Serge Bouaziz

Structure of the human telomere in K+ solution: a stable basket-type G-quadruplex with only two G-tetrad layers

Previously, it has been reported that human telomeric DNA sequences could adopt in different experimental conditions four different intramolecular G-quadruplexes each involving three G-tetrad layers, namely, Na(+) solution antiparallel-stranded basket form, K(+) crystal parallel-stranded propeller form, K(+) solution (3 + 1) Form 1, and K(+) solution (3 + 1) Form 2. Here we present a new intramolecular G-quadruplex adopted by a four-repeat human telomeric sequence in K(+) solution (Form 3). This structure is a basket-type G-quadruplex with only two G-tetrad layers: loops are successively edgewise, diagonal, and edgewise; glycosidic conformations of guanines are syn x syn x anti x anti around each tetrad. Each strand of the core has both a parallel and an antiparallel adjacent strands; there are one narrow, one wide, and two medium grooves. Despite the presence of only two G-tetrads in the core, this structure is more stable than the three-G-tetrad intramolecular G-quadruplexes previously observed for human telomeric sequences in K(+) solution. Detailed structural elucidation of Form 3 revealed extensive base pairing and stacking in the loops capping both ends of the G-tetrad core, which might explain the high stability of the structure. This novel structure highlights the conformational heterogeneity of human telomeric DNA. It establishes a new folding principle for G-quadruplexes and suggests new loop sequences and structures for targeting in human telomeric DNA.

NMR Structure of the HIV-1 Regulatory Protein Vpr

The human immunodeficiency virus type 1 (HIV-1) genome encodes a highly conserved regulatory gene product, Vpr (96 residues, 14kDa), which is incorporated into virions. In the infected cells, Vpr, expressed late in the virus cycle, is believed to function in the early phases of HIV-1 replication, such as nuclear migration of pre-integration complex, transcription of the proviral genome, viral multiplication by blocking cells in G2 phase and regulation of apoptosis phenomenon. Vpr has a critical role in long term AIDS disease by inducing infection in non-dividing cells such as monocytes and macrophages. To gain insight into the structure-function relationships of Vpr, the (1-96)Vpr protein was synthesized with 22 labeled amino acids. Its 3D structure was analyzed in the presence of CD(3)CN and in pure water at low pH and refined by restrained simulated annealing. The structure of the protein is characterized by three well-defined alpha-helices: 17-33, 38-50 and 56-77 surrounded by flexible N and C-terminal domains. In contrast to the structure obtained in the presence of TFE, the three alpha-helices are folded around a hydrophobic core constituted of Leu, Ile, Val and aromatic residues as illustrated by numerous long range NOEs. This structure accounts for the interaction of Vpr with different targets.

APOBEC3A Is a Specific Inhibitor of the Early Phases of HIV-1 Infection in Myeloid Cells

Myeloid cells play numerous roles in HIV-1 pathogenesis serving as a vehicle for viral spread and as a viral reservoir. Yet, cells of this lineage generally resist HIV-1 infection when compared to cells of other lineages, a phenomenon particularly acute during the early phases of infection. Here, we explore the role of APOBEC3A on these steps. APOBEC3A is a member of the APOBEC3 family that is highly expressed in myeloid cells, but so far lacks a known antiviral effect against retroviruses. Using ectopic expression of APOBEC3A in established cell lines and specific silencing in primary macrophages and dendritic cells, we demonstrate that the pool of APOBEC3A in target cells inhibits the early phases of HIV-1 infection and the spread of replication-competent R5-tropic HIV-1, specifically in cells of myeloid origins. In these cells, APOBEC3A affects the amount of vDNA synthesized over the course of infection. The susceptibility to the antiviral effect of APOBEC3A is conserved among primate lentiviruses, although the viral protein Vpx coded by members of the SIVSM/HIV-2 lineage provides partial protection from APOBEC3A during infection. Our results indicate that APOBEC3A is a previously unrecognized antiviral factor that targets primate lentiviruses specifically in myeloid cells and that acts during the early phases of infection directly in target cells. The findings presented here open up new venues on the role of APOBEC3A during HIV infection and pathogenesis, on the role of the cellular context in the regulation of the antiviral activities of members of the APOBEC3 family and more generally on the natural functions of APOBEC3A.

Localization of HIV-1 Vpr to the nuclear envelope: Impact on Vpr functions and virus replication in macrophages

