Marie-Line Andreola

G-quadruplexes in viruses: function and potential therapeutic applications

G-rich nucleic acids can form non-canonical G-quadruplex structures (G4s) in which four guanines fold in a planar arrangement through Hoogsteen hydrogen bonds. Although many biochemical and structural studies have focused on DNA sequences containing successive, adjacent guanines that spontaneously fold into G4s, evidence for their in vivo relevance has recently begun to accumulate. Complete sequencing of the human genome highlighted the presence of ∼300 000 sequences that can potentially form G4s. Likewise, the presence of putative G4-sequences has been reported in various viruses genomes [e.g., Human immunodeficiency virus (HIV-1), Epstein–Barr virus (EBV), papillomavirus (HPV)]. Many studies have focused on telomeric G4s and how their dynamics are regulated to enable telomere synthesis. Moreover, a role for G4s has been proposed in cellular and viral replication, recombination and gene expression control. In parallel, DNA aptamers that form G4s have been described as inhibitors and diagnostic tools to detect viruses [e.g., hepatitis A virus (HAV), EBV, cauliflower mosaic virus (CaMV), severe acute respiratory syndrome virus (SARS), simian virus 40 (SV40)]. Here, special emphasis will be given to the possible role of these structures in a virus life cycle as well as the use of G4-forming oligonucleotides as potential antiviral agents and innovative tools.

Topology of a DNA G-quadruplex structure formed in the HIV-1 promoter: a potential target for anti-HIV drug development.

Nucleic acid sequences containing guanine tracts are able to adopt noncanonical four-stranded nucleic acid structures called G-quadruplexes (G4s). These structures are based on the stacking of two or more G-tetrads; each tetrad is a planar association of four guanines held together by eight hydrogen bonds. In this study, we analyzed a conserved G-rich region from HIV-1 promoter that is known to regulate the transcription of the HIV-1 provirus. Strikingly, our analysis of an alignment of 1684 HIV-1 sequences from this region showed a high conservation of the ability to form G4 structures despite a lower conservation of the nucleotide primary sequence. Using NMR spectroscopy, we determined the G4 topology adopted by a DNA sequence from this region (HIV-PRO1: 5 TGGCCTGGGCGGGACTGGG 3). This DNA fragment formed a stable two G-tetrad antiparallel G4 with an additional Watson-Crick CG base pair. This hybrid structure may be critical for HIV-1 gene expression and is potentially a novel target for anti-HIV-1 drug development.

Intasome architecture and chromatin density modulate retroviral integration into nucleosome

BackgroundRetroviral integration depends on the interaction between intasomes, host chromatin and cellular targeting cofactors as LEDGF/p75 or BET proteins. Previous studies indicated that the retroviral integrase, by itself, may play a role in the local integration site selection within nucleosomal target DNA. We focused our study on this local association by analyzing the intrinsic properties of various retroviral intasomes to functionally accommodate different chromatin structures in the lack of other cofactors.ResultsUsing in vitro conditions allowing the efficient catalysis of full site integration without these cofactors, we show that distinct retroviral integrases are not equally affected by chromatin compactness. Indeed, while PFV and MLV integration reactions are favored into dense and stable nucleosomes, HIV-1 and ASV concerted integration reactions are preferred into poorly dense chromatin regions of our nucleosomal acceptor templates. Predicted nucleosome occupancy around integration sites identified in infected cells suggests the presence of a nucleosome at the MLV and HIV-1 integration sites surrounded by differently dense chromatin. Further analyses of the relationships between the in vitro integration site selectivity and the structure of the inserted DNA indicate that structural constraints within intasomes could account for their ability to accommodate nucleosomal DNA and could dictate their capability to bind nucleosomes functionally in these specific chromatin contexts.ConclusionsThus, both intasome architecture and compactness of the chromatin surrounding the targeted nucleosome appear important determinants of the retroviral integration site selectivity. This supports a mechanism involving a global targeting of the intasomes toward suitable chromatin regions followed by a local integration site selection modulated by the intrinsic structural constraints of the intasomes governing the target DNA bending and dictating their sensitivity toward suitable specific nucleosomal structures and density.

