The human immunodeficiency virus (HIV) requires a programmed −1 ribosomal frameshift for Pol gene expression. The HIV frameshift site consists of a heptanucleotide slippery sequence (UUUUUUA) followed by a spacer region and a downstream RNA stem–loop structure. Here we investigate the role of the RNA structure in promoting the −1 frameshift. The stem–loop was systematically altered to decouple the contributions of local and overall thermodynamic stability towards frameshift efficiency. No correlation between overall stability and frameshift efficiency is observed. In contrast, there is a strong correlation between frameshift efficiency and the local thermodynamic stability of the first 3–4 bp in the stem–loop, which are predicted to reside at the opening of the mRNA entrance channel when the ribosome is paused at the slippery site. Insertion or deletions in the spacer region appear to correspondingly change the identity of the base pairs encountered 8 nt downstream of the slippery site. Finally, the role of the surrounding genomic secondary structure was investigated and found to have a modest impact on frameshift efficiency, consistent with the hypothesis that the genomic secondary structure attenuates frameshifting by affecting the overall rate of translation.

HIV-1 requires a −1 translational frameshift to properly synthesize the viral enzymes required for replication. The frameshift mechanism is dependent upon two RNA elements, a seven-nucleotide slippery sequence (UUUUUUA) and a downstream RNA structure. Frameshifting occurs with a frequency of ~5%, and increasing or decreasing this frequency may result in a decrease in viral replication. Here, we report the results of a high-throughput screen designed to find small molecules that bind to the HIV-1 frameshift site RNA. Out of 34,500 compounds screened, 202 were identified as positive hits. We show that one of these compounds, doxorubicin, binds the HIV-1 RNA with low micromolar affinity (K d = 2.8 μM). This binding was confirmed and localized to the RNA using NMR. Further analysis revealed that this compound increased the RNA stability by approximately 5 °C and decreased translational frameshifting by 28% (±14%), as measured in vitro.Twenty-five years after its initial identification, human immunodeficiency virus type 1 (HIV-1) continues to be a major health concern across much of the world. Although many anti-HIV-1 drugs have been developed, the virus is often able to evade these therapies owing to its high mutation rate. In addition to new classes of drugs being designed against the traditional targets (protease and reverse transcriptase) (1,2), recent research has focused on targeting the −1 programmed translational frameshift that occurs between the gag and pol reading frames (3)(4)(5)(6)(7)(8). This frameshift allows for translation of the pol genes (encoding the protease, reverse transcriptase, and integrase enzymes) in the form of a Gag-Pol fusion polyprotein via the evasion of a stop codon located at the end of the gag gene. Without this frameshift, only the Gag polyprotein (matrix, capsid, nucleocapsid, and p6 structural proteins) will be produced. The structural and enzymatic proteins are found in approximately a 20:1 molar ratio as a result of a frameshift efficiency of approximately 5% (9-15). This stoichiometry is required for appropriate packaging of virus particles, and an increase or decrease in the frameshift efficiency has been found to significantly decrease the production of infectious virions (4,15,16).The frameshift event is programmed by two cis-acting RNA elements between the gag and pol genes: a seven-nucleotide slippery sequence (UUUUUUA) and a highly conserved stemloop structure immediately downstream (9,17). This stem-loop structure has been shown to induce ribosomal pausing, which is required for frameshifting, and its stability has been correlated with the efficiency of ribosomal frameshifting (18,19). At the base of the stem-loop, the frameshift site RNA contains a conserved GGA bulge. This sequence has been hypothesized to interact with the translational machinery during frameshifting (20), by virtue of its predicted positioning near the mRNA entrance channel, which is located 13-15 nucleotides of the P site codon (21). Due to the distance the mRNA must traverse through the r...

The HIV-1 ribosomal frameshift element is highly structured, regulates translation of all virally encoded enzymes, and is a promising therapeutic target. The prior model for this motif contains two helices separated by a three-nucleotide bulge. Modifications to this model were suggested by SHAPE chemical probing of an entire HIV-1 RNA genome. Novel features of the SHAPE-directed model include alternate helical conformations and a larger, more complex structure. These structural elements also support the presence of a secondary frameshift site within the frameshift domain. Here, we use oligonucleotide-directed structure perturbation, probing in the presence of formamide, and in-virion experiments to examine these models. Our data support a model in which the frameshift domain is anchored by a stable helix outside the conventional domain. Less stable helices within the domain can switch from the SHAPE-predicted to the two-helix conformation. Translational frameshifting assays with frameshift domain mutants support a functional role for the interactions predicted by and specific to the SHAPE-directed model. These results reveal that the HIV-1 frameshift domain is a complex, dynamic structure and underscore the importance of analyzing folding in the context of full-length RNAs.

