Initiation of HIV Reverse Transcription.

Reverse transcription of retroviral genomes into double stranded DNA is a key event for viral replication. The very first stage of HIV reverse transcription, the initiation step, involves viral and cellular partners that are selectively packaged into the viral particle, leading to an RNA/protein complex with very specific structural and functional features, some of which being, in the case of HIV-1, linked to particular isolates. Recent understanding of the tight spatio-temporal regulation of reverse transcription and its importance for viral infectivity further points toward reverse transcription and potentially its initiation step as an important drug target.

Structured mRNAs regulate translation initiation by binding to the platform of the ribosome.

Gene expression can be regulated at the level of initiation of protein biosynthesis via structural elements present at the 5' untranslated region of mRNAs. These folded mRNA segments may bind to the ribosome, thus blocking translation until the mRNA unfolds. Here, we report a series of cryo-electron microscopy snapshots of ribosomal complexes directly visualizing either the mRNA structure blocked by repressor protein S15 or the unfolded, active mRNA. In the stalled state, the folded mRNA prevents the start codon from reaching the peptidyl-tRNA (P) site inside the ribosome. Upon repressor release, the mRNA unfolds and moves into the mRNA channel allowing translation initiation. A comparative structure and sequence analysis suggests the existence of a universal stand-by site on the ribosome (the 30S platform) dedicated for binding regulatory 5' mRNA elements. Different types of mRNA structures may be accommodated during translation preinitiation and regulate gene expression by transiently stalling the ribosome.

Mg2+ dependency of HIV-1 reverse transcription, inhibition by nucleoside analogues and resistance.

Metal ions are essential for DNA polymerase and RNase H activities of HIV-1 reverse transcriptase (RT). RT studies are routinely performed at 6-8 mM Mg2+, despite the fact that the in vivo concentration might be as low as 0.2 mM. We studied the influence of MgCl2 and ATP, which likely binds a significant fraction of the magnesium pool in vivo, on the DNA polymerase and RNase H activities of HIV-1 RT, its inhibition by nucleoside RT inhibitors (NRTIs) and primer unblocking by AZT-resistant RT. At low Mg2+ concentration, reverse transcription of a natural template strongly increased despite a dramatically reduced intrinsic polymerase activity under such conditions. Low Mg2+ concentrations affected the RNA stability and indirectly decreased its degradation by the RNase H activity. The reduced RNA degradation prevented premature dissociation of the template and primer strands that otherwise generated dead-end DNA products. In addition, low Mg2+ dramatically decreased the incorporation of NRTIs into DNA and increased nucleotide excision by AZT-resistant RT. The latter effect is also most likely owing to the diminished cleavage of the RNA template. Thus, differences in the free Mg2+ concentration between different cell types or during the cell cycle might strongly affect HIV-1 replication and its inhibition.

Cooperative and specific binding of Vif to the 5' region of HIV-1 genomic RNA.

The viral infectivity factor (Vif) protein of human immunodeficiency virus type 1 (HIV-1) is essential for viral replication in vivo. Packaging of Vif into viral particles is mediated by an interaction with viral genomic RNA and association with viral nucleoprotein complexes. Despite recent findings on the RNA-binding properties of Vif suggesting that Vif could be involved in retroviral assembly, no RNA sequence or structure specificity has been determined so far. To gain further insight into the mechanisms by which Vif might regulate viral replication, we studied the interactions of Vif with HIV-1 genomic RNA in vitro. Using extensive biochemical analysis, we have measured the affinity of recombinant Vif proteins for synthetic RNAs corresponding to various regions of the HIV-1 genome. We found that recombinant Vif proteins bind specifically to HIV-1 viral RNA fragments corresponding to the 5'-untranslated region (5'-UTR), gag and the 5' part of pol (K(d) between 45 nM and 65 nM). RNA encompassing nucleotides 1-497 or 499-996 of the HIV-1 genomic RNA bind 9+/-2 and 21+/-3 Vif molecules, respectively, and at least some of these proteins bind in a cooperative manner (Hill constant alpha(H) = 2.3). In contrast, RNAs corresponding to other parts of the HIV-1 genome or heterologous RNAs showed poor binding capacity and weak cooperativity (K(d) > 200 nM). Moreover, RNase T1 footprinting revealed a hierarchical binding of Vif, pointing to TAR and the poly(A) stem-loop structures as primary strong affinity targets, and downstream structures as secondary sites with moderate affinity. Taken together, our findings suggest that Vif may assist other proteins to maintain a correct folding of the genomic RNA in order to facilitate its packaging and further steps such as reverse transcription. Interestingly, our results suggest also that Vif could bind the viral RNA in order to protect it from the action of the antiviral factor APOBEC-3G/3F.

