Targeting the dimerization initiation site of HIV-1 RNA with aminoglycosides: from crystal to cell

Abstract

The kissing-loop complex that initiates dimerization of genomic RNA is crucial for Human Immunodeficiency Virus Type 1 (HIV-1) replication. We showed that owing to its strong similitude with the bacterial ribosomal A site it can be targeted by aminoglycosides. Here, we present its crystal structure in complex with neamine, ribostamycin, neomycin and lividomycin. These structures explain the specificity for 4,5-disubstituted 2-deoxystreptamine (DOS) derivatives and for subtype A and subtype F kissing-loop complexes, and provide a strong basis for rational drug design. As a consequence of the different topologies of the kissing-loop complex and the A site, these aminoglycosides establish more contacts with HIV-1 RNA than with 16S RNA. Together with biochemical experiments, they showed that while rings I, II and III confer binding specificity, rings IV and V are important for affinity. Binding of neomycin, paromomycin and lividomycin strongly stabilized the kissing-loop complex by bridging the two HIV-1 RNA molecules. Furthermore, in situ footprinting showed that the dimerization initiation site (DIS) of HIV-1 genomic RNA could be targeted by these aminoglycosides in infected cells and virions, demonstrating its accessibility.

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

“…were located with SHELX (Sheldrick 2008), and the structure was solved using SHARP (de la Fortelle and Bricogne 1997). For radiation damage studies, 5-iodo-and 5-bromo-U267 DIS loop-loop complex bound to lividomycin aminoglycoside were crystallized as described earlier (Ennifar et al 2006) and frozen in liquid ethane. The 27-nt SRL hairpin was crystallized according to the method described by Correll et al (1999).…”

“…Data were collected at 90 K on a Pilatus 6M detector (Dectris AG) on five U2656-SeCH 3 SRL crystals, two U267-SeCH 3 DIS duplex/ Ribostamycin crystals (Freisz et al 2008), four U267-Bromo DIS kissing-complex/Lividomycin crystals (Ennifar et al 2006), and three U267-Iodo DIS kissing-complex/Lividomycin crystals (Ennifar et al 2007a). Data for seleno and iodo derivatives were collected at 12.6647 keV and at 13.4895 keV for bromo derivatives.…”

SHAPE analysis of the 5′ end of the Mason-Pfizer monkey virus (MPMV) genomic RNA reveals structural elements required for genome dimerization

“…The pal in the DIS loop of one gRNA has been shown to interact with the complementary sequence in the DIS loop on the second gRNA, resulting in the "kissing loop" interaction (Paillart et al 1994(Paillart et al , 1996a(Paillart et al , 1997Clever et al 1996;Haddrick et al 1996;Lodmell et al 2000Lodmell et al , 2001. Due to its central role in gRNA dimerization process that has been shown to be important for gRNA packaging and the continuity of viral life cycle, DIS has been an attractive target for antiretroviral drugs specifically aminoglycosides (Ennifar et al 2003(Ennifar et al , 2006. The formation of "kissing loop" complex is likely to be further stabilized by the long-range interactions (LRIs) between the 5 ′ end (R/U5) and 3 ′ end (gag) sequences of the retroviral packaging signal RNA (for review, see Paillart et al 1996b).…”

LigandRNA: computational predictor of RNA–ligand interactions

“…Many antibiotics act by binding specifically to the most important sites of the ribosome that are largely formed by the RNA: the peptidyl transferase center (PCT) in the large subunit, the decoding center in the small subunit, or the protein exit channel (for review, see Poehlsgaard and Douthwaite 2005). Viral RNA can also be a drug target; e.g., aminoglycosides can also act as inhibitors of the dimerization initiation site of HIV-1 RNA (Ennifar et al 2006), self-splicing group I introns (Park et al 2000), ribozymes of hepatitis delta virus (Chen et al 1997), and hammerhead ribozymes of several plant viroids (Borda and Sigurdsson 2004). Considerable effort has been directed at finding compounds that target HIV-1 TAR RNA (Bannwarth and Gatignol 2005).…”