Alteration of Nucleic Acid Structure and Stability Modulates the Efficiency of Minus-Strand Transfer Mediated by the HIV-1 Nucleocapsid Protein

Susan L Heilman-Miller,Tiyun Wu,Judith G Levin

doi:10.1074/jbc.m401646200

Abstract

During human immunodeficiency virus type 1 minus-strand transfer, the nucleocapsid protein (NC) facilitates annealing of the complementary repeat regions at the 3'-ends of acceptor RNA and minus-strand strong-stop DNA ((-) SSDNA). In addition, NC destabilizes the highly structured complementary trans-activation response element (TAR) stem-loop (TAR DNA) at the 3'-end of (-) SSDNA and inhibits TAR-induced self-priming, a dead-end reaction that competes with minus-strand transfer. To investigate the relationship between nucleic acid secondary structure and NC function, a series of truncated (-) SSDNA and acceptor RNA constructs were used to assay minus-strand transfer and self-priming in vitro. The results were correlated with extensive enzymatic probing and mFold analysis. As the length of (-) SSDNA was decreased, self-priming increased and was highest when the DNA contained little more than TAR DNA, even if NC and acceptor were both present; in contrast, truncations within TAR DNA led to a striking reduction or elimination of self-priming. However, destabilization of TAR DNA was not sufficient for successful strand transfer: the stability of acceptor RNA was also crucial, and little or no strand transfer occurred if the RNA was highly stable. Significantly, NC may not be required for in vitro strand transfer if (-) SSDNA and acceptor RNA are small, relatively unstructured molecules with low thermodynamic stabilities. Collectively, these findings demonstrate that for efficient NC-mediated minus-strand transfer, a delicate thermodynamic balance between the RNA and DNA reactants must be maintained.

Highlights

nucleocapsid protein (NC) nucleic acid chaperone activity is critical for the minus-strand and plus-strand [18, 20, 22, 42] transfer events that are required to complete elongation of minus- and plus-strand DNAs and to generate the long terminal repeats that flank the ends of proviral DNA [43]
In the absence of NC, the trans-activation response element (TAR) DNA structure in HIV-1 (Ϫ) SSDNA (Fig. 1B) induces nonproductive self-priming (Fig. 1A), which dramatically reduces the efficiency of minus-strand transfer [21, 23, 25, 51, 53, 55,56,57]
We have performed a systematic analysis to understand how the nucleic acid chaperone activity of NC is affected by varying degrees of stable secondary structure in either (Ϫ) SSDNA or acceptor RNA during minusstrand transfer

Summary

THE JOURNAL OF BIOLOGICAL CHEMISTRY

Vol 279, No 42, Issue of October 15, pp. 44154 –44165, 2004 Printed in U.S.A. Alteration of Nucleic Acid Structure and Stability Modulates the Efficiency of Minus-Strand Transfer Mediated by the HIV-1 Nucleocapsid Protein*. NC destabilizes the highly structured complementary trans-activation response element (TAR) stem-loop (TAR DNA) at the 3؅-end of (؊) SSDNA and inhibits TAR-induced self-priming, a deadend reaction that competes with minus-strand transfer. In vitro studies with mutant HIV-1 NC proteins have demonstrated that the zinc finger motifs are required for NC-dependent inhibition of self-priming and destabilization of highly structured nucleic acids (21, 23, 64 – 67), including the TAR stem-loops in (Ϫ) SSDNA (HIV-1 [21, 23, 60] and feline immunodeficiency virus [68]) and in viral RNA [69]. These results demonstrate that a delicate balance must exist between the stabilities of intramolecular secondary structures in (Ϫ) SSDNA and acceptor RNA and the stability of the intermolecular strand transfer duplex

EXPERIMENTAL PROCEDURES

Nucleic Acid Structure and NC Function in Strand Transfer

RNA length

DISCUSSION