New insights into the formation of HIV-1 reverse transcription initiation complex

doi:10.1016/j.biochi.2007.01.016

Biochimie

Volume 89, Issue 10, October 2007, Pages 1204-1210

https://doi.org/10.1016/j.biochi.2007.01.016 Get rights and content

Abstract

HIV-1 reverse transcriptase uses the host tRNA₃^Lys as a primer for the synthesis of the minus DNA strand. The first event in viral replication is thus the annealing of tRNA to the primer binding site (PBS) in the 5′ UTR of the viral RNA. This event requires a major RNA rearrangement which is chaperoned by the viral NC protein. The binding of NC to nucleic acids is essentially non-specific, however, NC is known to bind selectively to hairpins located in the 5′ region of the viral RNA. In a previous study, using an NMR approach in which the reaction is slowed down by controlling temperature, we were able to follow details in this RNA unfolding/refolding process and to uncover an intermediate state. We showed that annealing initiates at the junction between the acceptor and the TΨC stems, and that, at physiological temperature, complete annealing is reached only in the presence of NC, probably when the zinc fingers contact the TΨC/D loops.

In the present work, we have refined our model of the formation of the tRNA₃^Lys/PBS duplex. First, we show that annealing can initiate both from the single-stranded CCA 3′-end bases of the acceptor stem and from the bases in the TΨC stem. Secondly, by NMR and fluorescence spectroscopy, we have studied the complex between the NC protein and RNA hairpins that mimic the D and T arms of the tRNA₃^Lys. Interestingly, the NC protein shows strong and specific binding to the D arm of tRNA₃^Lys, which could explain the overall annealing mechanism.

Introduction

In retroviruses, initiation of reverse transcription is primed by a cellular tRNA that is encapsidated in viral particles. tRNA₃^Lys is the natural primer of all immunodeficiency viruses, including the type 1 human immunodeficiency virus (HIV-1) [1], [2], [3]. The HIV-1 nucleocapsid protein (NC) is a short, basic, nucleic acid binding protein with two zinc finger domains, each containing the invariant CCHC metal-ion binding motif. The mature protein (55 amino acid residues) is produced by proteolytic cleavage of the Gag precursor and is found in the interior of the virus particle, where it is tightly associated with genomic RNA. NC or the NC domain in Gag is involved in multiple functions during the virus replication cycle, including genomic RNA packaging, primer placement on viral RNA, reverse transcription, and integration. Many of these functions rely on the nucleic acid chaperone activity of NC, i.e. its ability to catalyse nucleic acid conformational rearrangements that lead to the most thermodynamically stable structure (for recent reviews [4], [5], [6]).

In a previous study [7], we were able to observe the progressive formation of the HIV-1 reverse transcription initiation complex using heteronuclear NMR of RNA–RNA–protein complexes under controlled temperature conditions. In particular, we have identified a nucleation site, at the end of the acceptor stem, where the viral RNA starts invading the tRNA₃^Lys structure. In addition, we were also able to characterize the different roles of the nucleocapsid protein during the formation of the initiation complex. In our model, the viral NC protein plays a key role by “unlocking” stable 3D structure of the primer tRNA through specific interaction with the D/TΨC loops. Therefore, at the end of this study, the question of the involvement of the 3′-end unpaired CCA bases of tRNA₃^Lys primer in the annealing process was still open. In addition, the mechanism of the specific 3D structure opening was still not clearly understood.

In the present report, we show that the annealing can proceed from two starting points in the tRNA₃^Lys primer, one at the beginning of the acceptor stem and the second at the end of the T stem. Both sites can be used by the viral RNA to begin its annealing with the 18 complementary bases of tRNA₃^Lys acceptor and T stems. The presence of NC protein is not necessary at this step, at least in vitro, but is essential to open the 3D structure of tRNA. The melting of the interaction at the level of the D/TΨC loops that locks the structure of the tRNA is possible through a specific interaction between the D loop and the NC protein.

Section snippets

Samples

The PBS (18 nucleotides) was ¹⁵N-labelled on its guanine nucleotides by in vitro transcription from an oligonucleotide template containing a 2′-O-methyl-G in position 2 (DNA template: 5′-G(2′OmeG)TCCCTGTTCGGGCGCCACTATAGTGAGTCGTATT-3′) [8]. Labelled nucleotides were purchased from Spectra Gases Inc. The 20 nucleotides RNA obtained was then purified by electrophoresis. After electroelution and ethanol precipitation, the RNA was resuspended, micro-dialysed against a first buffer (10 mM sodium

Where are the nucleation sites for tRNA₃^Lys/PBS duplex formation?

