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Laboratory of Molecular Virology.

(Department Molecular Bases of Human Diseases)

 

Leader: Monsef BENKIRANE

 

Human Immunodeficiency Virus type 1 (HIV-1), the causative agent of AIDS, is a retrovirus that primarily infects cells of the immune system. The outcome of HIV-1 infection is the result of complex interactions between viral proteins and host cell factors. In most cases, HIV-1 successfully hijacks cellular pathways and bypasses cellular restriction factors for optimal replication, leading to continuous rounds of infection, replication and cell death. Ongoing viral replication causes the loss of CD4+ T cells and progression to immunodeficiency in infected individuals. However, in certain situations, the virus replication can be successfully controlled. First, HAART (Highly Active Anti-Retroviral Therapy) treatment revealed the existence of a pool of resting memory CD4+ T cells harboring integrated, but silent HIV-1 proviruses. Although this situation occurs in a small number of cells, it suggests that the intracellular defense mechanisms can be effective against HIV. This long lived viral reservoir is believed to be the major obstacle against HIV-1 eradication by HAART. Second, HIV-infected individuals, who can control the virus to undetectable levels for many years in the absence of any treatment, have been identified and referred to as Elite HIV Controllers, "EC". Again, this is a rare situation observed in 0.5% of infected patients. Still, it demonstrates that it is possible to naturally and effectively control HIV replication and disease progression. A common feature of these two situations is that viral replication is controlled at the gene expression level. A major challenge in the HIV field is to understand how, in these naturally occurring situations, the intracellular defense and/or immune response win the battle against HIV. Our main objectives are to identify the host factors and define the molecular mechanisms involved in the regulation of HIV-1 gene expression and to explore the involvement of cellular small non-coding RNAs in virus replication. We also use viruses as tools to understand important cellular processes, such as transcription and RNAi.

1- Understanding HIV-1 gene expression through the identification of key regulatory host factors involved in activating or repressing the viral promoter.
HIV-1 Tat assembles a multifunctional transcription elongation complex and stably associates with the 7SK snRNP (Sobhian et al. 2010. Mol Cell 38, 439-451). Studying the HIV-1 transcriptional activator Tat has led to important progress in our understanding of transcription elongation by RNAPII, a key regulatory step of gene expression. In this study, we purified HIV-1 Tat-associated factors from HeLa nuclear extracts and showed by biochemical analysis that HIV-1 Tat forms two distinct and stable complexes. The first one is Tatcom1, which consists of the core active P-TEFb, MLL-fusion partners involved in leukemia (AF9, AFF4, AFF1, ENL and ELL) and PAF1/CDC73. Importantly, Tatcom1 formation relies on Cyclin T1 and CDK9, while optimal CDK9 CTD-kinase activity depends on the presence of AF9. MLL-fusion partners and PAF1 are required for Tat-mediated transactivation of the HIV-1 promoter.

The second complex, Tatcom2, is composed of CDK9, Cyclin T1 and 7SK snRNP containing LARP7 and MEPCE. Tat remodels 7SK snRNP by interacting directly with 7SKRNA in vivo, leading to the formation of stress-resistant 7SK snRNP particles. Besides the identification of new factors that are important for P-TEFb function and are required for Tat transcriptional activity, our data show a coordinated control of RNAPII elongation by different classes of transcription elongation factors acting at the same promoter.

2- Understanding the crosstalk between HIV-1 replication and RNAi.
Suppression of HIV-1 replication by microRNA effectors (Chable-Bessia, Meziane et al. 2009. Retrovirology 6, 26). The rate of HIV-1 gene expression is a key step that determines the kinetics of virus spread and AIDS progression. Viral entry and gene expression are considered to be the key determinants for cell permissiveness to HIV. Recent reports highlighted the involvement of miRNAs in regulating HIV-1 replication post-transcriptionally. In this study we explored the role of cellular factors required for miRNA-mediated mRNA translational inhibition in regulating HIV-1 gene expression. We showed that HIV-1 mRNAs associate and co-localize with components of the RNA Induced Silencing Complex (RISC), and we characterized some of the proteins required for miRNA-mediated silencing (miRNA effectors). RCK/p54, GW182, LSm-1 and XRN1 negatively regulate HIV-1 gene expression by preventing viral mRNA association with polysomes. Interestingly, knockdown of RCK/p54 or DGCR8 resulted in virus reactivation in peripheral blood mononuclear cells (PBMCs) isolated from HIV-infected patients treated with suppressive HAART.
Competition between Dicer mRNA, pre-miRNA, viral RNA for Exportin-5 binding strikes a regulatory balance in cellular miRNA levels. (Bennasser et al. 2011. Nat Struct Mol Biol 18, 323-327).
microRNAs (miRNAs) are a class of small non-coding RNAs (sncRNAs) that function by regulating gene expression post-transcriptionally. Alterations in miRNA expression can dramatically influence cellular physiology and are associated with human diseases, including cancer. Here, we demonstrated cross-regulation between two components of the RNA interference machinery. Specific inhibition of Exportin-5, the karyopherin responsible for pre-miRNA export, down-regulates Dicer expression, the RNase III required for pre-miRNA maturation. This effect is post-transcriptional and results from increased nuclear localization of Dicer mRNA. In vitro assays and cellular RNA immunoprecipitation experiments showed that Exportin-5 directly interacts with Dicer mRNA. Titration of Exportin-5 by over-expressing either pre-miRNA or the adenoviral VA1 RNA resulted in loss of the Dicer mRNA/Exportin-5 interaction and reduction of Dicer level. This saturation also occurs during adenoviral infection and enhances viral replication. Our study reveals an important cross-regulatory mechanism between pre-miRNA or viral small RNAs and Dicer through XPO5.

