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R Marquet

Publications and source records attributed to R Marquet.

At least 19 recordsLinked to original sources
PubMed2006

Nucleoside and nucleotide inhibitors of HIV-1 replication.

HIV-1 reverse transcriptase (RT) is one of the main targets for antiviral therapy. Two classes of RT inhibitors can be distinguished: those that are nucleoside or nucleotide analogues (sharing the common NRTIs abbreviation) and those that are not. This review focuses on the NRTIs, which are highly efficient in slowing down viral replication and are used in combination regimens. Unfortunately, the current inhibitors do not completely suppress viral replication and due to the high capacity of adaptation of HIV, allow the selection of drug-resistant viruses. Resistance mechanisms to NRTIs have been extensively investigated and can be divided into two types: improved discrimination of a nucleotide analogue relative to the natural substrate or increased phosphorolytic cleavage of an analogue-blocked primer. This knowledge is important both for the development of new drugs designed to target resistant strains and for the development of new antiviral strategies. The NRTIs currently in clinical trials and new developments in this area are also reviewed.

Drug Resistance, Viral↗
PubMed2001

The emergence of different resistance mechanisms toward nucleoside inhibitors is explained by the properties of the wild type HIV-1 reverse transcriptase.

Nucleoside reverse transcriptase inhibitors (NRTIs) represent one of the main drug families used against AIDS. Once incorporated in DNA, they act as chain terminators, due to the lack of a 3'-hydroxyl group. As for the other anti-human immunodeficiency virus type 1 drugs, their efficiency is limited by the emergence of resistant viral strains. Unexpectedly, previous studies indicated that resistance toward NRTIs is achieved via two distinct and generally exclusive mechanisms. Resistance mutations either decrease the efficiency of NRTIs incorporation or increase their excision from the extended primer. To understand the emergence of different resistance mechanisms toward a single inhibitor class, we compared the incorporation and the pyrophosphorolysis of several NRTIs using wild type reverse transcriptase (WT RT). We found that the efficiency of discrimination or excision by pyrophosphorolysis in the presence of nucleotides of a given NRTI is a key determinant in the emergence of one or the other resistance pathway. Indeed, our results suggest that the pathway by which RT become resistant toward a given NRTI can be predicted by studying the inhibition of WT RT, because the resistance mutations do not confer new properties to the mutant enzyme, but rather exacerbate pre-existing properties of the WT enzyme. They also help to understand the low cross-resistance toward d4T observed with the 3'-azido-3'-deoxythymidine (AZT or zidovudine)-resistant RT.

Acquired Immunodeficiency Syndrome↗
PubMed2001

Structure and dimerization of HIV-1 kissing loop aptamers.

Dimerization of two homologous strands of genomic RNA is an essential feature of the retroviral replication cycle. In HIV-1, genomic RNA dimerization is facilitated by a conserved stem-loop structure located near the 5' end of the viral RNA called the dimerization initiation site (DIS). The DIS loop is comprised of nine nucleotides, six of which define an autocomplementary sequence flanked by three conserved purine residues. Base- pairing between the loop sequences of two copies of genomic RNA is necessary for efficient dimerization. We previously used in vitro evolution to investigate a possible structural basis for the marked sequence conservation of the DIS loop. In this study, chemical structure probing, measurements of the apparent dissociation constants, and computer structure analysis of dimerization-competent aptamers were used to analyze the dimers' structure and binding. The selected aptamers were variants of the naturally occurring A and B subtypes. The data suggest that a sheared base-pair closing the loop of the DIS is important for dimerization in both subtypes. On the other hand, the open or closed state of the last base-pair in the stem differed in the two subtypes. This base-pair appeared closed in the subtype A DIS dimer and open in subtype B. Finally, evidence for a cross-talk between nucleotides 2, 5, and 6 was found in some, but not all, loop contexts, indicating some structural plasticity depending on loop sequence. Discriminating between the general rules governing dimer formation and the particular characteristics of individual DIS aptamers helps to explain the affinity and specificity of loop-loop interactions and could provide the basis for development of drugs targeted against the dimerization step during retroviral replication.

Base Pairing↗
PubMed2000

Identification of the in vitro HIV-2/SIV RNA dimerization site reveals striking differences with HIV-1.

