Nucleic Acid Conformational Changes Essential for HIV-1 Nucleocapsid Protein-mediated Inhibition of Self-priming in Minus-strand Transfer

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Abstract

Reverse transcription of the HIV-1 genome is a complex multi-step process. HIV-1 nucleocapsid protein (NC) is a nucleic acid chaperone protein that has been shown to greatly facilitate the nucleic acid rearrangements that precede the minus-strand transfer step in reverse transcription. NC destabilizes the highly structured transactivation response region (TAR) present in the R region of the RNA genome, as well as a complementary hairpin structure (“TAR DNA”) at the 3′-end of the newly synthesized minus-strand strong-stop DNA ((−) SSDNA). Melting of the latter structure inhibits a self-priming (SP) reaction that competes with the strand transfer reaction. In an in vitro minus-strand transfer system consisting of a (−) SSDNA mimic and a TAR-containing acceptor RNA molecule, we find that when both nucleic acids are present, NC facilitates formation of the transfer product and the SP reaction is greatly reduced. In contrast, in the absence of the acceptor RNA, NC has only a small inhibitory effect on the SP reaction. To further investigate NC-mediated inhibition of SP, we developed a FRET-based assay that allows us to directly monitor conformational changes in the TAR DNA structure upon NC binding. Although the majority (∼71%) of the TAR DNA molecules assume a folded hairpin conformation in the absence of NC, two minor “semi-folded” and “unfolded” populations are also observed. Upon NC binding to the TAR DNA alone, we observe a modest shift in the population towards the less-folded states. In the presence of the RNA acceptor molecule, NC binding to TAR DNA results in a shift of the majority of molecules to the unfolded state. These measurements help to explain why acceptor RNA is required for significant inhibition of the SP reaction by NC, and support the hypothesis that NC-mediated annealing of nucleic acids is a concerted process wherein the unwinding step occurs in synchrony with hybridization.

Introduction

Reverse transcription in retroviruses is a complex process that results in synthesis of a linear double-stranded DNA from the single-stranded genomic RNA template.1 The HIV nucleocapsid protein (NC) stimulates this process, which involves a number of nucleic acid rearrangements, including tRNA primer annealing and two strand transfer events.1., 2., 3., 4., 5., 6., 7., 8., 9. Thus, NC is a nucleic acid “chaperone” protein that facilitates nucleic acid conformational changes to form the most thermodynamically stable interactions in an ATP-independent fashion.10., 11., 12., 13., 14.

HIV-1 NC is a 55 amino acid, highly basic, nucleic acid-binding protein. The mature form, also known as NCp7, is generated by cleavage of the viral Gag precursor protein.15., 16., 17. NC contains two CCHC metal-binding motifs, which form tandem zinc finger structures connected by a short linker sequence.18., 19. We have recently shown that the zinc finger structures are critical for NC's nucleic acid chaperone activity.20., 21., 22., 23. The CCHC zinc fingers are also required for full chaperone activity of FIV NCp8 in vitro.24 In addition, NC's zinc fingers appear to impart preferential binding to single-stranded nucleic acids.21., 25., 26., 27.

Despite NC's apparent preference for binding single-stranded nucleic acids, the preference is modest,28 consistent with its role in interacting with both single and double-stranded RNA and DNA molecules during reverse transcription. NC greatly facilitates tRNA primer annealing to the HIV genome. However, it has been shown that NC binding to tRNA alone does not induce global unwinding of the tRNA structure,29., 30. but rather causes more subtle changes in the tertiary and secondary structural motifs.30., 31. Thus, at least in the case of tRNA annealing, NC binding does not facilitate tRNA unwinding in the absence of the complementary primer binding site, i.e. unwinding occurs simultaneously with annealing.

Following tRNA primer annealing, minus-strand strong-stop DNA ((−) SSDNA) is synthesized and then translocated to the 3′ terminus of the viral RNA genome, in a reaction mediated by base pairing of the complementary repeat (R) regions in (−) SSDNA and viral RNA. NC has been shown to stimulate minus-strand transfer by increasing the rate of intermolecular annealing and by blocking a competing intramolecular self-priming (SP) reaction.4., 7., 32., 33. SP occurs due to the presence of a highly structured sequence at the 3′-end of the (−) SSDNA.1., 4., 7., 34. This sequence, which is complementary to the TAR element at the 5′-end of the genome, will hereafter be referred to as “TAR DNA”. The TAR DNA sequence has the potential to fold back onto itself and form a hairpin, which can self-prime DNA synthesis and form a heterogeneous mixture of minus-strand DNA products with plus-strand extensions.7

