Biomedical subjects

D Muriaux

Publications and source records attributed to D Muriaux.

10 recordsLinked to original sources

PubMed2006

Mutant murine leukemia virus Gag proteins lacking proline at the N-terminus of the capsid domain block infectivity in virions containing wild-type Gag.

We have investigated the properties of murine leukemia virus Gag mutants in which the p12-CA cleavage site is altered. In one mutant, the cleavage is blocked; in the other, the conserved proline at the N-terminus of CA has been replaced with glycine. No infectivity was detected in either mutant. Mutant particles cannot synthesize full-length DNA upon infecting permissive cells. Particles composed of a mixture of wild-type and mutant proteins have severely impaired infectivity. These mixed particles are defective in their ability to synthesize DNA upon infection, but this defect is less severe than the loss of infectivity. Thus, proteins lacking the correct N-terminus of CA inhibit DNA synthesis and also interfere with formation or integration of a full-length, normal provirus. The results imply that CA proteins function as part of a large, highly organized structure in reverse transcription and apparently at a later step as well.

Animals↗

PubMed2004

Targeting the assembly of the human immunodeficiency virus type I.

In the rush to develop anti-viral drugs against the human immunodeficiency virus type I (HIV-1), all the steps of the viral life cycle are potential targets of therapeutic intervention. In this review, we will explore the recent advances on strategies that aim at obstructing the formation, the release and the infectivity of newly formed virion particles from HIV-1 infected cells.

Animals↗

PubMed2001

RNA is a structural element in retrovirus particles.

A single retroviral protein, Gag, is sufficient for virus particle assembly. While Gag is capable of specifically packaging the genomic RNA into the particle, this RNA species is unnecessary for particle assembly in vivo. In vitro, nucleic acids profoundly enhance the efficiency of assembly by recombinant Gag proteins, apparently by acting as "scaffolding" in the particle. To address the participation of RNA in retrovirus assembly in vivo, we analyzed murine leukemia virus particles that lack genomic RNA because of a deletion in the packaging signal of the viral RNA. We found that these particles contain cellular mRNA in place of genomic RNA. This result was particularly evident when Gag was expressed by using a Semliki Forest virus-derived vector: under these conditions, the Semliki Forest virus vector-directed mRNA became very abundant in the cells and was readily identified in the retroviral virus-like particles. Furthermore, we found that the retroviral cores were disrupted by treatment with RNase. Taken together, the data strongly suggest that RNA is a structural element in retrovirus particles.

Animals↗

PubMed1998

The annealing of tRNA3Lys to human immunodeficiency virus type 1 primer binding site is critically dependent on the NCp7 zinc fingers structure.

The nucleocapsid protein NCp7 of the human immunodeficiency virus type 1 contains two zinc fingers of the CX2CX4HX4C type, flanked by several basic residues, and plays a major role in viral infectivity. Thus, NCp7 was shown to promote annealing of the tRNA3Lys to the primer binding site, a key step in reverse transcription. However, previous in vitro experiments were unable to clarify the role of the zinc fingers in this process, due to nucleic acid aggregation induced by the basic N- and C-terminal domains of NCp7. We show here that deletion of these sequences in (12-53)NCp7 strongly reduces the formation of aggregates and allows a direct visualization of the binary or ternary complexes between NCp7 and nucleic acids by gel electrophoresis. (12-53)NCp7 is able to induce hybridization of the 33P tRNA3Lys and the human immunodeficiency virus type 1 viral RNA-(77-257), which contains the primer binding site. Modification of the proximal zinc finger conformation in Cys23(12-53)NCp7 led to a large reduction in this hybridization process, while replacement of Trp37 by Leu in the distal zinc fingers resulted in a complete absence of annealing activity. These data account for the in vivo loss of viral infectivity following these mutations and emphasize the critical role of the structure of the zinc finger domain of NCp7. This could facilitate a rational approach to new antiviral agents directed toward NCp7.

Amino Acid Sequence↗

PubMed1996

NCp7 activates HIV-1Lai RNA dimerization by converting a transient loop-loop complex into a stable dimer.