Although their genomes cannot be aligned at the nucleotide level, the HIV-1/SIVcpz and the HIV-2/SIVsm viruses are closely related lentiviruses that contain homologous functional and structural RNA elements in their 5'-untranslated regions. In both groups, the domains containing the trans-activating region, the 5'-copy of the polyadenylation signal, and the primer binding site (PBS) are followed by a short stem-loop (SL1) containing a six-nucleotide self-complementary sequence in the loop, flanked by unpaired purines. In HIV-1, SL1 is involved in the dimerization of the viral RNA, in vitro and in vivo. Here, we tested whether SL1 has the same function in HIV-2 and SIVsm RNA. Surprisingly, we found that SL1 is neither required nor involved in the dimerization of HIV-2 and SIV RNA. We identified the NarI sequence located in the PBS as the main site of HIV-2 RNA dimerization. cis and trans complementation of point mutations indicated that this self-complementary sequence forms symmetrical intermolecular interactions in the RNA dimer and suggested that HIV-2 and SIV RNA dimerization proceeds through a kissing loop mechanism, as previously shown for HIV-1. Furthermore, annealing of tRNA(3)(Lys) to the PBS strongly inhibited in vitro RNA dimerization, indicating that, in vivo, the intermolecular interaction involving the NarI sequence must be dissociated to allow annealing of the primer tRNA.

Binding Sites↗
PubMed2000

Inhibition of the initiation of HIV-1 reverse transcription by 3'-azido-3'-deoxythymidine. Comparison with elongation.

Initiation of human immunodeficiency virus-1 reverse transcription requires formation of a complex containing the viral RNA, primer tRNA(3)(Lys), and reverse transcriptase. Initiation, corresponding to addition of the first six nucleotides to tRNA(3)(Lys), is distinguished from elongation by its high specificity and low efficiency (processivity). Here, we compared the inhibition of initiation and elongation of reverse transcription by 3'-azido-3'-deoxythymidine 5'-triphosphate (AZTTP), the active form of 3'-azido-3'-deoxythymidine. We report the first detailed study of nucleotide binding, discrimination, and pyrophosphorolysis by the authentic initiation complex. We showed that the initiation and elongation complexes bound AZTTP and dTTP with the same affinity, while the polymerization rates were reduced by 148-160-fold during initiation. The pyrophosphorolysis rate of dTTP was reduced by the same extent, indicating that the polymerization equilibrium is the same in the two phases. The efficient unblocking of the 3'-azido-3'-deoxythymidine 5'-monophosphate (AZTMP)-terminated primer by pyrophosphorolysis significantly relieved inhibition of DNA synthesis during elongation in the presence of physiological pyrophosphate concentrations. Remarkably, although pyrophosphorolysis of dTMP and AZTMP were equally efficient during elongation, reverse transcriptase was almost totally unable to unblock the AZTMP-terminated primer during initiation. As a result, inhibition of reverse transcription by AZTTP was more efficient during initiation than elongation of reverse transcription, despite a reduced selectivity of incorporation.

Base Sequence↗
PubMed2000

Dynamics of the HIV-1 reverse transcription complex during initiation of DNA synthesis.

Initiation of human immunodeficiency virus-1 (HIV-1) reverse transcription requires formation of a complex containing the viral RNA (vRNA), tRNA(3)(Lys) and reverse transcriptase (RT). The vRNA and the primer tRNA(3)(Lys) form several intermolecular interactions in addition to annealing of the primer 3' end to the primer binding site (PBS). These interactions are crucial for the efficiency and the specificity of the initiation of reverse transcription. However, as they are located upstream of the PBS, they must unwind as DNA synthesis proceeds. Here, the dynamics of the complex during initiation of reverse transcription was followed by enzymatic probing. Our data revealed reciprocal effects of the tertiary structure of the vRNA.tRNA(3)(Lys) complex and reverse transcriptase (RT) at a distance from the polymerization site. The structure of the initiation complex allowed RT to interact with the template strand up to 20 nucleotides upstream from the polymerization site. Conversely, nucleotide addition by RT modified the tertiary structure of the complex at 10-14 nucleotides from the catalytic site. The viral sequences became exposed at the surface of the complex as they dissociated from the tRNA following primer extension. However, the counterpart tRNA sequences became buried inside the complex. Surprisingly, they became exposed when mutations prevented the intermolecular interactions in the initial complex, indicating that the fate of the tRNA depended on the tertiary structure of the initial complex.