Evolution of 2-long terminal repeat (2-LTR) episomal HIV-1 DNA in raltegravir-treated patients and in in vitro infected cells

OBJECTIVESnOur aim was to analyse the evolution of HIV-1 2-long terminal repeat (2-LTR) circular DNA in vitro and ex vivo in the presence of raltegravir.nnnPATIENTS AND METHODSnTwenty-five patients starting a raltegravir-based regimen were included. Total HIV-1 DNA and 2-LTR DNA were quantified at baseline and in follow-up samples up to month 12. The effect of raltegravir on the formation of 2-LTR circles was evaluated in HeLa P4 cells. The effect of raltegravir was also investigated by sequence analysis of the 2-LTR circle junctions.nnnRESULTSnAmong 21 patients with undetectable 2-LTR DNA at baseline, 7 had detectable 2-LTR DNA during the follow-up. Three of four patients with detectable 2-LTR DNA at baseline had undetectable 2-LTR DNA during the follow-up (P = 0.27). The mean 2-LTR level increased significantly (+0.07 log(10)/month, P = 0.02) in raltegravir-treated patients, and a 2-LTR increase was also observed in raltegravir-treated HeLa P4 cells, with a peak at 3 days post-infection. 2-LTR DNA showed a high prevalence of deletions ex vivo (64.5%) and in vitro (50%) in the presence of raltegravir, which was not statistically different from the prevalence in untreated patients or cells.nnnCONCLUSIONSnIn antiretroviral-experienced patients receiving raltegravir, 2-LTR DNA increased while total HIV-1 DNA decreased over time. The frequent rearrangements found in 2-LTR sequences warrant further investigations to determine the dynamics of evolution of unintegrated HIV-1 DNA.

Anticancer molecule AS1411 exhibits low nanomolar antiviral activity against HIV-1

During clinical trials, a number of fully characterized molecules are dropped along the way because they do not provide enough benefit for the patient. Some of them show limited side effects and might be of great use for other applications. AS1411 is a nucleolin-targeting aptamer that underwent phase II clinical trials as anticancer agent. Here, we show that AS1411 exhibits extremely potent antiviral activity and is therefore an attractive new lead as anti-HIV agent.

Yeast and the AIDS Virus: The Odd Couple

Despite being simple eukaryotic organisms, the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe have been widely used as a model to study human pathologies and the replication of human, animal, and plant viruses, as well as the function of individual viral proteins. The complete genome of S. cerevisiae was the first of eukaryotic origin to be sequenced and contains about 6,000 genes. More than 75% of the genes have an assigned function, while more than 40% share conserved sequences with known or predicted human genes. This strong homology has allowed the function of human orthologs to be unveiled starting from the data obtained in yeast. RNA plant viruses were the first to be studied in yeast. In this paper, we focus on the use of the yeast model to study the function of the proteins of human immunodeficiency virus type 1 (HIV-1) and the search for its cellular partners. This human retrovirus is the cause of AIDS. The WHO estimates that there are 33.4 million people worldwide living with HIV/AIDS, with 2.7 million new HIV infections per year and 2.0 million annual deaths due to AIDS. Current therapy is able to control the disease but there is no permanent cure or a vaccine. By using yeast, it is possible to dissect the function of some HIV-1 proteins and discover new cellular factors common to this simple cell and humans that may become potential therapeutic targets, leading to a long-lasting treatment for AIDS.

Modulation of chromatin structure by the FACT histone chaperone complex regulates HIV-1 integration

BackgroundInsertion of retroviral genome DNA occurs in the chromatin of the host cell. This step is modulated by chromatin structure as nucleosomes compaction was shown to prevent HIV-1 integration and chromatin remodeling has been reported to affect integration efficiency. LEDGF/p75-mediated targeting of the integration complex toward RNA polymerase II (polII) transcribed regions ensures optimal access to dynamic regions that are suitable for integration. Consequently, we have investigated the involvement of polII-associated factors in the regulation of HIV-1 integration.ResultsUsing a pull down approach coupled with mass spectrometry, we have selected the FACT (FAcilitates Chromatin Transcription) complex as a new potential cofactor of HIV-1 integration. FACT is a histone chaperone complex associated with the polII transcription machinery and recently shown to bind LEDGF/p75. We report here that a tripartite complex can be formed between HIV-1 integrase, LEDGF/p75 and FACT in vitro and in cells. Biochemical analyzes show that FACT-dependent nucleosome disassembly promotes HIV-1 integration into chromatinized templates, and generates highly favored nucleosomal structures in vitro. This effect was found to be amplified by LEDGF/p75. Promotion of this FACT-mediated chromatin remodeling in cells both increases chromatin accessibility and stimulates HIV-1 infectivity and integration.ConclusionsAltogether, our data indicate that FACT regulates HIV-1 integration by inducing local nucleosomes dissociation that modulates the functional association between the incoming intasome and the targeted nucleosome.