BackgroundHIV-1 Vpr is a dynamic protein that primarily localizes in the nucleus, but a significant fraction is concentrated at the nuclear envelope (NE), supporting an interaction between Vpr and components of the nuclear pore complex, including the nucleoporin hCG1. In the present study, we have explored the contribution of Vpr accumulation at the NE to the Vpr functions, including G2-arrest and pro-apoptotic activities, and virus replication in primary macrophages.ResultsIn order to define the functional role of Vpr localization at the NE, we have characterized a set of single-point Vpr mutants, and selected two new mutants with substitutions within the first α-helix of the protein, Vpr-L23F and Vpr-K27M, that failed to associate with hCG1, but were still able to interact with other known relevant host partners of Vpr. In mammalian cells, these mutants failed to localize at the NE resulting in a diffuse nucleocytoplasmic distribution both in HeLa cells and in primary human monocyte-derived macrophages. Other mutants with substitutions in the first α-helix (Vpr-A30L and Vpr-F34I) were similarly distributed between the nucleus and cytoplasm, demonstrating that this helix contains the determinants required for localization of Vpr at the NE. All these mutations also impaired the Vpr-mediated G2-arrest of the cell cycle and the subsequent cell death induction, indicating a functional link between these activities and the Vpr accumulation at the NE. However, this localization is not sufficient, since mutations within the C-terminal basic region of Vpr (Vpr-R80A and Vpr-R90K), disrupted the G2-arrest and apoptotic activities without altering NE localization. Finally, the replication of the Vpr-L23F and Vpr-K27M hCG1-binding deficient mutant viruses was also affected in primary macrophages from some but not all donors.ConclusionThese results indicate that the targeting of Vpr to the nuclear pore complex may constitute an early step toward Vpr-induced G2-arrest and subsequent apoptosis; they also suggest that Vpr targeting to the nuclear pore complex is not absolutely required, but can improve HIV-1 replication in macrophages.

Infectious bursal disease virus, a non-enveloped virus, possesses a capsid-associated peptide that deforms and perforates biological membranes

Double-stranded RNA (dsRNA) virions constitute transcriptionally competent machines that must translocate across cell membranes to function within the cytoplasm. The entry mechanism of such non-enveloped viruses is not well described. Birnaviruses are unique among dsRNA viruses because they possess a single shell competent for entry. We hereby report how infectious bursal disease virus, an avian birnavirus, can disrupt cell membranes and enter into its target cells. One of its four structural peptides, pep46 (a 46-amino acid amphiphilic peptide) deforms synthetic membranes and induces pores visualized by electron cryomicroscopy, having a diameter of less than 10 nm. Using both biological and synthetic membranes, the pore-forming domain of pep46 was identified as its N terminus moiety (pep22). The N and C termini of pep22 are shown to be accessible during membrane destabilization and pore formation. NMR studies show that pep46 inserted into micelles displays a cis-trans proline isomerization at position 16 that we propose to be associated to the pore formation process. Reverse genetic experiments confirm that the amphiphilicity and proline isomerization of pep46 are both essential to the viral cycle. Furthermore, we show that virus infectivity and its membrane activity (probably because of the release of pep46 from virions) are controlled differently by calcium concentration, suggesting that entry is performed in two steps, endocytosis followed by endosome permeabilization. Our findings reveal a possible entry pathway of infectious bursal disease virus: in endosomes containing viruses, the lowering of the calcium concentration promotes the release of pep46 that induces the formation of pores in the endosomal membrane.

Direct Vpr-Vpr interaction in cells monitored by two photon fluorescence correlation spectroscopy and fluorescence lifetime imaging.

BackgroundThe human immunodeficiency virus type 1 (HIV-1) encodes several regulatory proteins, notably Vpr which influences the survival of the infected cells by causing a G2/M arrest and apoptosis. Such an important role of Vpr in HIV-1 disease progression has fuelled a large number of studies, from its 3D structure to the characterization of specific cellular partners. However, no direct imaging and quantification of Vpr-Vpr interaction in living cells has yet been reported. To address this issue, eGFP- and mCherry proteins were tagged by Vpr, expressed in HeLa cells and their interaction was studied by two photon fluorescence lifetime imaging microscopy and fluorescence correlation spectroscopy.ResultsResults show that Vpr forms homo-oligomers at or close to the nuclear envelope. Moreover, Vpr dimers and trimers were found in the cytoplasm and in the nucleus. Point mutations in the three α helices of Vpr drastically impaired Vpr oligomerization and localization at the nuclear envelope while point mutations outside the helical regions had no effect. Theoretical structures of Vpr mutants reveal that mutations within the α-helices could perturb the leucine zipper like motifs. The ΔQ44 mutation has the most drastic effect since it likely disrupts the second helix. Finally, all Vpr point mutants caused cell apoptosis suggesting that Vpr-mediated apoptosis functions independently from Vpr oligomerization.ConclusionWe report that Vpr oligomerization in HeLa cells relies on the hydrophobic core formed by the three α helices. This oligomerization is required for Vpr localization at the nuclear envelope but not for Vpr-mediated apoptosis.

Bombyx mori single repeat telomeric DNA sequence forms a G-quadruplex capped by base triads.

