Cation-dependent cleavage of the duplex form of the subtype-B HIV-1 RNA dimerization initiation site

The crystal structure of subtype-B HIV-1 genomic RNA Dimerization Initiation Site duplex revealed chain cleavage at a specific position resulting in 3′-phosphate and 5′-hydroxyl termini. A crystallographic analysis showed that Ba2+, Mn2+, Co2+ and Zn2+ bind specifically on a guanine base close to the cleaved position. The crystal structures also point to a necessary conformational change to induce an ‘in-line’ geometry at the cleavage site. In solution, divalent cations increased the rate of cleavage with pH/pKa compensation, indicating that a cation-bound hydroxide anion is responsible for the cleavage. We propose a ‘Trojan horse’ mechanism, possibly of general interest, wherein a doubly charged cation hosted near the cleavage site as a ‘harmless’ species is further transformed in situ into an ‘aggressive’ species carrying a hydroxide anion

HIV-1 DIS stem loop forms an obligatory bent kissing intermediate in the dimerization pathway.

The HIV-1 dimerization initiation sequence (DIS) is a conserved palindrome in the apical loop of a conserved hairpin motif in the 5′-untranslated region of its RNA genome. DIS hairpin plays an important role in genome dimerization by forming a ‘kissing complex’ between two complementary hairpins. Understanding the kinetics of this interaction is key to exploiting DIS as a possible human immunodeficiency virus (HIV) drug target. Here, we present a single-molecule Förster resonance energy transfer (smFRET) study of the dimerization reaction kinetics. Our data show the real-time formation and dissociation dynamics of individual kissing complexes, as well as the formation of the mature extended duplex complex that is ultimately required for virion packaging. Interestingly, the single-molecule trajectories reveal the presence of a previously unobserved bent intermediate required for extended duplex formation. The universally conserved A272 is essential for the formation of this intermediate, which is stabilized by Mg(2+), but not by K(+) cations. We propose a 3D model of a possible bent intermediate and a minimal dimerization pathway consisting of three steps with two obligatory intermediates (kissing complex and bent intermediate) and driven by Mg(2+) ions

2′-Methylseleno-modified oligoribonucleotides for X-ray crystallography synthesized by the ACE RNA solid-phase approach

Site-specifically modified 2′-methylseleno RNA represents a valuable derivative for phasing of X-ray crystallographic data. Several successful applications in three-dimensional structure determination of nucleic acids, such as the Diels–Alder ribozyme, have relied on this modification. Here, we introduce synthetic routes to 2′-methylseleno phosphoramidite building blocks of all four standard nucleosides, adenosine, cytidine, guanosine and uridine, that are tailored for 2′-O-bis(acetoxyethoxy)methyl (ACE) RNA solid-phase synthesis. We additionally report on their incorporation into oligoribonucleotides including deprotection and purification. The methodological expansion of 2′-methylseleno labeling via ACE RNA chemistry is a major step to make Se-RNA generally accessible and to receive broad dissemination of the Se-approach for crystallographic studies on RNA. Thus far, preparation of 2′-methylseleno-modified oligoribonucleotides has been restricted to the 2′-O-[(triisopropylsilyl)oxy]methyl (TOM) and 2′-O-tert-butyldimethylsilyl (TBDMS) RNA synthesis methods

Conformations of Flanking Bases in HIV-1 RNA DIS Kissing Complexes Studied by Molecular Dynamics

Explicit solvent molecular dynamics simulations (in total almost 800 ns including locally enhanced sampling runs) were applied with different ion conditions and with two force fields (AMBER and CHARMM) to characterize typical geometries adopted by the flanking bases in the RNA kissing-loop complexes. We focus on flanking base positions in multiple x-ray and NMR structures of HIV-1 DIS kissing complexes and kissing complex from the large ribosomal subunit of Haloarcula marismortui. An initial x-ray open conformation of bulged-out bases in HIV-1 DIS complexes, affected by crystal packing, tends to convert to a closed conformation formed by consecutive stretch of four stacked purine bases. This is in agreement with those recent crystals where the packing is essentially avoided. We also observed variants of the closed conformation with three stacked bases, while nonnegligible populations of stacked geometries with bulged-in bases were detected, too. The simulation results reconcile differences in positions of the flanking bases observed in x-ray and NMR studies. Our results suggest that bulged-out geometries are somewhat more preferred, which is in accord with recent experiments showing that they may mediate tertiary contacts in biomolecular assemblies or allow binding of aminoglycoside antibiotics

