DNA polymerase were determined by using microsequence analysis in the subnanomole range and were found to be identical up to 17 residues. The common sequence was as follows: Thr-Val-Ala-Leu-His-Leu-Ala-Ile-Pro-Leu-Lys-Trp-Lys-Pro-Asn-His-Thr-. This result provides convincing chemical evidence that the α chain is derived from the NH₂-terminal region of the β chain by proteolytic cleavage, whereas the amino acid composition for these α and β subunits and p32 DNA endonuclease suggests that the latter is derived from the carboxyl-terminal region of the β chain.     

Surface-Active Agents for Isolation of the Core Component of Avian Myeloblastosis Virus

Sixty-one surface-active agents were evaluated in a procedure designed to assess their ability to remove the envelope from the core component of avian myeloblastosis virus (AMV). The procedure consisted of centrifugation of intact AMV through a series of sucrose gradients each containing an upper layer of agent at one of eight concentrations between 0.01 and 10%. The effectiveness of an agent in producing AMV cores was indicated by (i) the appearance of light-scattering bands in the region of core buoyant density in gradient tubes; (ii) the range of surfactant concentration over which these bands appeared; and (iii) an electron microscopy assessment by the negative-staining technique of the relative proportion of core to non-core material in each of these bands. Six nonionic surfactants were selected by this screening method for comparison in regard to recovery of core protein and endogenous ribonucleic acid (RNA)-dependent deoxyribonucleic acid (DNA) polymerase activity, as well as further morphologic evaluation by electron microscopy. The nonionic surfactants of the polyoxyethylene alcohol class (particularly, Sterox SL) were most effective. Nonionic surfactants of the polyoxyethylene alkylphenol class (particularly, Nonidet P-40) were also effective. Sterox SL and Nonidet P-40 each gave a more than fivefold increase in specific activity of endogenous RNA-dependent DNA polymerase, and each gave a low recovery of core protein. Sterox SL did not interfere to the extent that Nonidet P-40 did in procedures which involved spectrophotometric assay at 260 nm. The use of Sterox SL resulted in the least envelope contamination of core preparations by electron microscopy examination, the most recovery of protein and endogenous RNA-dependent DNA polymerase activity, and a core buoyant density in sucrose of 1.27 g/ml.     

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.     

Ribonuclease H/DNA Polymerase HIV-1 Reverse Transcriptase Dual Inhibitor: Mechanistic Studies on the Allosteric Mode of Action of Isatin-Based Compound RMNC6

The DNA polymerase and ribonuclease H (RNase H) activities of human immunodeficiency virus type 1 (HIV-1) are needed for the replication of the viral genome and are validated drug targets. However, there are no approved drugs inhibiting RNase H and the efficiency of DNA polymerase inhibitors can be diminished by the presence of drug resistance mutations. In this context, drugs inhibiting both activities could represent a significant advance towards better anti-HIV therapies. We report on the mechanisms of allosteric inhibition of a newly synthesized isatin-based compound designated as RMNC6 that showed IC₅₀ values of 1.4 and 9.8 μM on HIV-1 RT-associated RNase H and polymerase activities, respectively. Blind docking studies predict that RMNC6 could bind two different pockets in the RT: one in the DNA polymerase domain (partially overlapping the non-nucleoside RT inhibitor [NNRTI] binding pocket), and a second one close to the RNase H active site. Enzymatic studies showed that RMNC6 interferes with efavirenz (an approved NNRTI) in its binding to the RT polymerase domain, although NNRTI resistance-associated mutations such as K103N, Y181C and Y188L had a minor impact on RT susceptibility to RMNC6. In addition, despite being naturally resistant to NNRTIs, the polymerase activity of HIV-1 group O RT was efficiently inhibited by RMNC6. The compound was also an inhibitor of the RNase H activity of wild-type HIV-1 group O RT, although we observed a 6.5-fold increase in the IC₅₀ in comparison with the prototypic HIV-1 group M subtype B enzyme. Mutagenesis studies showed that RT RNase H domain residues Asn474 and Tyr501, and in a lesser extent Ala502 and Ala508, are critical for RMNC6 inhibition of the endonuclease activity of the RT, without affecting its DNA polymerization activity. Our results show that RMNC6 acts as a dual inhibitor with allosteric sites in the DNA polymerase and the RNase H domains of HIV-1 RT.     

Presence in Leukemic Cells of Avian Myeloblastosis Virus-Specific DNA Sequences Absent in Normal Chicken Cells

(3)H-labeled 35S RNA from purified avian myeloblastosis virus (AMV) was exhaustively hybridized with an excess of normal chicken DNA to remove all viral RNA sequences which are complementary to DNA from uninfected cells. The [(3)H]RNA which failed to hybridize was isolated by hydroxylapatite column chromatography which separates DNA-RNA hybrids from single-stranded [(3)H]RNA. The residual RNA hybridized to leukemic chicken DNA but did not rehybridize with normal chicken DNA. This demonstrates conclusively that DNA from AMV-induced leukemic cells contain viral-specific sequences which are absent in DNA from normal cells.     

Structural Studies of Avian Myeloblastosis Virus: Comparison of Polypeptides in Virion and Core Component by Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis

Two different systems of dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in separate laboratories detected analogous patterns of dye bands in virions of avian myeloblastosis virus (AMV). At least 11 of the dye bands co-migrated with the major polypeptides reported in Rous sarcoma virus. Particles with the morphology of the AMV core component, obtained after exposure of AMV to the nonionic surfactant Sterox SL, contained major polypeptides p12, p27, p60, p64, p91, and p98. The polypeptide p12 has been previously shown to be the major constituent of the inner ribonucleoprotein (RNP) of the AMV core, and has been designated p12(N). Two RNP polypeptides, p64 and p91, co-electrophoresed with purified AMV DNA polymerase and have now been designated p64(P) and p91(P). The polypeptide p27 has been identified as a probable constituent of the core shell, and has accordingly now been designated p27(C). In comparison to virions of AMV, the AMV core component contained a greatly reduced amount of polypeptide p15 and appeared to lack a major polypeptide, p19. Consequently, these polypeptides may be associated either with the exterior of the core shell or the interior of the viral envelope. Glycopeptides were not detected in AMV cores, in agreement with earlier reports that they reside in external projections from the viral envelope.