Dual and Opposite Effects of hRAD51 Chemical Modulation on HIV-1 Integration

The cellular DNA repair hRAD51 protein has been shown to restrict HIV-1 integration both inxa0vitro and inxa0vivo. To investigate its regulatory functions, we performed a pharmacological analysis of the retroviral integration modulation by hRAD51. We found that, inxa0vitro, chemical activation of hRAD51 stimulates its integration inhibitory properties, whereas inhibition of hRAD51 decreases the integration restriction, indicating that the modulation of HIV-1 integration depends on the hRAD51 recombinase activity. Cellular analyses demonstrated that cells exhibiting high hRAD51 levels prior to de novo infection are more resistant to integration. On the other hand, when hRAD51 was activated during integration, cells were more permissive. Altogether, these data establish the functional link between hRAD51 activity and HIV-1 integration. Our results highlight the multiple and opposite effects of the recombinase during integration and provide new insights into the cellular regulation of HIV-1 replication.

Hepatitis C virus intragenomic interactions are modulated by the SLVI RNA structure of the core coding sequence

Several RNA interactions are thought to play a role in the regulation of the hepatitis C virus (HCV) life cycle. Most of these interactions involve the 5BSL3.2 domain and therefore occur at the 3 end of the viral genomic RNA. A long-range interaction has also been described between 5BSL3.2 and the 5 untranslated region (UTR). Another interaction involves the SLVI stem loop of the core coding region and the 5UTR. We aimed to analyse the role of this SLVI domain, which likely interferes with others interactions. By evaluating RNA stability, translation and RNA synthesis, we showed that the SLVI stem loop extensively modulates the effect of the interactions mediated by the 5BSL3.2 domain and strongly affects the IIId/5BSL3.2 interaction. Numerous interactions in HCV genomic RNA have been described in the UTRs and the coding sequence but their roles are poorly understood. We showed that the SLVI domain located in the core coding sequence plays an important role in the translation of the polyprotein, but also in the modulation of long-range RNA interactions centred on the 5BSL3.2 domain. The SLVI domain has been absent from most studies, especially from the extensively used subgenomic replicon; our data highlight the importance of this domain in the studies of these long-range interactions in the HCV life cycle.

Modulation of the functional association between the HIV-1 intasome and the nucleosome by histone amino-terminal tails

BackgroundStable insertion of the retroviral DNA genome into host chromatin requires the functional association between the intasome (integrase·viral DNA complex) and the nucleosome. The data from the literature suggest that direct protein–protein contacts between integrase and histones may be involved in anchoring the intasome to the nucleosome. Since histone tails are candidates for interactions with the incoming intasomes we have investigated whether they could participate in modulating the nucleosomal integration process.ResultsWe show here that histone tails are required for an optimal association between HIV-1 integrase (IN) and the nucleosome for efficient integration. We also demonstrate direct interactions between IN and the amino-terminal tail of human histone H4 in vitro. Structure/function studies enabled us to identify amino acids in the carboxy-terminal domain of IN that are important for this interaction. Analysis of the nucleosome-binding properties of catalytically active mutated INs confirmed that their ability to engage the nucleosome for integration in vitro was affected. Pseudovirus particles bearing mutations that affect the IN/H4 association also showed impaired replication capacity due to altered integration and re-targeting of their insertion sites toward dynamic regions of the chromatin with lower nucleosome occupancy.ConclusionsCollectively, our data support a functional association between HIV-1 IN and histone tails that promotes anchoring of the intasome to nucleosomes and optimal integration into chromatin.