HIV-1 RNA Dimerization Initiation Site Is Structurally Similar to the Ribosomal A Site and Binds Aminoglycoside Antibiotics

Abstract

Human immunodeficiency virus (HIV) genomic RNA is packaged into virions as a dimer. The first step of dimerization is the formation of a kissing-loop complex at the so-called dimerization initiation site (DIS). We found an unexpected and fortuitous resemblance between the HIV-1 DIS kissing-loop complex and the eubacterial 16 S ribosomal aminoacyl-tRNA site (A site), which is the target of aminoglycoside antibiotics. Similarities exist not only at the primary and secondary structure level but also at the tertiary structure level, as revealed by comparison of the respective DIS and A site crystal structures. Gel shift, inhibition of lead-induced cleavage, and footprinting experiments showed that paromomycin and neomycin specifically bind to the kissing-loop complex formed by the DIS, with an affinity and a geometry similar to that observed for the A site. Modeling of the aminoglycoside-DIS complex allowed us to identify antibiotic modifications likely to increase the affinity and/or the specificity for the DIS. This could be a starting point for designing antiviral drugs against HIV-1 RNA dimerization.The fight against HIV 1 relies essentially on multitherapies targeting two viral enzymes: reverse transcriptase and protease. Due to a highly variable genome leading to rapid selection of mutations that confer resistance to enzymatic inhibitors, there is a strong need for targeting new viral molecules. New potential targets such as viral and cellular proteins involved in viral adsorption, fusion, and integration have been proposed (1). Functional sites within the genomic RNA, such as the Tat-responsive element (2) and the Rev-responsive element (3), have also been described as potential targets for the aminoglycoside antibiotic neomycin B.Another interesting viral RNA target is the dimerization initiation site (DIS) of HIV-1 genomic RNA. This stem-loop is highly conserved among the different HIV-1 subtypes identified by sequence alignment. It initiates dimerization by forming a kissing-loop complex through base-pairing of a 6-nucleotide self-complementary sequence in each loop (4 -8) (Fig. 1). It has been shown that this kissing-loop complex is converted in vitro by the nucleocapsid protein into a more stable complex, assumed to correspond to an extended duplex (9). Stabilization of the RNA dimer was also observed during maturation of the viral particles (10). Genome dimerization facilitates recombination (11) and is required for efficient RNA packaging (12-15) and reverse transcription (13, 16). Alteration of the DIS dramatically reduces viral infectivity (12-15).We recently solved the crystal structures of the DIS kissingloop complexes of HIV-1 subtypes A and B (17). In the present study, we show that, unexpectedly, these structures are very similar to the ribosomal aminoacyl decoding site (A site) complexed with paromomycin (18, 19), an aminoglycoside antibiotic interfering with translation in bacteria. Based on this similarity, we modeled a DIS kissing-loop complex binding two paromomycin molecules. This model is...

A fast selenium derivatization strategy for crystallization and phasing of RNA structures

“…This RNA/drug complex involves a 23-nt RNA homodimer, which promotes viral genome dimerization and is recognized by aminoglycosides (Ennifar et al 2003(Ennifar et al , 2007bBernacchi et al 2007;Freisz et al 2008). In parallel to an extensive but unsuccessful search for heavy atom derivatives, we attempted to determine the structure using 5-halogen-pyrimidine derivatives.…”

Conformational transitions of flanking purines in HIV‐1 RNA dimerization initiation site kissing complexes studied by CHARMM explicit solvent molecular dynamics

“…The relevance of bulged-out conformations in solution is supported by the binding of aminoglycoside antibiotics to the DIS kissing complex with bulged-out 5 0 flanking bases. 15,71,72 This binding was first predicted based on the structural similarity between DIS kissing complex and bacterial 16S ribosomal A site, and then it was confirmed by the experiments. It is however possible that, in analogy with ribosomal A site, the binding of the antibiotics can shift the bulged-in/bulged-out equilibrium of flanking bases toward the bulged-out form.…”

Structure of the cytosine–cytosine mismatch in the thymidylate synthase mRNA binding site and analysis of its interaction with the aminoglycoside paromomycin

“…This widening confers the ability of the CC mismatch-containing helix to bind paromomycin, while the fully Watson-Crick counterpart with a narrower major groove shows no binding. Other RNA molecules that bind aminoglycosides and aminoglycoside analogs also bind at a site that is distorted from standard A-form RNA geometry (Fourmy et al 1996;Carter et al 2000;Vicens and Westhof 2001;Ennifar et al 2003;Raghunathan et al 2006) or have been found to widen its major groove upon binding (Staple et al 2008).…”