Translational operator of mRNA on the ribosome: how repressor proteins exclude ribosome binding.

The ribosome of Thermus thermophilus was cocrystallized with initiator transfer RNA (tRNA) and a structured messenger RNA (mRNA) carrying a translational operator. The path of the mRNA was defined at 5.5 angstroms resolution by comparing it with either the crystal structure of the same ribosomal complex lacking mRNA or with an unstructured mRNA. A precise ribosomal environment positions the operator stem-loop structure perpendicular to the surface of the ribosome on the platform of the 30S subunit. The binding of the operator and of the initiator tRNA occurs on the ribosome with an unoccupied tRNA exit site, which is expected for an initiation complex. The positioning of the regulatory domain of the operator relative to the ribosome elucidates the molecular mechanism by which the bound repressor switches off translation. Our data suggest a general way in which mRNA control elements must be placed on the ribosome to perform their regulatory task.

Staphylococcus aureus RNAIII and the endoribonuclease III coordinately regulate spa gene expression.

Staphylococcus aureus RNAIII is one of the largest regulatory RNAs, which controls several virulence genes encoding exoproteins and cell-wall-associated proteins. One of the RNAIII effects is the repression of spa gene (coding for the surface protein A) expression. Here, we show that spa repression occurs not only at the transcriptional level but also by RNAIII-mediated inhibition of translation and degradation of the stable spa mRNA by the double-strand-specific endoribonuclease III (RNase III). The 3' end domain of RNAIII, partially complementary to the 5' part of spa mRNA, efficiently anneals to spa mRNA through an initial loop-loop interaction. Although this annealing is sufficient to inhibit in vitro the formation of the translation initiation complex, the coordinated action of RNase III is essential in vivo to degrade the mRNA and irreversibly arrest translation. Our results further suggest that RNase III is recruited for targeting the paired RNAs. These findings add further complexity to the expression of the S. aureus virulon.

First snapshots of the HIV-1 RNA structure in infected cells and in virions.

With the increasing interest of RNAs in regulating a range of cell biological processes, very little is known about the structure of RNAs in tissue culture cells. We focused on the 5'-untranslated region of the human immunodeficiency virus type 1 RNA genome, a highly conserved RNA region, which contains structural domains that regulate key steps in the viral replication cycle. Up until now, structural information only came from in vitro studies. Here, we developed chemical modification assays to test nucleotide accessibility directly in infected cells and viral particles, thus circumventing possible biases and artifacts linked to in vitro assays. The secondary structure of the 5'-untranslated region in infected cells points to the existence of the various stem-loop motifs associated to distinct functions, proposed from in vitro probing, mutagenesis, and phylogeny. However, compared with in vitro data, subtle differences were observed in the dimerization initiation site hairpin, and none of the proposed long range interactions were observed between the functional domains. Moreover, no global RNA rearrangement was observed; structural differences between infected cells and viral particles were limited to the primer binding site, which became protected against chemical modification upon tRNA(3) (Lys) annealing in virions and to the main packaging signal. In addition, our data suggested that the genomic RNA could already dimerize in the cytoplasm of infected cells. Taken together, our results provided the first analysis of the dynamic of RNA structure of the human immunodeficiency virus type 1 RNA genome during virus assembly ex vivo.

Structural variability of the initiation complex of HIV-1 reverse transcription.

HIV-1 reverse transcription is initiated from a tRNA(3)(Lys) molecule annealed to the viral RNA at the primer binding site (PBS), but the structure of the initiation complex of reverse transcription remains controversial. Here, we performed in situ structural probing, as well as in vitro structural and functional studies, of the initiation complexes formed by highly divergent isolates (MAL and NL4.3/HXB2). Our results show that the structure of the initiation complex is not conserved. In MAL, and according to sequence analysis in 14% of HIV-1 isolates, formation of the initiation complex is accompanied by complex rearrangements of the viral RNA, and extensive interactions with tRNA(3)(Lys) are required for efficient initiation of reverse transcription. In NL4.3, HXB2, and most isolates, tRNA(3)(Lys) annealing minimally affects the viral RNA structure and no interaction outside the PBS is required for optimal initiation of reverse transcription. We suggest that in MAL, extensive interactions with tRNA(3)(Lys) are required to drive the structural rearrangements generating the structural elements ultimately recognized by reverse transcriptase. In NL4.3 and HXB2, these elements are already present in the viral RNA prior to tRNA(3)(Lys) annealing, thus explaining that extensive interactions with the primer are not required. Interestingly, such interactions are required in HXB2 mutants designed to use a non-cognate tRNA as primer (tRNA(His)). In the latter case, the extended interactions are required to counteract a negative contribution associate with the alternate primer.