In a previous study [7], we showed that the melting process starts at the junction between the acceptor and T stems, at the level of weak AU and GU base pairs which can be easily disrupted and hence allow subsequent invasion of the tRNA₃^Lys structure by the viral RNA. However, our experimental approach, which relied on the NMR observation of imino protons, did not allow observation of PBS binding to the unpaired 3′-end of the tRNA, since the imino protons of the first three base pairs are

Conclusion

In this paper, the use of selective labelling of the primer binding site allowed us to characterize the nucleation points of the annealing process between the PBS and the tRNA₃^Lys. Moreover, we used small hairpins to mimic the D and T arms of tRNA₃^Lys to determine the specific role of NC in the formation of tRNA₃^Lys/PBS duplex recognized by the HIV reverse transcriptase. Studying separate hairpins cannot obviously mimic the entire tRNA, in particular at the level of the TΨC/D loops interaction.

Acknowledgements

P. Barraud is supported by a studentship from Ministère de la Recherche. This work was supported by the French AIDS national Agency (ANRS) and ‘Ensemble contre le SIDA’ (Sidaction).

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To have time to record NMR spectra, we slowed down the reaction of annealing of tRNALys3 to the PBS by using a low amount of NC (only two equivalents compared to the RNA concentration) and by playing with the temperature used to record NMR experiments (15 °C). A model mechanism consistent with kinetic studies (Hargittai et al., 2004) and our NMR data (Barraud et al., 2007; Tisne et al., 2004) is presented in Fig. 6. First, we identified the nucleation point of the annealing and showed that nucleation does not require the presence of the NC, even if it strongly facilitates it.
HIV-1 reverse transcription is initiated from a tRNA^Lys₃ molecule annealed to the viral RNA at the primer binding site (PBS). The annealing of tRNA^Lys₃ requires the opening of its three-dimensional structure and RNA rearrangements to form an efficient initiation complex recognized by the reverse transcriptase. This annealing is mediated by the nucleocapsid protein (NC). In this paper, we first review the actual knowledge about HIV-1 viral RNA and tRNA^Lys₃ structures. Then, we summarize the studies explaining how NC chaperones the formation of the tRNA^Lys₃/PBS binary complex. Additional NMR data that investigated the NC interaction with tRNA^Lys₃ D-loop are presented. Lastly, we focused on the additional interactions occurring between tRNA^Lys₃ and the viral RNA and showed that they are dependent on HIV-1 isolates, i.e. the sequence and the structure of the viral RNA.
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The tRNALys,3 is selectively packaged into virions and used as a primer for reverse transcriptase (RT) to initiate minus-strand strong stop DNA synthesis ((−) ssDNA) (reviewed in Isel et al., 2010). HIV-1 NCp chaperones the annealing of the tRNALys,3 primer onto the PBS by facilitating structural changes in the tRNALys,3 as well as in the viral RNA (Barraud et al., 2007; Brule et al., 2002; Hargittai et al., 2004; Isel et al., 1995; Tisné, 2005; Tisné et al., 2004). Henriet and collaborators showed that Vif can also promote the annealing of tRNALys,3 to the PBS, which generates a functional tRNALys,3/RNA complex, even though Vif was less efficient than the NC proteins used in this study (NCp15, NCp9 and NCp7) (Henriet et al., 2007).
The viral infectivity factor (Vif) is essential for the productive infection and dissemination of HIV-1 in non-permissive cells that involve most natural HIV-1 target cells. Vif counteracts the packaging of two cellular cytidine deaminases named APOBEC3G (A3G) and A3F by diverse mechanisms including the recruitment of an E3 ubiquitin ligase complex and the proteasomal degradation of A3G/A3F, the inhibition of A3G mRNA translation or by a direct competition mechanism. In addition, Vif appears to be an active partner of the late steps of viral replication by participating in virus assembly and Gag processing, thus regulating the final stage of virion formation notably genomic RNA dimerization and by inhibiting the initiation of reverse transcription. Vif is a small pleiotropic protein with multiple domains, and recent studies highlighted the importance of Vif conformation and flexibility in counteracting A3G and in binding RNA. In this review, we will focus on the oligomerization and RNA chaperone properties of Vif and show that the intrinsic disordered nature of some Vif domains could play an important role in virus assembly and replication. Experimental evidence demonstrating the RNA chaperone activity of Vif will be presented.
Comparative nucleic acid chaperone properties of the nucleocapsid protein NCp7 and Tat protein of HIV-1