The presence of HIV NC in in vitro minus-strand transfer assay systems has been shown to reduce the extent of TAR-dependent self-priming.7., 32., 33., 35. However, whether NC inhibition of SP is due to direct destabilization of the TAR DNA hairpin, or whether the reduction of SP in these in vitro systems is dependent on the presence of the TAR RNA acceptor strand is an open question. Lapadat-Tapolsky et al. originally reported that SP is inhibited by NC in the absence of an RNA acceptor.32 More recently, Hughes and co-workers have shown that NC can significantly inhibit SP from a 100-nt synthetic (−) SSDNA oligonucleotide only in the presence of a 17-nt DNA oligomer complementary to the 3′-end of the (−) SSDNA.33 The size of the DNA oligomer is similar to that of short RNA oligomers formed as a result of RNase H degradation of the RNA template during (−) SSDNA synthesis.33., 36.

Recently, using both absorbance and fluorescence spectroscopy, Mély and co-workers have directly examined NC's effect on the structure of a 55-nt TAR DNA hairpin mimic.37 This work showed that binding of an NC-derived peptide containing the zinc finger motifs, (12-55)NCp7, enhances the intrinsic fraying of the ends of the TAR DNA hairpin, and results in melting of 7–8 bp. However, the effect of NC binding on the TAR DNA structure in the presence of acceptor RNA was not probed. Nevertheless, taken together with the data of Hughes and co-workers discussed above, these results imply that partial melting of the TAR DNA hairpin may not be sufficient to reduce SP, at least in the absence of a complementary nucleic acid.

To examine this hypothesis and to gain further insights into NC's role as a nucleic acid chaperone protein in the minus-strand transfer step of reverse transcription, we carried out both biochemical and FRET-based assays. Using a modified in vitro minus-strand transfer assay system,7 we investigated NC's effect on the amount of SP products formed in the absence and presence of acceptor RNA. In addition, we used a FRET-based approach to directly monitor structural changes in the TAR DNA hairpin in the presence and absence of both NC and acceptor RNA. We found that NC binding to the TAR DNA hairpin alone causes partial unfolding of the hairpins. Under these conditions, very little NC-dependent inhibition of SP is observed. We also established that the acceptor RNA is required for significant inhibition of SP in this system. Moreover, in the presence of the acceptor RNA, the majority of the TAR DNA molecules are present in an unfolded state. Taken together, our data support the conclusion that the conformational changes observed upon NC binding to the TAR DNA alone are not sufficient to inhibit SP and promote strand transfer, and that substantial unwinding of the TAR DNA hairpin occurs only in the presence of the acceptor RNA.

Section snippets

NC effects on TAR-dependent self-priming

Previously, we showed that NC has the ability to promote the annealing step in minus-strand transfer20., 22., 38. and to reduce the formation of undesired SP products.7 In these assays, the acceptor RNA was always present and the effect of NC on the SP reaction in the absence of the complementary RNA was not reported. To address this question, we assayed DNA synthesis in the absence and presence of acceptor RNA. As shown in Figure 1(a), in the absence of NC, the predominant band observed

Discussion

In the present work, we have used both biochemical and FRET-based assays to investigate NC's activity in a system that models minus-strand transfer, an essential step in reverse transcription of the HIV-1 retroviral genome. We demonstrate that both NC and the acceptor RNA must be present in order to facilitate strand transfer and to inhibit a competing SP reaction, i.e. NC alone is insufficient to block SP of the TAR hairpin in (−) SSDNA (Figure 1). These results are in general agreement with

Protein purification

All of the proteins used in this work were obtained or prepared according to previously published procedures as follows: HIV-1 RT,7 wild-type NC,43 and T7 RNA polymerase.47

Nucleic acid preparation

Singly-labeled DNA oligonucleotides containing either 5′-Cy3 or 3′-FL (Cy3–DNA and DNA–FL, respectively), as well as the doubly-labeled DNA (Cy3–DNA–FL), were obtained from TriLink Biotechnologies (San Diego, CA). The oligonucleotides were purified on 12% denaturing polyacrylamide gels and determined to be greater than 95%

Acknowledgements

We thank Drs Natalia Tretyakova and Soobong Park for carrying out the mass spectrometry of the dye-labeled DNA oligonucleotides. We also thank Dr Wai-Tak Yip for assistance with data analysis as well as helpful discussion, and Dr Susan Heilman-Miller for valuable discussion and critical reading of the manuscript. We are grateful to Dr. Robert Gorelick for supplying some of the pure recombinant NC protein used in this work. This research was supported by NIH grant AI65056 (K.M.-F), NIH

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