Sandra Da Silva Amaral's thesis defence

Jury

• Rapporteur and Examiner: Jean-Christophe Paillart, directeur de recherche, Université de Strasbourg
• Rapporteur and Examiner: Hugues de Rocquigny, directeur de Recherche, Université de Tours
• Examiner: Carine Tisné, directrice de recherche, Université Paris Cité
• Examiner: Nelly Morellet, chargée de recherche, Université de Paris-Saclay

Management

• Thesis Director: Philippe Fossé, directeur de la Recherche, CNRS

Abstract

Title:  Role of TAR and cTAR stem-loops on HIV-1 first strand transfer

The HIV-1 life cycle requires the reverse transcription process. This process allows the synthesis of a double-stranded DNA from its genome, which consists of two copies of single-stranded RNA of positive polarity used as a template. Two events, called terminal strand transfers, are necessary for reverse transcription to occur and each results in the synthesis of one of the two DNA strands. My thesis work focuses on the first strand transfer. This occurs from the 5' end to the 3' end of the viral genomic RNA (gRNA) at the duplicated R sequences after the synthesis of the strong-stop DNA (ssDNA) by the reverse transcriptase (RT). As ssDNA is a copy of the R and U5 regions of the 5' end́ of the gRNA, it can pair with the R region also present in the 3' of the viral RNA and allow synthesis of the negative polarity DNA strand. The sequences of the R regions form two stem-loops (cTAR and cpoly(A) in ssRNA, 3'TAR and 3'poly(A) in gRNA). These structures must be open to allow hybridization between the R regions of the ssDNA and RNA.

Several in vitro studies suggest that the HIV-1 nucleocapsid protein (NC) is partly responsible for the opening of these stem-loops and their pairing. This protein is known for its chaperone nucleic activity and is involved in several steps of the viral cycle. The works studying the role of NC on reverse transcription showed in vitro that this protein facilitates the first strand transfer and destabilizes the TAR and cTAR stem-loops by binding to certain guanines. Furthermore, the destabilisation reaction by NC is dependent on the number of consecutive base pairs involved in the stem-loop.

The thesis work determines whether the first strand transfer depends on the destabilisation of the ssDNA cTAR and the gRNA TAR stem-loops by NC ex vivo. To this end, different types of mutations were designed and introduced in these stem-loops to prevent their destabilisation by NC and to observe an effect on reverse transcription. Replication of mutant viruses and more precisely the reverse transcription of gRNA are analysed and discussed.