Nucleocapsid protein 7 (NCp7), the human immunodeficiency virus type 1 (HIV-1) nucleocapsid protein, was shown to strongly potentiate the dimerization of the retroviral genomic RNA. This process involves the interaction of two retroviral RNA monomer subunits near their 5'-ends. A region located upstream from the splice donor site was recently identified as being responsible for the formation of dimeric HIV-1 RNA. This region appeared to be confined within a stem-loop structure, with an autocomplementary sequence in the loop. In an in vitro study of spontaneous dimer formation, we reported that the 77-402 RNA transcript forms two distinct dimers differing in their thermostability: D37 and D55. We identified D37 as a "kissing" complex structure, formed via a loop-loop interaction between the two monomers, and D55 as a double stranded structure involving all nucleotides of the stem-loop via canonical base pairing. In this report, we have characterized the role of NCp7 in the HIV-1Lai RNA dimerization process by using in vitro dimerization assays with RNA transcripts of different lengths and dimer thermal dissociation. Our results show that the nucleocapsid protein NCp7 activates RNA dimerization very likely through interaction with the kissing complex and converts it into a stable dimer. Furthermore, this NCp7-promoted conversion only occurs if the 240-280 stem-loop structure is present in HIV-1Lai RNA molecules and contains the autocomplementary G257CGCGC262 sequence. This study suggests that, under physiological conditions, an NCp7-mediated RNA conformational change is involved in the maturation of the HIV-1 RNA dimer.

Base Sequence↗

PubMed1996

A short autocomplementary sequence plays an essential role in avian sarcoma-leukosis virus RNA dimerization.

Retroviral genomes consist of two identical RNA molecules joined noncovalently near their 5'-ends. Recently, two models have been proposed for RNA dimer formation on the basis of results obtained in vitro with human immunodeficiency virus type 1 RNA and Moloney murine leukemia virus RNA. It was first proposed that viral RNA dimerizes by forming an interstrand quadruple helix with purine tetrads. The second model postulates that RNA dimerization is initiated by a loop-loop interaction between the two RNA molecules. In order to better characterize the dimerization process of retroviral genomic RNA, we analyzed the in vitro dimerization of avian sarcoma-leukosis virus (ASLV) RNA using different transcripts. We determined the requirements for heterodimer formation, the thermal dissociation of RNA dimers, and the influence of antisense DNA oligonucleotides on dimer formation. Our results strongly suggest that purine tetrads are not involved in dimer formation. Data show that an autocomplementary sequence located upstream from the splice donor site and within a major packaging signal plays a crucial role in ASLV RNA dimer formation in vitro. This sequence is able to form a stem-loop structure, and phylogenetic analysis reveals that it is conserved in 28 different avian sarcoma and leukosis viruses. These results suggest that dimerization of ASLV RNA is initiated by a loop-loop interaction between two RNA molecules and provide an additional argument for the ubiquity of the dimerization process via loop-loop interaction.

Alpharetrovirus↗

PubMed1996

Nucleocapsid protein 10 activates dimerization of the RNA of Moloney murine leukaemia virus in vitro.

Short RNA species that encompass the psi domain of the retroviral genome spontaneously form dimers in vitro, and the retroviral nucleocapsid protein activates this dimerization in vitro. Addition of gag RNA sequences downstream of the 3' end of the psi domain decreases the level of spontaneous dimerization. Here, we report the effects of RNA length on dimerization in vitro, studied with RNA fragments from Moloney murine leukaemia virus that contain the psi domain and all or part of the gag sequence. Extension of the RNA leads to progressive inhibition of the in vitro dimerization process. Sequences located downstream of the 3' end of the psi domain seem to stabilize the monomeric structures. This stabilization participates in dimerization of the RNA sequences involved in the recognition of two RNA molecules. We studied the ability of nucleocapsid protein 10 to promote dimerization of such long RNA fragments, and found that the protein greatly enhances their dimerization in vitro. We propose that nucleocapsid protein 10 stimulates the overall dimerization process by reduction of the energy barrier that must be overcome to allow dimer formation. Our results show that dimerization of RNA form Moloney murine leukaemia virus in vitro is enhanced by nucleocapsid protein 10. This finding is in agreement with the involvement of the nucleocapsid protein in RNA dimerization in vivo.