The dicistrovirus intergenic region internal ribosome entry site (IRES) adopts a triple-pseudoknotted RNA structure and occupies the core ribosomal E, P, and A sites to directly recruit the ribosome and initiate translation at a non-AUG codon. A subset of dicistrovirus IRESs directs translation in the 0 and +1 frames to produce the viral structural proteins and a +1 overlapping open reading frame called ORFx, respectively. Here we show that specific mutations of two unpaired adenosines located at the core of the threehelical junction of the honey bee dicistrovirus Israeli acute paralysis virus (IAPV) IRES PKI domain can uncouple 0 and +1 frame translation, suggesting that the structure adopts distinct conformations that contribute to 0 or +1 frame translation. Using a reconstituted translation system, we show that ribosomes assembled on mutant IRESs that direct exclusive 0 or +1 frame translation lack reading frame fidelity. Finally, a nuclear magnetic resonance/small-angle X-ray scattering hybrid approach reveals that the PKI domain of the IAPV IRES adopts an RNA structure that resembles a complete tRNA. The tRNA shape-mimicry enables the viral IRES to gain access to the ribosome tRNA-binding sites and form intermolecular contacts with the ribosome that are necessary for initiating IRES translation in a specific reading frame.translation | virus | RNA | ribosome | internal ribosome entry site F idelity of protein synthesis and the transmission of genetic information from mRNA into a nascent protein rely on the accurate selection and maintenance of the translational reading frame. In canonical eukaryotic translation, after recruitment and scanning of ribosomes on an mRNA, the translational reading frame is initially established by methionyl-tRNA i anticodon: codon pairing in the ribosomal P site. Although the mechanisms that specify reading frame selection and maintenance during translation are not completely understood, programmed recoding mechanisms that have been identified in some viral and cellular mRNAs have yielded significant insights into the cis-acting signals that increase coding capacity or allow translation using alternate reading frames (1, 2).We recently demonstrated that a subset of viruses within the Dicistroviridae family harbors an intergenic region internal ribosome entry site (IGR IRES) that can direct translation in alternative reading frames (3), providing an excellent model for studying RNA-ribosome interactions that influence reading frame selection. An IRES is generally a structured RNA element that can recruit the ribosome in a 5′ end-independent manner and without the full complement of canonical translation initiation factors (4, 5). Among the diverse types of IRES elements found in both viral and messenger RNAs, the IGR IRES uses the most streamlined mechanism, dispensing the need for all canonical initiation factors to directly recruit the ribosome and initiate translation at a non-AUG codon (6-8). The IGR IRES adopts an RNA structure comprising two independently folded domains; ps...

The HIV-1 frameshift site (FS) plays a critical role in viral replication. During translation, the HIV-1 FS transitions from a 3-helix to a 2-helix junction RNA secondary structure. The 2-helix junction structure contains a GGA bulge, and purine-rich bulges are common motifs in RNA secondary structure. Here, we investigate the dynamics of the HIV-1 FS 2-helix junction RNA. Interhelical motions were studied under different ionic conditions using NMR order tensor analysis of residual dipolar couplings. In 150 mM potassium, the RNA adopts a 43°(±4°) interhelical bend angle (β) and displays large amplitude, anisotropic interhelical motions characterized by a 0.52(±0.04) internal generalized degree of order (GDOint) and distinct order tensor asymmetries for its two helices (η = 0.26(±0.04) and 0.5(±0.1)). These motions are effectively quenched by addition of 2 mM magnesium (GDOint = 0.87(±0.06)), which promotes a near-coaxial conformation (β = 15°(±6°)) of the two helices. Base stacking in the bulge was investigated using the fluorescent purine analog 2-aminopurine. These results indicate that magnesium stabilizes extrahelical conformations of the bulge nucleotides, thereby promoting coaxial stacking of helices. These results are highly similar to previous studies of the HIV transactivation response RNA, despite a complete lack of sequence similarity between the two RNAs. Thus, the conformational space of these RNAs is largely determined by the topology of their interhelical junctions.