Comparison of the thermal stabilities of the αß heterodimer and the α subunit of avian myeloblastosis virus reverse transcriptase.

Avian myeloblastosis virus reverse transcriptase (AMV RT) is a heterodimer consisting of a 63-kDa α subunit and a 95-kDa ß subunit. In this study, we explored the role of the interaction between the α and ß subunits on AMV RT stability. The recombinant AMV RT α subunit was expressed in insect cells and purified. It exhibited lower thermal stability than the native AMV RT αß heterodimer. Unlike the αß heterodimer, the α subunit was not stabilized by template-primer. These results suggest that interaction between the α and ß subunits is important for AMV RT stability.

Avian retrovirus DNA internal attachment site requirements for full-site integration in vitro.

Concerted integration of retrovirus DNA termini into the host chromosome in vivo requires specific interactions between the cis-acting attachment (att) sites at the viral termini and the viral integrase (IN) in trans. In this study, reconstruction experiments with purified avian myeloblastosis virus (AMV) IN and retrovirus-like donor substrates containing wild-type and mutant termini were performed to map the internal att DNA sequence requirements for concerted integration, here termed full-site integration. The avian retrovirus mutations were modeled after internal att site mutations studied at the in vivo level with human immunodeficiency virus type 1 (HIV-1) and murine leukemia virus (MLV). Systematic overlapping 4-bp deletions starting at nucleotide positions 7, 8, and 9 in the U3 terminus had a decreasing detrimental gradient effect on full-site integration, while more internal 4-bp deletions had little or no effect. This decreasing detrimental gradient effect was measured by the ability of mutant U3 ends to interact with wild-type U3 ends for full-site integration in trans. Modification of the highly conserved C at position 7 on the catalytic strand to either A or T resulted in the same severe decrease in full-site integration as the 4-bp deletion starting at this position. These studies suggest that nucleotide position 7 is crucial for interactions near the active site of IN for integration activity and for communication in trans between ends bound by IN for full-site integration. The ability of AMV IN to interact with internal att sequences to mediate full-site integration in vitro is similar to the internal att site requirements observed with MLV and HIV-1 in vivo and with their preintegration complexes in vitro.

Oncogenic point mutations induce altered conformation, redox sensitivity, and DNA binding in the minimal DNA binding domain of avian myeloblastosis virus v-Myb.

c-Myb is the founder member of a class of transcription factors with tryptophan-rich repeats responsible for DNA binding. Activated oncogenic forms of Myb are encoded by the avian retroviruses, avian myeloblastosis virus (AMV) and E26. AMV v-Myb encodes a truncated protein with 11 point mutations relative to c-Myb. The mutations in the DNA binding domain (DBD) were reported to impose distinct phenotypes of differentiation on transformed myeloid cells (Introna, M., Golay, J., Frampton, J., Nakano, T., Ness, S. A., and Graf, T. (1990) Cell 63, 1287-1297). The molecular mechanism operating has remained elusive since no change in sequence specificity has been found. We introduced AMV-specific point mutations in the minimal DBD of chicken c-Myb and studied their effect on structure and function of the purified protein. Fluorescence emission spectra and fluorescence quenching experiments showed that the AMV-specific point mutations had a significant effect on the conformation of the DBD, giving rise to a more compact structure, a change that was accompanied by a reduced sensitivity toward cysteine-specific alkylation and oxidation. The DNA binding properties were also altered by the AMV-specific point mutations, leading to protein-DNA complexes with highly reduced stability. This reduction in stability was, however, more severe with certain subtypes of binding sequences than with others. This differential behavior was also observed in an in vivo model system where DBD-VP16 fusions were coexpressed with various reporters. These findings imply that different subsets of Myb-responsive promoters may react differentially toward the AMV-specific mutations, a phenomenon that could contribute to the altered patterns of gene expression induced by the AMV v-Myb relative to wild type c-Myb.

Okazaki fragments, a constant component of avian myeloblastosis virus core-bound 7 S DNA.

We have shown that the unusual CsCl-buoyant density and velocity sedimentation properties of the isolated host 7 S DNA species associated with the core fraction of avian myeloblastosis virus (AMV) are made mainly by tight association of RNA pieces prevalently joined to the single-stranded portion of this material. It was shown indirectly on sedimentation patterns of [methyl-3H]thymidine and [14C]uridine double-labelled and glyoxylated total AMV DNA, and directly in phosphorylation experiments with T4 polynucleotide kinase performed on the single-stranded portion of AMV DNA that the RNA-DNA link in AMV DNA is of a covalent nature and that the 5'-terminal end of DNA at the RNA-DNA junction is occupied by all four common deoxyribonucleotides. This first evidence of the presence of Okazaki fragments in 7 S AMV DNA clearly indicates that this DNA does not represent a randomly fragmented host DNA included by chance into virions but special fragments of host DNA having the properties of DNA replicative structures with possible consequences for some viral function(s) including those involved in virus-cell interactions.