Base Sequence↗

PubMed1996

A kissing complex together with a stable dimer is involved in the HIV-1Lai RNA dimerization process in vitro.

Retroviruses contain a dimeric RNA consisting of two identical molecules of genomic RNA. The interaction between the two monomers is thought to occur near their 5'ends. We previously identified a region upstream from the splice donor site, comprising an autocomplementary sequence, responsible for the formation of dimeric HIV-1Lai RNA [Muriaux, D., Girard, P.-M., Bonnet-Mathonire, B., & Paoletti, J.(1995) J. Biol. Chem. 270,8209-8216]. This region appeared to be confined within a putative stem-loop structure. Here we report an in vitro model under conditions of low inioc strength. Two dimers of RNA 77-402 were identified as a function of temperature, and a significant difference was found in their thermostability. Dimer D55, formed at 55 degrees Celsius, is more stable than dimer D37, formed at 37 degrees C. RNase probing experiments confirm the involvement of a stem-loop structure in the dimerization process. In the monomer, the free G257-CGCGC262 sequence forms a loop in the 240-280 region of RNA 77-402, whereas this sequence is engaged in base pairing when D55 and D37 dimers are formed. Our results show that the loop-loop interaction of the autocomplementary G257CGCGC262 sequence, though hydrogen bonding, is responsible for the formation of dimer D37 and strongly suggest that D37 is a "kissing" complex. In contrast, in dimer D55, all the nucleotides of the two hairpin stems, 243-254/264-277, are involved in a complete interstrand interaction.

Base Sequence↗

PubMed1995

A short autocomplementary sequence in the 5' leader region is responsible for dimerization of MoMuLV genomic RNA.

Previous work has shown that a region of Moloney murine leukemia virus (MoMuLV) RNA located between nucleotides 280 and 330 in the PSI region (nt 215-565) is implicated in the dimerization process. We show with a deletion from nucleotides 290-299 in PSI RNA transcripts and through an antisense oligonucleotide complementary to nucleotides 275-291 that the 283-298 region is involved in RNA dimer formation in vitro. In an attempt to further characterize the mechanism of dimer formation, a series of short RNA transcripts was synthesized which overlapps the PSI region of MoMuLV RNA. The dimerization of these RNAs is temperature dependent. The predicted secondary structure of the 278-303 region, as a function of temperature, reveals that this sequence is able to adopt two conformations: (1) the U288 AGCUA293 sequence in a loop; (2) part of the same nucleotides implicated in a stem. These results, together with thermodynamic analysis, strongly suggest that (1) the loop conformation of the UAGCUA sequence modulates the relative amount of RNA dimer and (2) a 16 bp long Watson-Crick base pairing is involved in RNA dimer formation. We propose that loop-loop recognition via the U288 AGCUA293 sequence leads to a stable structure induced by a stem-loop opening. Furthermore, our results do not support purine quartet formation as necessary for the dimerization of the 5' leader MoMuLV RNA.

Base Composition↗

PubMed1995

Dimerization of HIV-1Lai RNA at low ionic strength. An autocomplementary sequence in the 5' leader region is evidenced by an antisense oligonucleotide.

Genomic human immunodeficiency virus type 1 (HIV-1) RNA consists of two identical RNA molecules joined noncovalently near their 5' ends in a region called the dimer linkage structure (DLS). Previous work has shown that the putative DLS is localized in a 113-nucleotide domain encompassing the 5' end of the gag gene. This region contains conserved purine tracks that are thought to mediate dimerization through purine quartets. However, recently, an HIV-1Mal RNA dimerization model was proposed as the HIV-1Mal RNA dimerization initiation site, involving another region upstream from the splice donor site and possibly confined within a stem-loop. In the present study, we have investigated the dimerization of HIV-1Lai RNA, using in vitro dimerization assays under conditions of low ionic strength, predictive RNA secondary structures determined by computer folding, and antisense DNA oligonucleotides in order to discriminate between these two models. Our results suggest that purine quartets are not involved in the dimer structure of HIV-1Lai RNA and have led to the identification of a region upstream from the splice donor site. This region, comprising an autocomplementary sequence in a possible stem-loop structure, is responsible for the formation of dimeric HIV-1Lai RNA.

Base Sequence↗