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RNA chaperones are proteins able to rearrange nucleic acid structures towards their most stable conformations. In retroviruses, the reverse transcription of the viral RNA requires multiple and complex nucleic acid rearrangements that need to be chaperoned. HIV-1 has evolved different viral-encoded proteins with chaperone activity, notably Tat and the well described nucleocapsid protein NCp7. We propose here an overview of the recent reports that examine and compare the nucleic acid chaperone properties of Tat and NCp7 during reverse transcription to illustrate the variety of mechanisms of action of the nucleic acid chaperone proteins.
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The reverse transcriptase binds to the tRNALys3/PBS complex and starts the reverse transcription. We were interested in deciphering the different steps of the formation of the initiation complex made between tRNALys3 and the PBS and in defining the role of the viral partners [88–93]. In parallel, we performed screening of tRNALys3 binders.
Secondary structure of the HIV reverse transcription initiation complex by NMR
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Citation Excerpt :
The first step in viral replication after entry into an infected cell is reverse transcription, which is catalyzed by a viral enzyme, reverse transcriptase (RT).1 RT must initiate from a specific RNA assembly and then navigate the complex secondary structure of genomic RNA while copying RNA to DNA.2 RT is a major target of therapeutic intervention in treatment of acquired immune deficiency syndrome.
Initiation of reverse transcription of genomic RNA is a key early step in replication of the human immunodeficiency virus (HIV) upon infection of a host cell. Viral reverse transcriptase initiates from a specific RNA–RNA complex formed between a host transfer RNA (tRNA^Lys₃) and a region at the 5′ end of genomic RNA; the 3′ end of the tRNA acts as a primer for reverse transcription of genomic RNA. We report here the secondary structure of the HIV genomic RNA–human tRNA^Lys₃ initiation complex using heteronuclear nuclear magnetic resonance methods. We show that both RNAs undergo large-scale conformational changes upon complex formation. Formation of the 18-bp primer helix with the 3′ end of tRNA^Lys₃ drives large conformational rearrangements of the tRNA at the 5′ end while maintaining the anticodon loop for potential loop–loop interactions. HIV RNA forms an intramolecular helix adjacent to the intermolecular primer helix. This helix, which must be broken by reverse transcription, likely acts as a kinetic block to reverse transcription.
Functional recognition of the modified human tRNA<sup>Lys3</sup><inf>UUU</inf> anticodon domain by HIV's nucleocapsid protein and a peptide mim
2011, Journal of Molecular Biology
The HIV-1 nucleocapsid protein, NCp7, facilitates the use of human tRNA^Lys3_UUU as the primer for reverse transcription. NCp7 also remodels the htRNA's amino acid accepting stem and anticodon domains in preparation for their being annealed to the viral genome. To understand the possible influence of the htRNA's unique composition of post-transcriptional modifications on NCp7 recognition of htRNA^Lys3_UUU, the protein's binding and functional remodeling of the human anticodon stem and loop domain (hASL^Lys3) were studied. NCp7 bound the hASL^Lys3_UUU modified with 5-methoxycarbonylmethyl-2-thiouridine at position-34 (mcm⁵s²U₃₄) and 2-methylthio-N⁶-threonylcarbamoyladenosine at position-37 (ms²t⁶A₃₇) with a considerably higher affinity than the unmodified hASL^Lys3_UUU (K_d = 0.28 ± 0.03 and 2.30 ± 0.62 μM, respectively). NCp7 denatured the structure of the hASL^Lys3_UUU-mcm⁵s²U₃₄;ms²t⁶A₃₇;Ψ₃₉ more effectively than that of the unmodified hASL^Lys3_UUU. Two 15 amino acid peptides selected from phage display libraries demonstrated a high affinity (average K_d = 0.55 ± 0.10 μM) and specificity for the ASL^Lys3_UUU-mcm⁵s²U₃₄;ms²t⁶A₃₇ comparable to that of NCp7. The peptides recognized a t⁶A₃₇-modified ASL with an affinity (K_d = 0.60 ± 0.09 μM) comparable to that for hASL^Lys3_UUU-mcm⁵s²U₃₄;ms²t⁶A₃₇, indicating a preference for the t⁶A₃₇ modification. Significantly, one of the peptides was capable of relaxing the hASL^Lys3_UUU-mcm⁵s²U₃₄;ms²t⁶A₃₇;Ψ₃₉ structure in a manner similar to that of NCp7, and therefore could be used to further study protein recognition of RNA modifications. The post-transcriptional modifications of htRNA^Lys3_UUU have been found to be important determinants of NCp7's recognition prior to the tRNA^Lys3_UUU being annealed to the viral genome as the primer of reverse transcription.

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New insights into the formation of HIV-1 reverse transcription initiation complex

Abstract

Introduction

Section snippets

Samples

Where are the nucleation sites for tRNA3Lys/PBS duplex formation?

Conclusion

Acknowledgements

Cell

Biochem. Biophys. Res. Commun.

Biochimie

Prog. Nucleic Acid Res. Mol. Biol.

J. Biol. Chem.

J. Mol. Biol.

J. Mol. Biol.

J. Mol. Biol.

Methods Enzymol.

J. Mol. Biol.

Biochim. Biophys. Acta

The selective packaging and annealing of primer tRNA3Lys in HIV-1

Curr. HIV Res.

Where are the nucleation sites for tRNA₃^Lys/PBS duplex formation?

The selective packaging and annealing of primer tRNA₃^Lys in HIV-1