HuR interacts with human immunodeficiency virus type 1 reverse transcriptase, and modulates reverse transcription in infected cells

Reverse transcription of the genetic material of human immunodeficiency virus type 1 (HIV-1) is a critical step in the replication cycle of this virus. This process, catalyzed by reverse transcriptase (RT), is well characterized at the biochemical level. However, in infected cells, reverse transcription occurs in a multiprotein complex – the reverse transcription complex (RTC) – consisting of viral genomic RNA associated with viral proteins (including RT) and, presumably, as yet uncharacterized cellular proteins. Very little is known about the cellular proteins interacting with the RTC, and with reverse transcriptase in particular. We report here that HIV-1 reverse transcription is affected by the levels of a nucleocytoplasmic shuttling protein – the RNA-binding protein HuR. A direct protein-protein interaction between RT and HuR was observed in a yeast two-hybrid screen and confirmed in vitro by homogenous time-resolved fluorescence (HTRF). We mapped the domain interacting with HuR to the RNAse H domain of RT, and the binding domain for RT to the C-terminus of HuR, partially overlapping the third RRM RNA-binding domain of HuR. HuR silencing with specific siRNAs greatly impaired early and late steps of reverse transcription, significantly inhibiting HIV-1 infection. Moreover, by mutagenesis and immunoprecipitation studies, we could not detect the binding of HuR to the viral RNA. These results suggest that HuR may be involved in and may modulate the reverse transcription reaction of HIV-1, by an as yet unknown mechanism involving a protein-protein interaction with HIV-1 RT

A proposal for a new HIV-1 DLS structural model

The dimer initiation site/dimer linkage sequence (DIS/DLS) region of the human immunodeficiency virus type 1 (HIV-1) RNA genome is suggested to play essential roles at various stages of the viral life cycle. Through a novel assay we had recently developed, we reported on the necessary and sufficient region for RNA dimerization in the HIV-1 virion. Using this system, we performed further detailed mapping of the functional base pairs necessary for HIV-1 DLS structure. Interestingly, the study revealed a previously unnoticed stem formation between two distantly positioned regions. Based on this and other findings on functional base pairing in vivo, we propose new 3D models of the HIV-1 DLS which contain a unique pseudoknot-like conformation. Since this pseudoknot-like conformation appears to be thermodynamically stable, forms a foundational skeleton for the DLS and sterically restricts the spontaneous diversification of DLS conformations, its unique shape may contribute to the viral life cycle and potentially serve as a novel target for anti-HIV-1 therapies

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

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

HIV-1 RT Inhibitors with a Novel Mechanism of Action: NNRTIs that Compete with the Nucleotide Substrate

HIV-1 reverse transcriptase (RT) inhibitors currently used in antiretroviral therapy can be divided into two classes: (i) nucleoside analog RT inhibitors (NRTIs), which compete with natural nucleoside substrates and act as terminators of proviral DNA synthesis, and (ii) non-nucleoside RT inhibitors (NNRTIs), which bind to a hydrophobic pocket close to the RT active site. In spite of the efficiency of NRTIs and NNRTIs, the rapid emergence of multidrug-resistant mutations requires the development of new RT inhibitors with an alternative mechanism of action. Recently, several studies reported the discovery of novel non-nucleoside inhibitors with a distinct mechanism of action. Unlike classical NNRTIs, they compete with the nucleotide substrate, thus forming a new class of RT inhibitors: nucleotide-competing RT inhibitors (NcRTIs). In this review, we discuss current progress in the understanding of the peculiar behavior of these compounds

Structural snapshots of Xer recombination reveal activation by synaptic complex remodeling and DNA bending

Bacterial Xer site-specific recombinases play an essential genome maintenance role by unlinking chromosome multimers, but their mechanism of action has remained structurally uncharacterized. Here, we present two high-resolution structures of Helicobacter pylori XerH with its recombination site DNA difH, representing pre-cleavage and post-cleavage synaptic intermediates in the recombination pathway. The structures reveal that activation of DNA strand cleavage and rejoining involves large conformational changes and DNA bending, suggesting how interaction with the cell division protein FtsK may license recombination at the septum. Together with biochemical and in vivo analysis, our structures also reveal how a small sequence asymmetry in difH defines protein conformation in the synaptic complex and orchestrates the order of DNA strand exchanges. Our results provide insights into the catalytic mechanism of Xer recombination and a model for regulation of recombination activity during cell division