Anticodon↗
PubMed2000

In vitro evidence for the interaction of tRNA(3)(Lys) with U3 during the first strand transfer of HIV-1 reverse transcription.

Over the course of its evolution, HIV-1 has taken maximum advantage of its tRNA(3)(Lys)primer by utilizing it in several steps of reverse transcription. Here, we have identified a conserved nonanucleotide sequence in the U3 region of HIV-1 RNA that is complementary to the anticodon stem of tRNA(3)(Lys). In order to test its possible role in the first strand transfer reaction, we applied an assay using a donor RNA corresponding to the 5'-part and an acceptor RNA spanning the 3'-part of HIV-1 RNA. In addition, we constructed two acceptor RNAs in which the nonanucleotide sequence complementary to tRNA(3)(Lys)was either substituted (S) or deleted (Delta). We used either natural tRNA(3)(Lys)or an 18 nt DNA as primer and measured the efficiency of (-) strand strong stop DNA transfer in the presence of wild-type, S or Delta acceptor RNA. Mutations in U3 did not decrease the transfer efficiency when reverse transcription was primed with the 18mer DNA. However, they significantly reduced the strand transfer efficiency in the tRNA(3)(Lys)-primed reactions. This reduction was also observed in the presence of nucleocapsid protein. These results suggest that tRNA(3)(Lys)increases (-) strand strong stop transfer by interacting with the U3 region of the genomic RNA. Sequence comparisons suggest that such long range interactions also exist in other lentiviruses.

Base Sequence↗
PubMed2000

Convergence of natural and artificial evolution on an RNA loop-loop interaction: the HIV-1 dimerization initiation site.

Loop-loop interactions among nucleic acids constitute an important form of molecular recognition in a variety of biological systems. In HIV-1, genomic dimerization involves an intermolecular RNA loop-loop interaction at the dimerization initiation site (DIS), a hairpin located in the 5' noncoding region that contains an autocomplementary sequence in the loop. Only two major DIS loop sequence variants are observed among natural viral isolates. To investigate sequence and structural constraints on genomic RNA dimerization as well as loop-loop interactions in general, we randomized several or all of the nucleotides in the DIS loop and selected in vitro for dimerization-competent sequences. Surprisingly, increasing interloop complementarity above a threshold of 6 bp did not enhance dimerization, although the combinations of nucleotides forming the theoretically most stable hexanucleotide duplexes were selected. Noncanonical interactions contributed significantly to the stability and/or specificity of the dimeric complexes as demonstrated by the overwhelming bias for noncanonical base pairs closing the loop and covariations between flanking and central loop nucleotides. Degeneration of the entire loop yielded a complex population of dimerization-competent sequences whose consensus sequence resembles that of wild-type HIV-1. We conclude from these findings that the DIS has evolved to satisfy simultaneous constraints for optimal dimerization affinity and the capacity for homodimerization. Furthermore, the most constrained features of the DIS identified by our experiments could be the basis for the rational design of DIS-targeted antiviral compounds.

Codon, Initiator↗
PubMed2000

The crystal structure of HIV reverse-transcription primer tRNA(Lys,3) shows a canonical anticodon loop.

We have solved to 3.3 A resolution the crystal structure of the HIV reverse-transcription primer tRNA(Lys,3). The overall structure is exactly comparable to the well-known L-shape structure first revealed by yeast tRNA(Phe). In particular, it unambiguously shows a canonical anticodon loop. This contradicts previous results in short RNA fragment studies and leads us to conclude that neither frameshifting specificities of tRNA(Lys) nor tRNA(Lys,3) primer selection by HIV are due to a specific three-dimensional anticodon structure. Comparison of our structure with the results of an NMR study on a hairpin representing a nonmodified anticodon stem-loop makes plausible the conclusion that chemical modifications of the wobble base U34 to 5-methoxycarbonyl-methyl-2-thiouridine and of A37 to 2-methylthio-N-6-threonylcarbamoyl-adenosine would be responsible for a canonical 7-nt anticodon-loop structure, whereas the unmodified form would result in a noncanonical UUU short triloop. The hexagonal crystal packing is remarkable and shows tight dimers of tRNAs forming a right-handed double superhelix. Within the dimers, the tRNAs are associated head-to-tail such that the CCA end of one tRNA interacts with the anticodon of the symmetry-related tRNA. This provides us with a partial view of a codon-anticodon interaction and gives insights into the positioning of residue 37, and of its posttranscriptional modifications, relative to the first base of the codon.