Avian myeloblastosis virus core-bound 7 S DNA, highly bent minute structures with sequence-directed curvature.

Structural properties and length distribution profile of 7 S avian myeloblastosis virus (AMV) DNA were studied by means of electron microscopy using two different techniques. This DNA represents mostly double strands, the single strands being in minority. We have shown directly that this DNA forms a bent structure typical of the majority of molecules. These bends are sensitive to the distamycin treatment which stretches most of the bent molecules. Some amount (up to 30%) of circular DNA molecules was detected also in DNA preparations, the nature and the size of which are reminiscent of electron microscopic data on microbubbles of replicating DNA. No specific AMV DNA structural features were found using osmium-tetroxide treatment. The basic size of AMV DNA was estimated to be approximately 150 bp, but its multimers were also detected. Their presence and significance is discussed.

Interactions of avian myeloblastosis virus nucleocapsid protein with nucleic acids.

The retroviral nucleocapsid protein (NC) associates, histone-like, with genomic RNA within the viral capsid. NC, an essential component of replication competent retroviruses, is also associated with events leading both to virus assembly and to reverse transcription. The nucleic acid binding properties of NC are key to understanding these properties, yet only a minimal biochemical description of NC-nucleic acid interactions is available. We have used the anisotropy of the intrinsic fluorescence of NC from avian myeloblastosis virus to quantify its binding to a variety of nucleic acids. Using salt back-titrations, the intrinsic equilibrium association constant per nucleic acid site, K(obs), was determined for NC binding to single- and double-stranded RNAs and DNAs. In 0.125 M NaCl, 40 mM HEPES at pH 7.0 and 27 degrees C, the log K(obs) ranged from 3.3 to 4.0 (average 3.7) for these nucleic acids. From the salt dependence of K(obs), it was estimated that, on balance, 1 ion was displaced upon formation of each complex; it is likely that cation displacement from nucleic acid is offset by anion binding by protein during complex formation. The logarithm of the mean intrinsic affinity in the absence of polyelectrolyte effects, log KT, was 3.1, corresponding to a delta G of -4.2 kcal/mol. K(obs), KT, and the number of displaced ions were independent of pH between pH 5.6 and 8.9, indicating that NC residues that titrate in this pH range are not contributing to binding. K(obs) and KT increase with temperature, in the range 15 to 47 degrees C. From van't Hoff analysis, entropy was found to be the driving force for formation of the NC-poly(rA) complex, even in the absence of the polyelectrolyte effect. The general nature of NC interactions with nucleic acids is shown by the similarity of the K(obs) values for RNAs and DNAs in both single-stranded and double-stranded structures. This ability of NC to interact with all types of nucleic acids may provide it with the necessary versatility to function like a histone in facilitating the packaging of viral RNA and yet function early in infection, where it has been ascribed a role in facilitating reverse transcription.

Cloning and nucleotide sequence of the 5' part of v-myb cDNA.

cDNA of a subgenomic v-myb mRNA from AMV-transformed BM-2 cells was cloned. Sequencing of the 5' end of this cDNA revealed the structure of both AMV leader and the splice junction in v-myb message. The leader is a novel variant of known avian retrovirus leaders. The long open reading frame in the cloned cDNA starts with six gag-derived codons spliced to the myb-specific sequence.

Retroviral nucleocapsid protein specifically recognizes the base and the ribose of mononucleotides and mononucleotide components.

The interaction of the retroviral nucleocapsid protein (NC) with nucleic acids forms the basis of its varied roles in the replication cycle, which include binding and condensing the viral RNA within the virion, stimulation of the early steps in reverse transcription, and dissociation from RNA in the replication complex. As part of an investigation of the NC binding site and of the forces that drive its interaction with nucleic acids, the relative affinities of NC from avian myeloblastosis virus were determined for a series of mononucleotides and mononucleotide components using a competitive displacement assay utilizing the extrinsic fluorescent probe bis-ANS [Secnik, J., Wang, Q., Chang, C.-M., & Jentoft, J.E. (1990) Biochemistry 29, 7991-7997]. The estimated binding affinities were unexpectedly similar for nucleotides, nucleosides, and bases (Ka greater than 10(6) M-1). AMP, UMP, GMP, and CMP bound to NC with essentially equal affinity, indicating that NC does not discriminate between bases. This is consistent with its role in coating, condensing, and packaging the RNA within virions. Nucleosides, bases, riboses, and ribose phosphate bind to NC with 1000-fold higher affinity than inorganic phosphate, indicating that the NC binding site includes elements that recognize nucleotide base and ribose components in addition to phosphate ions. However, the binding affinities of components are not additive, i.e., the Kapp values for adenine and deoxyribose are very similar to that for deoxyadenosine, indicating that the interaction between the NC subsite and the base and the sugar components is complex. The stoichiometry of the complex between bis-ANS and NC was established to be NC.(bis-ANS)3.(ABSTRACT TRUNCATED AT 250 WORDS)