Specific recognition of rpsO mRNA and 16S rRNA by Escherichia coli ribosomal protein S15 relies on both mimicry and site differentiation.

The ribosomal protein S15 binds to 16S rRNA, during ribosome assembly, and to its own mRNA (rpsO mRNA), affecting autocontrol of its expression. In both cases, the RNA binding site is bipartite with a common subsite consisting of a G*U/G-C motif. The second subsite is located in a three-way junction in 16S rRNA and in the distal part of a stem forming a pseudoknot in Escherichia coli rpsO mRNA. To determine the extent of mimicry between these two RNA targets, we determined which amino acids interact with rpsO mRNA. A plasmid carrying rpsO (the S15 gene) was mutagenized and introduced into a strain lacking S15 and harbouring an rpsO-lacZ translational fusion. Analysis of deregulated mutants shows that each subsite of rpsO mRNA is recognized by a set of amino acids known to interact with 16S rRNA. In addition to the G*U/G-C motif, which is recognized by the same amino acids in both targets, the other subsite interacts with amino acids also involved in contacts with helix H22 of 16S rRNA, in the region adjacent to the three-way junction. However, specific S15-rpsO mRNA interactions can also be found, probably with A(-46) in loop L1 of the pseudoknot, demonstrating that mimicry between the two targets is limited.

Secondary structure of the 3' UTR of bicoid mRNA.

Formation of the Bicoid morphogen gradient in early Drosophila embryos requires the pre-localization of bicoid mRNA to the anterior pole of the egg. The program of bcd mRNA localization involves multiples steps and proceeds from oogenesis until early embryogenesis. This process requires cis-elements in the 3' UTR of bcd mRNA and successive and/or concomitant critical protein interactions. Furthermore, numerous RNA elements and binding proteins contribute to regulate bcd expression. In the present paper, we investigated the secondary structure of the full length 3' UTR of the bcd mRNA, using a variety of chemical and enzymatic structural probes. This RNA probing analysis allowed us to give a detailed description of the 3' UTR of the bcd mRNA and its organization into five well-defined and independent domains (I-V). One prominent result that emerges from our data is the unexpected high degree of flexibility of the different domains relative to each others. This plasticity relies upon the open conformation of the central hinge region interconnecting domains II, III, and IV + V. Otherwise, dimerization of the 3' UTR, which participates to anchoring bcd mRNA at the anterior pole of the embryo, only results in discrete and local change in domain III. Domain I that contains sites for trans-acting factors exhibiting single stranded RNA binding specificity is mainly unstructured. By contrast, each core domains (II-V) is highly organized and folds into helices interrupted by bulges and interior loops and closed by very exposed apical loops. These elements mostly built specific determinants for trans-acting factors. Besides, these findings provide a valuable database for structure/function studies.

Molecular mimicry in translational regulation: the case of ribosomal protein S15.

Ribosomal protein S15 is highly conserved among prokaryotes. It plays a pivotal role in the assembly of the central domain of the small ribosomal subunit and regulates its own expression by a feedback mechanism at the translational level. The protein recognizes two RNA targets (rRNA and mRNA) that share only partial similarity. Its interaction with 16S rRNA has been fully characterized, while mRNA interactions and regulatory mechanisms have been extensively studied in E. coli and in T. thermophilus. Recently, we have characterized which aminoacids are involved in E. coli mRNA recognition, using an in vivo assay allowing to identify S15 mutations affecting the S15-mRNA interactions without altering 30S subunit assembly. Here, we address the following questions: Are common determinants used by S15 to recognize its rRNA and mRNA targets? What is the extent of molecular mimicry? Is the regulatory mechanism conserved? Our results indicate that specific recognition of mRNA and rRNA relies on both mimicry and site differentiation. They also highlight the high plasticity of RNA to adapt to evolutionary constraints.

Mechanism of dimerization of bicoid mRNA: initiation and stabilization.