Animals↗
PubMed2000

NMR and biochemical characterization of recombinant human tRNA(Lys)3 expressed in Escherichia coli: identification of posttranscriptional nucleotide modifications required for efficient initiation of HIV-1 reverse transcription.

Reverse transcription of HIV-1 viral RNA uses human tRNA(Lys)3 as a primer. Some of the modified nucleotides carried by this tRNA must play a key role in the initiation of this process, because unmodified tRNA produced in vitro is only marginally active as primer. To provide a better understanding of the contribution of base modifications in the initiation complex, we have designed a recombinant system that allows tRNA(Lys)3 expression in Escherichia coli. Because of their high level of overexpression, some modifications are incorporated at substoichiometric levels. We have purified the two major recombinant tRNA(Lys)3 subspecies, and their modified nucleotide contents have been characterized by a combination of NMR and biochemical techniques. Both species carry psis, Ds, T, t6A, and m7G. Differences are observed at position 34, within the anticodon. One fraction lacks the 5-methylaminomethyl group, whereas the other lacks the 2-thio group. Although the s2U34-containing recombinant tRNA is a less efficient primer, it presents most of the characteristics of the mammalian tRNA. On the other hand, the mnm5U34-containing tRNA has a strongly reduced activity. Our results demonstrate that the modifications that are absent in E. coli (m2G10, psi27, m5C48, m5C49, and m1A58) as well as the mnm5 group at position 34 are dispensable for initiation of reverse transcription. In contrast, the 2-thio group at position 34 seems to play an important part in this process.

Base Sequence↗
PubMed1999

The crystal structure of the dimerization initiation site of genomic HIV-1 RNA reveals an extended duplex with two adenine bulges.

BACKGROUND: An important step in retroviral replication is dimerization of the genomic RNA prior to encapsidation. Dimerization is initiated by the formation of a transient 'kissing-loop complex' that is thought to be subsequently matured into an extended duplex by the nucleocapsid protein (NCp). Although chemical probing and nuclear magnetic resonance spectroscopy have provided insight into the structure of the kissing-loop structure, no structural information concerning the extended-duplex state is available so far. RESULTS: The structure of a minimal HIV-1 RNA dimerization initiation site has been solved at 2.3 A resolution in two different space groups. It reveals a 22 base pair extended duplex with two noncanonical Watson-Crick-like G-A mismatches, each adjacent to a bulged-out adenine. The structure shows significant asymmetry in deep groove width and G-A base-pair conformations. A network of eight magnesium cations was clearly identified, one being unusually chelated by the 3' phosphate of each bulge across an extremely narrowed deep major groove. CONCLUSIONS: These crystal structures represent the putative matured form of the initial kissing-loop complex. They show the ability of this self-complementary RNA hairpin loop to acquire a more stable extended duplex structure. Both bulged adenines form a striking 'base grip' that could be a recognition signal, either in cis for another viral RNA sequence, or in trans for a protein, possibly the NCp. Magnesium binding might be important to promote and stabilize the observed extrahelical conformation of these bulges.

Adenine↗
PubMed1999

Structural basis for the specificity of the initiation of HIV-1 reverse transcription.

