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Coevolution in the pol gene of HIV-1: a crossroad at the fronteers of the génération of new "species" of HIV

Research unit

UPR 9002 - Architecture et réactivité de l'ARN (IBMC)
15, rue Rene Descartes 67084 - Strasbourg Cedex

Group

Name: Rétrovirus et évolution moléculaire

Group leader: NEGRONI Matteo - m.negroni@ibmc-cnrs.unistra.fr

Group leader's phone: 0388417006

Website: Visit website

Group organization:
- Chercheurs: 2
- ITA: 1
- Doctorants: 1
- Post-Docs: 0
- Autres: 5

Publications of the team linked to the topic (3 last years):
1) Hamoudi M, Simon-Loriere E, Gasser R and Negroni M
Genetic diversity of the highly variable V1 region interferes with HIV-1 envelope functionality
Retrovirology, (2013) 10: 114

2) Rossolillo P, Winter F, Simon-Loriere E, Gallois-Montbrun S, and Negroni M
Retrovolution: HIV-driven Evolution of Cellular Genes and Improvement of Anticancer Drug Activation
PLoS Genetics (2012) 8(8): e1002904. doi:10.1371/journal.pgen.1002904.
3) Simon-Loriere E, Rossolillo P, and Negroni M
RNA structures, genomic organisation, and selection of recombinant HIV
RNA Biology (2011), 8(2), 280-286.

About PhD

PhD Director: NEGRONI Matteo - m.negroni@ibmc-cnr.unistra.fr

Phone: 0388417006

Junior advisor: LENER Daniela

Co-tutely: non

Co-Director: non

About PhD topic :

Title: Coevolution in the pol gene of HIV-1: a crossroad at the fronteers of the génération of new "species" of HIV

Project: Reverse transcriptase and integrase are two key enzymes of the life cycle of retroviruses. The reverse transcriptase (RT) converts the genomic RNA into a double stranded DNA that will be integrated into the host genome by the integrase (IN). Very rapidly, after the discovery that AIDS is caused by a human retrovirus, the human immunodeficiency virus (HIV), RT has become the primary target of anti-viral therapies and a large amount of information have been gathered from different studies. The crystal structure of HIV-1 RT has been solved either alone or in presence of a substrate/inhibitor and the mechanistic aspects of reverse transcription and its inhibition are well known. On the other hand, even if deeply studied, less is known on HIV-1 IN especially because we lack the crystal structure of the full-length protein. Recent studies by cryoelectromicroscopy have analysed functional integrase tetramer complexes (full length protein) assembled in presence of substrate (viral DNA + host genomic DNA) and cellular cofactors. The fitting of the available partial crystal structures (different IN domains) into the cryoelectromicroscopy density map has allowed the proposal of a tri-dimensional model for the process of integration. It appears evident that understanding the structural organisation of the integrase within the tetrameric complex is crucial for the development of new antiviral therapies. Moreover it has been shown that IN interacts not only with several cellular cofactors but also with the viral RT, interaction suggested to be important for the reverse transcription.
Thus, a lot is known about these two enzymes, though shaded zones are still present because we still lack detailed information on the reverse transcription complex (RTC) and on the pre-integration complex (PIC) compositions and on the interplay of the two enzymes within each complex.
Protein functionality often relies on the existence of coevolution networks, which indicates a cross talk between individual parts of proteins either at the intra- or inter-proteic level. The project consists in exploiting the natural sequence divergence existing among primary isolates of HIV to probe such networks, this with the ultimate goal of improving our understanding of the architecture of the IN and of the role that the RT/IN interaction plays in the infectious cycle, during reverse transcription, nuclear import, chromatin targeting and integration itself. The results we obtained in the laboratory show, for the first time (1) that the nature of the reverse transcriptase strongly influences the efficiency of the integration process, an observation potentially leading to the identification of a new parameter governing this process; (2) the existence of coevolution networks between and within individual domains of the IN that ensure its functionality. Generation and characterisation of chimeras between primary isolates of HIV-1 has lead to these observations. The first effect is observed either for chimeras between isolates of the group M or between isolates of the group M and O, while the second effect occurs mostly for the chimeras between M and O. Interestingly, M/O recombinants are found in increasing amount in the pandemic of AIDS, and constitute a potential boundary for the generation of "new species" of HIV with new properties.
The present project aims to characterise: a) the nature of the viral DNA after reverse transcription, in cell culture and in vitro, in order to get insights on the influence that the RT or reverse transcription products have on integration; b) the domains implicated in coevolution, which are crucial for IN activity. The results from the point a and b will be used for the structural analysis of chimerical proteins, through a collaboration with a leading group in the field.

Wished skills: Competences in virology and cell biology would be useful. Competences in structural biology would be an additional useful "tool".

Expertises which will be acquired during the training: Extensive cell culture techniques, molecular genetics. Knowledge for the study of molecular mechanisms in the context of cell culture, and of the principles of molecular evolution. Molecular biology.