Dimerization of bcd mRNA was shown to be important for the formation of ribonucleoprotein particles and their localization in Drosophila embryo. The cis-element responsible for dimerization is localized in a stem-loop domain (domain III) containing two essential complementary 6-nucleotide sequences in a hairpin loop (LIIIb) and an interior loop (LIIIa). Such an RNA element can potentially generate single or double "hand-by-arm" interactions leading to open and closed complexes, respectively. The former retains the possibility of forming multimers, whereas the latter does not. We showed previously that dimerization proceeds through a two-step mechanism, which includes a transition from the reversible initiation complex into a very stable one. Here we have addressed the nature of the initial interactions and the mechanism of transition. We engineered a series of different RNA fragments with the capacity to form defined open dimers, multimers, or closed dimers. We compared their thermodynamic and kinetic behavior and mapped nucleotides involved in intermolecular interactions by enzymatic and chemical footprinting experiments and chemical modification interference. Our results indicate that the initiation step leads to a reversible open dimer, involving a more limited number of intermolecular base pairs than expected. The two loops play distinct roles in this process, and the structure of loop IIIb is more constrained than that of loop IIIa. Thus, loop IIIa appears to be the driving element of the recognition process. The initial open dimer is then converted into a stable closed dimer, possibly through a kinetically controlled mechanism.

Structure-function relationships of the initiation complex of HIV-1 reverse transcription: the case of mutant viruses using tRNA(His) as primer.

Reverse transcription of HIV-1 RNA is initiated from the 3' end of a tRNA3Lys molecule annealed to the primer binding site (PBS). An additional interaction between the anticodon loop of tRNA3Lys and a viral A-rich loop is required for efficient initiation of reverse transcription of the HIV-1 MAL isolate. In the HIV-1 HXB2 isolate, simultaneous mutations of the PBS and the A-rich loop (mutant His-AC), but not of the PBS alone (mutant His) allows the virus to stably utilize tRNA(His) as primer. However, mutant His-AC selects additional mutations during cell culture, generating successively His-AC-GAC and His-AC-AT-GAC. Here, we wanted to establish direct relationships between the evolution of these mutants in cell culture, their efficiency in initiating reverse transcription and the structure of the primer/template complexes in vitro. The initiation of reverse transcription of His and His-AC RNAs was dramatically reduced. However, His-AC-GAC RNA, which incorporated three adaptative point mutations, was reverse transcribed more efficiently than the wild type RNA. Incorporation of two additional mutations decreased the efficiency of the initiation of reverse transcription, which remained at the wild type level. Structural probing showed that even though both His-AC and His-AC-GAC RNAs can potentially interact with the anticodon loop of tRNA(His), only the latter template formed a stable interaction. Thus, our results showed that the selection of adaptative mutations by HIV-1 mutants utilizing tRNA(His) as primer was initially dictated by the efficiency of the initiation of reverse transcription, which relied on the existence of a stable interaction between the mutated A-rich loop and the anticodon loop of tRNA(His).

The RNA binding protein FMRP: new connections and missing links.

The loss of the fragile X mental retardation protein (FMRP) is responsible for the most common cause of inherited mental retardation called the fragile X syndrome. FMRP is suspected to participate in the synaptic plasticity of neurons by acting on posttranscriptional control of gene expression. FMRP is an RNA binding protein that associates with mRNAs together with other proteins to form large ribonucleoprotein complexes. These complexes are proposed to participate in the transport, localization and translation of target mRNAs. Progress has been made recently in the identification of the mRNAs and the proteins present in these complexes and a possible connection with the micro-RNA dependent regulatory pathway has been established.

Effects of tRNA 3 Lys aminoacylation on the initiation of HIV-1 reverse transcription.

HIV-1 utilizes cellular tRNA(3)(Lys) to prime the initiation of reverse transcription. The selective incorporation of cytoplasmic tRNA(3)(Lys) into HIV-1 particles was recently shown to involve the lysyl-tRNA synthetase, and hence, the encapsidated tRNA(3)(Lys) is likely to be aminoacylated. Here, we tested the effect of aminoacylation on the initiation of reverse transcription. We show that HIV-1 reverse transcriptase is unable to extend lysyl-tRNA(3)(Lys). In addition, the viral polymerase does not significantly enhance the rate of tRNA deacylation, in contrast with previous studies on avian retroviruses. Thus, aminoacylation of the primer tRNA might prevent the initiation of HIV-1 reverse transcription from taking place before viral budding and maturation.

Ribosomal protein S15 represses its own translation via adaptation of an rRNA-like fold within its mRNA.