A combined NMR–molecular dynamics approach has been applied to determine the solution structure of a truncated analogue of the Bombyx mori telomeric d(TTAGG) single repeat sequence in Na+ cation-containing aqueous solution. The two-fold symmetric four-stranded d(TAGG) quadruplex contains two adjacent G(syn)·G(syn)·G(anti)·(anti) G-tetrads sandwiched between novel (T·A)·A triads with individual strands having both a parallel and antiparallel neighbour around the quadruplex. The (T·A)·A triad represents the first experimental verification of a base triad alignment which constitutes a key postulate in the recently proposed model of triad-DNA. Further, the (T·A)·A triad is generated by positioning an A residue through hydrogen bonding in the minor groove of a Watson–Crick T·A base pair and includes a T–A platform related to an A–A platform recently observed in the structure of the P4-P6 domain of the Tetrahymena self splicing group I ribozyme. The novel architecture of the truncated Bombyx mori quadruplex structure sets the stage for the design and potential identification of additional base tetrads and triads that could participate in pairing alignments of multi-stranded DNA structures during chromosome association and genetic recombination.

The C-terminal domain of the HIV-1 regulatory protein Vpr adopts an antiparallel dimeric structure in solution via its leucine-zipper-like domain

HIV-1 Vpr is a highly conserved accessory protein that is involved in many functions of the virus life cycle. Vpr facilitates the entry of the HIV pre-integration complex through the nuclear pore, induces G2 cell cycle arrest, regulates cell apoptosis, increases transcription from the long terminal repeat and enhances viral replication. Vpr contains a Leu/Ile-rich domain (amino acids 60-81) in its C-terminal part, which is critical for dimerization. The sequence comprising residues 52-96 is implicated in properties of the protein such as DNA interaction and apoptosis via interaction with the adenine nucleotide translocator. To understand the specific interactions of Vpr-(52-96), the ability of this peptide to dimerize via a leucine-zipper mechanism has been investigated, by NMR and fluorescence spectroscopy. In contrast with results from a study performed in the presence of trifluoroethanol, our results, obtained in 30% (v/v) [2H]acetonitrile, show that Vpr-(52-96) in solution still forms an a-helix spanning residues 53-75, but dimerizes in an antiparallel orientation, through hydrophobic interactions between leucine and isoleucine residues and stacking between His71 and Trp54. Moreover, to demonstrate the physiological relevance of the dimer structure, fluorescence spectroscopy experiments have been performed in a Mes buffer, which confirmed the formation of the dimer in aqueous solution and highlighted the spatial proximity between Trp54 and His71. Surprisingly, the leucine-zipper structure shown in the present work for Vpr-(52-96) mimics the structure of full-length Vpr-(1-96), and this could explain why some of the properties of Vpr-(52-96) and Vpr-(1-96) are identical, while some are even enhanced for Vpr-(52-96), particularly in the case of DNA transfection experiments.

Structure-Function Relationship of Vpr: Biological Implications

Vpr, incorporated into the HIV-1 virion, shows multiple activities including nuclear transport of the preintegration complex to the nucleus, activation of the transcription, cell cycle arrest at the G2/M transition and induction of apoptosis. Vpr controls many host cell functions through a variety of biological activities and by interaction with cellular biochemical pathways. Nuclear import of Vpr may be due to its interaction with nuclear transport factors and components of the nuclear pore complex. Cell cycle arrest has been correlated with the binding to DCAF1, a cullin 4A-associated factor and apoptosis may be facilitated by interaction with mitochondrial proteins, in a caspase-dependent mechanism. The structure of Vpr(1-96) and various fragments have been determined by NMR in diverse solvents. The different functions of Vpr can be classified according to their relationship with the different structural domains of the protein and appear to correlate with the partners interacting with these domains. Thus, virion packaging seems to be mediated by the first alpha-helix (17-33), activation of the transcription, regulation of apoptosis and subcellular transport appear to be dependent on the second alpha-helix (38-50) and cell cycle arrest seems to be induced by the carboxyl terminal alpha-helix (55-77). Mutational analysis performed by several groups have provided a strong basis to understand the structure-function relationship of Vpr. The aim of this review is to run through these mutations using the available information on sequences and discuss their effect on the functions of Vpr from the point of view of its structure.

Synthesis and biological evaluation of a new derivative of bevirimat that targets the Gag CA-SP1 cleavage site.

Bevirimat (2), the first-in-class HIV-1 maturation inhibitor, shows a low efficacy due essentially to the natural polymorphism of its target, the CA-SP1 junction. Moreover, its low hydrosolubility makes it difficult to study its interaction with the CA-SP1 junction. We have synthesized new derivatives of bevirimat by adding different hydrophilic substituents at the C-28 position to improve their hydrosolubility and perform the structural study of a complex by NMR. Synthesis of the new derivatives, the effect of substituents at the C-28 position and their hydrosolubility are discussed. The ability of these molecules to inhibit viral infection and their cytotoxicity is assessed. Compared to the well-known bevirimat (2), one of our compounds (16) shows a higher hydrosolubility associated with a 2.5 fold increase in activity, a higher selectivity index and a better antiviral profile. Moreover, for the first time a direct interaction between a derivative of bevirimat (16) and the domain CA-SP1-NC is shown by NMR. Information from this study should allow us to decipher the mechanism by which bevirimat inhibits HIV-1 maturation and how the natural polymorphism of the spacer peptide SP1 triggers resistance to inhibitors.