Endonuclease activity of purified RNA-directed DNA polymerase from avian myeloblastosis virus.

Highly purified preparations of RNA-directed DNA polymerase from avian myeloblastosis virus (AMV) contain a Mn2+-activated endonuclease activity capable of nicking supercoiled DNA. This endonuclease activity co-sediments in glycerol gradients with the alphabeta form of AMV DNA polymerase, and co-chromatographs with DNA polymerase activity on DEAE-cellulose, phosphocellulose, and heparin-Sepharose. It is also present in AMV alphabeta-DNA polymerase purified by electrophoresis through nondenaturing polyacrylamide gels and subsequently chromatographed on poly(C)-agarose. alphabeta-associated endonuclease is co-immunoprecipitated with DNA polymerase activity by antiserum directed against alphabeta holoenzyme. The alpha form of AMV DNA polymerase lacks this activity. In its enzymatic properties, alphabeta-associated endonuclease resembles the endodeoxyribonuclease activity associated with the AMV p32 protein, which has been shown to be structurally related to the beta (but not the alpha) subunit of AMV DNA polymerase.     

Isolation of two subgroup-specific leukemogenic viruses from standard avian myeloblastosis virus.

Two populations of virus having subgroup-specific homogeneity (A and B) were isolated from standard avian myeloblastosis virus stocks by passage in vivo through genetically defined chickens. Each possesses leukemogenic activity in vivo. Other properties and potential usefulness of these agents are discussed.     

Structural studies of the retroviral proteinase from avian myeloblastosis associated virus.

The structure of the retroviral proteinase from avian myeloblastosis associated virus (MAV) has been determined and refined at 2.2 A resolution. This structure is compared with those of homologous proteinases from Rous sarcoma virus (RSV) and human immunodeficiency type 1 virus (HIV). Through comparison with the structure of a proteinase-inhibitor complex from HIV, a model of a complex between MAV proteinase and a peptide substrate has been generated. Examination of this model suggests structural basis for the diverse specifications of viral proteinases.     

Characterization of the adenosine triphosphatase of avian myeloblastosis virus.

The ATPase of avian myeloblastosis virus (AMV) is not a recognizable cellular enzyme. It hydrolyzes ATP, GTP, ITP, UTP, and dCTP at equal rates, is inhibited by high concentrations of dithiothreitol, and is partially inhibited by 1 × 10^?5mp-chloromercuribenzoic acid (PCMB) and p-chloromercuribenzene sulfonate acid (PCMBS). The inhibition by the mercurials is reversed by increasing the concentration of PCMB or PCMBS to 1 × 10^?3m. The enzyme requires phospholipid for activity. Incubation with phospholipase C inhibits activity and subsequent addition of lecithin-containing saturated fatty acids partially restores activity, whereas lecithin-containing unsaturated fatty acids further inhibit activity.     

A cell-free mammalian protein-synthesizing system stimulated by RNA from avian myeloblastosis virus.

Carcinoma cells, oncornavirus-infected cells and fetal bovine tissue provide salt wash ribosomal factors capable of responding to avian myeloblastosis virus (AM virus)-RNA and stimulating the incorporation of amino acids into proteins as well as catalyzing the binding of N-acetylated (35S) methionyl-tRNA. The exogenously dependent amino acid incorporation system is stimulated by the high molecular weight species of AM virus-RNA only, particularly the fraction containing polyadenylate (poly(A)) residues; the system is also markedly inhibited by the low molecular weight AM virus-RNA species. Activity for the exogenous system displays very definite divalent/monovalent cation optima and requires the presence of mammalian transfer RNA.     

Primer recognition by avian myeloblastosis virus RNA-directed DNA polymerase.

Tryptophanyl-tRNA was specifically labeled at the 3' end with [3H]tryptophan and cleaved in half with RNase under denaturing conditions, and the 3' half was shown to hybridize exclusively at the 5' end of avian myeloblastosis virus RNA. The RNA-dependent DNA polymerase of avian myeloblastosis virus is capable of efficiently binding the 3' half of the primer molecule.     

