STUDIES OF RETROVIRAL PROTEIN-NUCLEIC ACID INTERACTIONS

Important note: Information in this article was accurate in 1992. The state of the art may have changed since the publication date.

STUDIES OF RETROVIRAL PROTEIN-NUCLEIC ACID INTERACTIONS

Diss Abstr Int [B]; 52(2):802 1991. Unique Identifier : AIDSLINE ICDB/92677992
Delahunty MD; Univ. of Maryland, Baltimore County

Abstract: The substrate interactions of two retroviral proteins were studied: the nucleocapsid (NC) protein and reverse transcriptase (RT). Retroviral NC proteins bind to retroviral RNA and contain one or two CysHis motifs which may be analogous to the CysHis motif of zinc fingers such as those found in the transcriptional activator TFIIIA. The gene for the full length NC protein from Moloney murine leukemia virus was cloned into an E coli expression vector in order to obtain sufficient quantities of protein to study the role of the CysHis motif in the binding of NC to nucleic acid. The resulting clone was shown by Northern blot analysis to be transcribed, but did not express NC protein in detectable levels. Nucleic acid binding studies were then pursued using a synthetic peptide that consists of the first CysHis motif of HIV-1 NC protein. The fluorescent polynucleotide, polyriboethenoadenylic acid (poly[eA]), was utilized to monitor peptide binding to nucleic acid. The apopeptide binds to poly(eA) with a larger site size and with less affinity at 1 mM sodium phosphate than the zinc-bound peptide does. The affinity is due to 1-2 electrostatic interactions as well as a nonelectrostatic contribution of about - 6 kcal/mol. The peptide binds to both single-stranded and double-stranded calf thymus DNA. Reverse transcriptase is the RNA-dependent DNA polymerase of the retrovirus, which plays an essential role in viral replication. An assay for binding at the primer site of HIV-1 RT was developed using the fluorescent probe, 2', 3'-trinitrophenyl-adenosine triphosphate. The Kd was found to be 21 uM. In fluorescence reversal assays, short oligodeoxynucleotides that act as primer analogs for RT showed increasing affinity with increasing length up to about 15 nucleotides. With polynucleotides, RT bound along the length of the nucleic acid and not just to the ends. Unfractionated tRNA and E coli tRNA(Trp) bound to RT with higher affinity than the synthetic oligodeoxynucleotides. (Full text available from University Microfilms International, Ann Arbor, MI, as Order No. AAD91-18002).

Keywords: Capsid/*METABOLISM DNA/*METABOLISM HIV-1/*METABOLISM Retroviridae Proteins/*METABOLISM RNA/*METABOLISM RNA-Directed DNA Polymerase/METABOLISM Viral Core Proteins/*METABOLISM THESIS
920228
M9220881

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