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    Regulation of HIV transcriptome

    Responsable du projet : Sarah Gallois-Montbrun (CRCN, Inserm)

    Contact : - Tel : +33 1 40 51 65 76

     

    The group

    Sarah Gallois-Montbrun (group leader, CRCN Inserm)

    Nam Nguyen Quang (post-doc, ANRS)

    Corentin Aubé (PhD student)

    Emilie Murigneux (IE Inserm)

    Louis Bergantz (M2)

     

    Objectives

    The Human Immunodeficiency Virus (HIV) genome is composed of two copies of single-stranded positive-sense RNA. Upon entry into the host cells, this viral genomic RNA is rapidly converted into cDNA by the reverse transcriptase and integrated into the host cells. Once integrated, the provirus is transcribed by the cellular machinery to generate an unspliced (US) viral pre-mRNA. A proportion of this full-length pre-mRNA is kept unspliced, exported to the cytoplasm and either used as genomic RNA or translated into viral structural proteins (Gag and Gag-Pol). The rest undergoes a complex alternative splicing driven by the spliceosome. This process gives rise to more than 100 different transcripts that are translated into the proteins needed for viral particles production. Imbalance in the levels of viral RNAs isoforms can have dramatic effects on viral particles production. Viral alternative splicing is therefore tightly regulated both temporally and spatially by a number of cellular and viral factors.

     

    The goal of our group is to decipher the different mechanisms involved in the regulation of HIV-1 transcriptome, from transcription to RNA processing and translation and how these steps impact viral replication.

     

    Research Interests

    • Quantitative analysis of HIV-1 transcriptome in infected cells.

    To decipher the complexity of the HIV-1 transcriptome and to better understand how cellular and viral proteins influence HIV-1 splicing regulation, we recently used a long-read sequencing approach developed by Oxford Nanopore Technology (ONT). ONT sequencing of HIV-1 infected CD4+ T cells generated reads long enough to reconstitute the cascade of splicing events leading to each particular viral isoform in a quantitative manner. This assay allowed us to produce the first dynamic picture of the cascade of splicing events taking place at early time points of infection in primary CD4+ T cells (Nguyan Quang, Retrovirology, 2020).

    Using this long read sequencing assay, we are now exploring the importance of viral and cellular factors in the regulation of HIV-1 transcriptome.

     

    • Roles of cellular factors in HIV-1 splicing regulation

    Argonaute protein and the miRNA pathway in HIV-1 RNA expression

    Argonaute (Ago) are the key proteins of the microRNA induced silencing complex (miRISC) which regulate more than 60% of cellular genes at a post-transcriptional level. In addition to their cytoplasmic roles, recent studies revealed that Ago1 and Ago2 can have nuclear functions, in particular in splicing regulation and transcription.

    Using High-Throughput Sequencing of RNA isolated by crosslinking immunoprecipitation (HITS-CLIP) of Ago in HIV-1 expressing cells, our team showed that Ago proteins interact with HIV-1 unspliced RNA, in particular in regions located nearby splice donor sites. Downregulation of both Ago1 and Ago2 increased the ratio of MS/Total viral RNAs as compared to control cells and decreased by 10 folds the production of viral particles. Interestingly, these functions were independent of the presence of miRNA.  Altogether our data suggest a non-canonical role of Ago in HIV-1 splicing independently of the miRNA pathway (Eckenfelder, Nucleic Acids Research, 2017).

     Our current research aims at exploring how Argonaute but also other cellular proteins play parts in this complex network regulating viral RNA production.

     

    •  Viral and host factors that interact with SARS-CoV-2 RNAs

    The beginning of 2020 has seen the emergence of COVID-19 pandemic, a respiratory disease caused by a novel coronavirus (CoV), the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). In the absence of efficient cure or vaccine currently available, there is an imminent need to better understand the molecular mechanisms supporting the replication of this new virus and to identify new targets to control its spread.

    CoV are positive RNA viruses and protein and viral RNAs interactions are crucial in determining the molecular switches towards progression of infection. However, our comprehension of the protein-viral RNA interaction network occurring during SARS-CoV-2 replication is limited.

    Strong of our expertise in cellular and molecular approaches to tackle host-virus interactions, our team is currently conducting a project to identify viral and host factors that interact with SARS-CoV-2 RNAs and that impact viral replication in cellular models of infection.

    The project is conducted in collaboration with the laboratory “Génomes, biologie cellulaire et thérapeutique" at hospital Saint Louis, Paris.

     

    Collaborations

    • Genomics Paris Centre, IBENS, Paris
    • Jean-Christophe Paillart, Institut de Biologie Moléculaire et Cellulaire, Strasbourg
    • Caroline Goujon, Irim, Montpellier
    • Claudine Pique, Institut Cochin, Paris
    • Florence Margottin, Institut Cochin, Paris

     

    Publications/References

    Argonaute proteins regulate HIV-1 multiply spliced RNA and viral production in a Dicer independent manner. Eckenfelder A, Ségéral E, Pinzón N, Ulveling D, Amadori C, Charpentier M, Nidelet S, Concordet JP, Zagury JF, Paillart JC, Berlioz-Torrent C, Seitz H, Emiliani S, Gallois-Montbrun S. Nucleic Acids Res. 2017 Apr 20;45(7):4158-4173. doi: 10.1093/nar/gkw1289.

    Dynamic nanopore long-read sequencing analysis of HIV-1 splicing events during the early steps of infection. Nguyen Quang N, Goudey S, Ségéral E, Mohammad A, Lemoine S, Blugeon C, Versapuech M, Paillart JC, Berlioz-Torrent C, Emiliani S, Gallois-Montbrun S. Retrovirology volume 17, Article number 25 (2020).

     

    Financial supports

    These programs are founded by ANRS, SIDACTION and ANR

    Equipes du département