Amino acid sequence of p15 from avian myeloblastosis virus complex

The complete amino acid sequence of the p15 gag protein from avian myeloblastosis virus (AMV) complex has been determined by sequential Edman degradation of the intact molecule and of peptide fragments generated by limited tryptic cleavage, cleavage with staphylococcal protease, and cyanogen bromide cleavage. AMV p15 is a single-chain protein containing 124 amino acids. The charged amino acids tend to be clustered in the primary structure. p15 contains a single cysteine at position 113 which may be essential for the p15 associated proteolytic activity. However, p15 shows no appreciable sequence homology with papain or other classical thiol proteases.     

Characterization of an adenosine triphosphatase from myeloblasts infected with the avian myeloblastosis virus.

An adenosine triphosphatase (ATPase EC 3.6.1.3) was partially purified from myeloblasts of chicken infected with the avian myeloblastosis virus and some of its molecular, catalytic and immunological properties were compared with that of the ATPase purified from the virus. Both the enzymes possessed almost same electrophoretic mobility, molecular weight, S20,w value, substrate specificity, metal-ion requirement, apparent Km value and sensitivity to inhibitors and activator. Evidence also indicated immunological identity of the two enzymes. The insensitivity of this enzyme to rutamycin or ouabain and extreme sensitivity to most of the detergents, trypsin and mercurials are the remarkable properties of this enzyme.     

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.     

Fractionation of two protein kinases from avian myeloblastosis virus and characterization of the protein kinase activity preferring basic phosphoacceptor proteins.

Two protein kinase activities were fractionated from purified virions of avian myeloblastosis virus. Distinguishing characteristics of these two protein kinases included: (i) their binding properties during purification by ion-exchange chromatography; (ii) their estimated molecular weights; and (iii) their phosphoacceptor protein specificities. The protein kinase that bound to the anion exchanger DEAE-cellulose (pH 7.2) had an estimated molecular weight of 60,000 to 64,000 and preferred basic phosphoacceptor proteins. The protein kinase that bound to the cation exchanger phosphocellulose (pH 7.2) had an estimated molecular weight of 42,000 to 46,000 and preferred acidic phosphoacceptor proteins. The protein kinase preferring basic phosphoacceptor proteins was further purified and characterized. Optimal transfer of phosphate catalyzed by this enzyme required a divalent metal ion, a sulfhydryl-reducing agent, and ATP as phosphate donor. GTP was not an effective phosphate donor at concentrations comparable to ATP; and the cyclic nucleotides cyclic AMP and cyclic GMP neither stimulated nor inhibited protein phosphorylation by the protein kinase. The specificity of the protein kinase for basic phosphoacceptor proteins extended to proteins from avian myeloblastosis virus, in that the neutral to basic virion proteins p12, p19, and p27 served as phosphate acceptors. In addition, the protein kinase also appeared to phosphorylate itself. The role(s) of this virion-associated protein kinase is discussed.     

Ribonuclease H activity present in purified DNA polymerase from avian myeloblastosis virus

The presence of ribonuclease H activity in the purified complex of DNA polymerase from avian myeloblastosis virus is described. Evidence includes co-chromatography of the two activities during all purification steps; the presence of ribonuclease H activity in the purified two-polypeptide complex of DNA polymerase; ion requirements for optimal activity of purified ribonuclease H are identical to those for the purified DNA polymerase; and monospecific antiserum against purified DNA polymerase neutralizes the ribonuclease H activity.     

Influence of phosphate on activity and stability of reverse transcriptase from avian myeloblastosis virus.

Activity of RNA-dependent DNA polymerase (RDDP) from avian myeloblastosis virus (AMV), either in purified form or in virus lysates, was increased by phosphorylation. Stability of RDDP in lysates buffered with phosphate was much greater (no loss of activity in 48 hours at 4 degrees) than that in lysates buffered with Tris-Cl (76% loss). Activity lost in the Tris-buffered extracts was completely restored by phosphorylation. The findings suggested that AMV RDDP activity is influenced by the degree of phosphorylation of the enzyme or enzyme-associated proteins and that this chemical modification is mediated by protein phosphokinase and phosphoprotein phosphatase present in crude extracts of purified AMV. Application of these results provided the basis of procedures whereby RDDP can be recovered in significantly higher yield and purity than formerly.