Initiation of human immunodeficiency virus type 1 (HIV-1) reverse transcription requires specific recognition of the viral genome, tRNA3Lys, which acts as primer, and reverse transcriptase (RT). The specificity of this ternary complex is mediated by intricate interactions between HIV-1 RNA and tRNA3Lys, but remains poorly understood at the three-dimensional level. We used chemical probing to gain insight into the three-dimensional structure of the viral RNA-tRNA3Lys complex, and enzymatic footprinting to delineate regions interacting with RT. These and previous experimental data were used to derive a three-dimensional model of the initiation complex. The viral RNA and tRNA3Lys form a compact structure in which the two RNAs fold into distinct structural domains. The extended interactions between these molecules are not directly recognized by RT. Rather, they favor RT binding by preventing steric clashes between the nucleic acids and the polymerase and inducing a viral RNA-tRNA3Lys conformation which fits perfectly into the nucleic acid binding cleft of RT. Recognition of the 3' end of tRNA3Lys and of the first template nucleotides by RT is favored by a kink in the template strand promoted by the short junctions present in the previously established secondary structure.

Base Sequence↗
PubMed1999

Crystallization of the dimerization-initiation site of genomic HIV-1 RNA: preliminary crystallographic results.

The genomic RNA of all retroviruses is encapsidated in virions as a dimer of single-stranded chains held together near their 5'-end. For HIV-1, the initial site of dimerization has been shown to be a hairpin with a nine-residue loop containing a self-complementary sequence of six residues. This structure is proposed to promote dimerization by loop-loop interaction and formation of a so-called 'kissing complex'. A 23-nucleotide RNA strand containing the loop enclosed by a seven base-pair stem has been synthesized. This oligomer was crystallized by the vapour-diffusion method at 310 K, pH 6.5, with methyl-pentanediol as the precipitant agent in the presence of MgCl2, KCl and spermine. Quasi-complete diffraction data were obtained at 2.7 A resolution with a conventional X-ray source and at 2.3 A resolution on a synchrotron beamline. The space group is P3121 or its enantiomorph P3221, with cell parameters a = b = 60. 1, c = 65.9 A at ambient temperature, or a = b = 59.0, c = 64.3 A in a nitrogen-gas stream. There are two oligomers per asymmetric unit as determined from absorbance measurements of a dissolved crystal whose volume was carefully determined. In some cases, either perfectly or partially twinned crystals were obtained. Perfect twinning is detected by an apparent hexagonal symmetry and yields unusable crystallographic data, whilst partial twinning yields usable data after adequate processing. Structure solution is under way by searching for heavy-atom derivatives and systematically substituting bromo- or iodo-uridines for uridines.

Base Sequence↗
PubMed1999

Dimerization of HIV-1 genomic RNA of subtypes A and B: RNA loop structure and magnesium binding.

Retroviruses encapsidate their genome as a dimer of homologous RNA molecules noncovalently linked close to their 5' ends. The dimerization initiation site (DIS) of human immunodeficiency virus type 1 (HIV-1) RNA is a hairpin structure that contains in the loop a 6-nt self-complementary sequence flanked by two 5' and one 3' purines. The self-complementary sequence, as well as the flanking purines, are crucial for dimerization of HIV-1 RNA, which is mediated by formation of a "kissing-loop" complex between the DIS of each monomer. Here, we used chemical modification interference, lead-induced cleavage, and three-dimensional modeling to compare dimerization of subtype A and B HIV-1 RNAs. The DIS loop sequences of these RNAs are AGGUGCACA and AAGCGCGCA, respectively. In both RNAs, ethylation of most but not all phosphate groups in the loop and methylation of the N7 position of the G residues in the self-complementary sequence inhibited dimerization. These results demonstrate that small perturbations of the loop structure are detrimental to dimerization. Conversely, methylation of the N1 position of the first and last As in the loop were neutral or enhanced dimerization, a result consistent with these residues forming a noncanonical sheared base pair. Phosphorothioate interference, lead-induced cleavage, and Brownian-dynamics simulation revealed an unexpected difference in the dimerization mechanism of these RNAs. Unlike subtype B, subtype A requires binding of a divalent cation in the loop to promote RNA dimerization. This difference should be taken into consideration in the design of antidimerization molecules aimed at inhibiting HIV-1 replication.

Base Sequence↗
PubMed1999

Protection against hyperacute xenograft rejection of transgenic rat hearts expressing human decay accelerating factor (DAF) transplanted into primates.