The 16S rRNA-binding ribosomal protein S15 is a key component in the assembly of the small ribosomal subunit in bacteria. We have shown that S15 from the extreme thermophile Thermus thermophilus represses the translation of its own mRNA in vitro, by interacting with the leader segment of its mRNA. The S15 mRNA-binding site was characterized by footprinting experiments, deletion analysis and site-directed mutagenesis. S15 binding triggers a conformational rearrangement of its mRNA into a fold that mimics the conserved three-way junction of the S15 rRNA-binding site. This conformational change masks the ribosome entry site, as demonstrated by direct competition between the ribosomal subunit and S15 for mRNA binding. A comparison of the T.thermophilus and Escherichia coli regulation systems reveals that the two regulatory mRNA targets do not share any similarity and that the mechanisms of translational inhibition are different. Our results highlight an astonishing plasticity of mRNA in its ability to adapt to evolutionary constraints, that contrasts with the extreme conservation of the rRNA-binding site.

Does the HIV-1 primer activation signal interact with tRNA3(Lys) during the initiation of reverse transcription?

Reverse transcription of HIV-1 RNA is primed by a tRNA3(Lys) molecule bound at the primer binding site (PBS). Complex intermolecular interactions were proposed between tRNA3(Lys) and the RNA of the HIV-1 Mal isolate. Recently, an alternative interaction was proposed between the TPsiC stem of tRNA3(Lys) and a primer activation signal (PAS) of the Lai and Hxb2 RNAs, suggesting major structural variations in the reverse transcription complex of different HIV-1 strains. Here, we analyzed mutants of the Hxb2 RNA that prevent the interaction between the PAS and tRNA3(Lys) or/and a complementary sequence in the viral RNA. We compared the kinetics of reverse transcription of the wild type and mutant Hxb2 RNAs, using either tRNA3(Lys) or an 18mer oligoribonucleotide complementary to the PBS, which cannot interact with the PAS, as primers. We also used chemical probing to test the structure of the mutant and wild type RNAs, as well as the complex formed between the later RNA and tRNA3(Lys). These experiments, together with the analysis of long term replication data of mutant viruses obtained by C. Morrow and coworkers (Birmingham, USA) that use alternate tRNAs as primers, strongly suggest that the interaction between the Hxb2 PAS and tRNA3(Lys) does not exist. Instead, the effects of the vRNA mutations on reverse transcription seem to be linked to incorrect folding of the mutant RNAs.

The modular structure of Escherichia coli threonyl-tRNA synthetase as both an enzyme and a regulator of gene expression.

In addition to its role in tRNA aminoacylation, Escherichia coli threonyl-tRNA synthetase is a regulatory protein which binds a site, called the operator, located in the leader of its own mRNA and inhibits translational initiation by competing with ribosome binding. This work shows that the two essential steps of regulation, operator recognition and inhibition of ribosome binding, are performed by different domains of the protein. The catalytic and the C-terminal domain of the protein are involved in binding the two anticodon arm-like structures in the operator whereas the N-terminal domain of the enzyme is responsible for the competition with the ribosome. This is the first demonstration of a modular structure for a translational repressor and is reminiscent of that of transcriptional regulators. The mimicry between the operator and tRNA, suspected on the basis of previous experiments, is further supported by the fact that identical regions of the synthetase recognize both the operator and the tRNA anticodon arm. Based on these results, and recent structural data, we have constructed a computer-derived molecular model for the operator-threonyl-tRNA synthetase complex, which sheds light on several essential aspects of the regulatory mechanism.

HIV-1 RNA dimerization initiation site is structurally similar to the ribosomal A site and binds aminoglycoside antibiotics.

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.

RNA loop-loop interactions as dynamic functional motifs.

RNA loop-loop interactions are frequently used to trigger initial recognition between two RNA molecules. In this review, we present selected well-documented cases that illustrate the diversity of biological processes using RNA loop-loop recognition properties. The first one is related to natural antisense RNAs that play a variety of regulatory functions in bacteria and their extra-chromosomal elements. The second one concerns the dimerization of HIV-1 genomic RNA, which is responsible for the encapsidation of a diploid RNA genome. The third one concerns RNA interactions involving double-loop interactions. These are used by the bicoid mRNA to form dimers, a property that appears to be important for mRNA localization in drosophila embryo, and by bacteriophage phi29 pRNA which forms hexamers that participate in the translocation of the DNA genome through the portal vertex of the capsid. Despite the high diversity of systems and mechanisms, some common features can be highlighted. (1) Efficient recognition requires rapid bi-molecular binding rates, regardless of the RNA pairing scheme. (2) The initial recognition is favored by particular conformations of the loops enabling a proper presentation of nucleotides (generally a restricted number) that initiate the recognition process. (3) The fate of the initial reversible loop-loop complex is dictated by both functional and structural constraints. RNA structures have evolved either to "freeze" the initial complex, or to convert it into a more stable one, which involves propagation of intermolecular interactions along topologically feasible pathways. Stabilization of the initial complex may also be assisted by proteins and/or formation of additional contacts.