BACKGROUND: Production of transgenic pigs for multiple transgenes is part of a potential strategy to prevent immunological events involved in xenograft rejection. Use of a genetically engineerable rodent as a donor in primates could allow testing in vivo of the effects of different transgenes on controlling xenograft rejection. As a first step in the development of a donor containing multiple transgenes, transgenic rats for human decay-accelerating factor (DAF) were used as heart donors to test their resistance against complement (C)-mediated rejection by non-human primates. MATERIALS AND METHODS: Transgenic rats were generated by using a construct containing the human DAF cDNA under the transcriptional control of the endothelial cell (EC)-specific human ICAM-2 promoter. DAF expression was evaluated by immunohistology and by FACS analysis of purified ECs. Resistance of transgenic hearts against C-mediated damage was evaluated by ex vivo perfusion with human serum and by transplantation into cynomolgus monkeys. RESULTS: Immunohistological analysis of DAF expression in several organs from two transgenic lines showed uniform expression on the endothelium of all blood vessels. ECs purified from transgenic hearts showed 50% DAF expression compared to human ECs and >70% reduction of C-dependent cell lysis compared to control rat ECs. Hemizygous transgenic hearts perfused with human serum showed normal function for >60 min vs. 11. 2 +/- 1.7 min in controls. Hemi- or homozygous transgenic hearts transplanted into cynomolgus monkeys showed longer survival (15.2 +/- 7 min and >4.5 hr, respectively) than controls (5.5 +/- 1.4 min). In contrast to hyperacutely rejected control hearts, rejected homozygous DAF hearts showed signs of acute vascular rejection (AVR) characterized by edema, hemorrhage, and an intense PMN infiltration. CONCLUSIONS: We demonstrate that endothelial-specific DAF expression increased heart transplant survival in a rat-to-primate model of xenotransplantation. This will aid in the analysis of AVR and of new genes that may inhibit this form of rejection, thus helping to define strategies for the production of transgenic pigs.

Animals↗
PubMed1998

Contacts between reverse transcriptase and the primer strand govern the transition from initiation to elongation of HIV-1 reverse transcription.

HIV-1 reverse transcriptase (RT) utilizes RNA oligomers to prime DNA synthesis. The initiation of reverse transcription requires specific interactions between HIV-1 RNA, primer tRNA3Lys, and RT. We have previously shown that extension of an oligodeoxyribonucleotide, a situation that mimicks elongation, is unspecific and differs from initiation by the polymerization rate and dissociation rate of RT from the primer-template complex. Here, we used replication intermediates to analyze the transition from the initiation to the elongation phases. We found that the 2'-hydroxyl group at the 3' end of tRNA had limited effects on the polymerization and dissociation rate constants. Instead, the polymerization rate increased 3400-fold between addition of the sixth and seventh nucleotide to tRNA3Lys. The same increase in the polymerization rate was observed when an oligoribonucleotide, but not an oligodeoxyribonucleotide, was used as a primer. In parallel, the dissociation rate of RT from the primer-template complex decreased 30-fold between addition of the 17th and 19th nucleotide to tRNA3Lys. The polymerization and dissociation rates are most likely governed by interactions of the primer strand with helix alphaH in the p66 thumb subdomain and the RNase H domain of RT, respectively.

Animals↗
PubMed1998

Solution studies of the dimerization initiation site of HIV-1 genomic RNA.

Dimerization of HIV-1 genomic RNA is an essential step of the viral cycle, initiated at a conserved stem-loop structure which forms a 'kissing complex' involving loop-loop interactions (dimerization initiation site, DIS). A 19mer RNA oligonucleotide (DIS-19) has been synthesized which forms a stable symmetrical dimer in solution at millimolar concentrations. Dimerization does not depend on addition of Mg2+. RNA ligation experiments unambiguously indicate that the formed dimer is a stable kissing complex under the NMR experimental conditions.1H NMR resonance assignments were obtained for DIS-19 at 290 K and pH 6.5. Analysis of the pattern of NOE connectivities reveals that the helix formed by loop-loop base pairing is stacked onto the two terminal stems. Non-canonical base pairs between two essential and conserved adenines are found at the junctions between the two intramolecular and the single intramolecular helices.

Binding Sites↗