3-Identification of host cell restriction factors.
Samhd1 is the dendritic and myeloid cell-specific HIV-1-restriction factor counteracted by Vpx. (Laguette et al. 2011. Nature http://dx.doi.org/10.1038/nature10117)
The primate lentivirus auxiliary protein Vpx counteracts an unknown restriction factor that renders human dendritic and myeloid cells largely refractory to HIV-1 infection. Here we identified Samhd1 as this restriction factor. Samhd1 is a protein involved in Aicardi-Goutière Syndrome (AGS), a genetic encephalopathy with symptoms mimicking congenital viral infections that has been proposed to act as a negative regulator of the interferon response 7. We show that Vpx induces proteasomal degradation of Samhd1. Silencing of Samhd1 in non-permissive cell lines alleviates HIV-1 restriction and is associated with a significant accumulation of viral DNA in infected cells. Concurrently, over-expression of Samhd1 in sensitive cells inhibits HIV-1 infection. The putative phosphohydrolase activity of Samhd1 is likely to be required for HIV-1 restriction. Vpx-mediated relief of restriction is abolished in Samhd1 negative cells. Finally, silencing of Samhd1 dramatically increases the susceptibility of monocytic-derived dendritic cells to infection. Altogether, our results demonstrate that Samhd1 is an anti-retroviral protein expressed in cells of the myeloid lineage that inhibits an early step of the viral life cycle. Our findings should be integrated in the development of DC-targeted vaccines against HIV/AIDS.

 

 

Group's News

 

Opportunities A 48 month postdoctoral position

A 48 month postdoctoral position is available starting from June 2013 to join a novel research program developed within the Molecular Virology Laboratory (Monsef Benkirane) at the Institute of Human Genetics (IGH), Montpellier, France (http://www.igh.cnrs.fr/).

The project is dedicated to the study of the interplay between HIV auxiliary proteins and cellular host factors. The laboratory has expertise in this field and is located within a stimulating research environment. The IGH provides state of the art facilities in microscopy, flow cytometry and is equipped with a biosafety level 3 (L3) laboratory.

Candidates should be highly motivated. Candidates must hold a PhD degree in Biological Sciences. Experience in biochemistry, cell biology and molecular biology is required. Candidates should have strong communication and organization skills.

Applicants should send a CV and contact information of two/three references to Nadine Laguette (nadine.laguette@igh.cnrs.fr).

News The HIT HIDDEN HIV website is now online!

Five organizations have decided to pool their resources in order to elaborate a stunning project funded by the European Commission: the Institute of Human Genetics (IGH), the Pasteur Institute, the University of Ulm, the Centre hospitalier universitaire vaudois and Pharis Biotec GmbH, a german firm, have launched HIT HIDDEN HIV in december 2012.

Read more

 

Group's publications of the year (2014):

Brégnard C, Benkirane M, Laguette N..
DNA damage repair machinery and HIV escape from innate immune sensing
(2014), Front Microbiol : 5:176. eCollection 2014. Review
Clifford R, Louis T, Robbe P, Ackroyd S, Burns A, Timbs AT, Colopy GW, Dreau H, Sigaux F, Judde JG, Rotger M, Telenti A, Lin YL, Pasero P, Maelfait J, Titsias M, Cohen DR, Henderson SJ, Ross M, Bentley D, Hillmen P, Pettitt A, Rehwinkel J, Knight SJ, Taylor JC, Crow YJ, Benkirane M, Schuh A..
SAMHD1 is mutated recurrently in chronic lymphocytic leukemia and is involved in response to DNA damage
(2014), BLOOD : 123(7):1021-31
Ducroux A, Benhenda S, Rivière L, Semmes OJ, Benkirane M, Neuveut C..
The Tudor Domain Protein Spindlin1 Is Involved in Intrinsic Antiviral Defense against Incoming Hepatitis B Virus and Herpes Simplex Virus Type 1
(2014), PLoS Pathog : 10(9):e1004343.
Laguette,N., Bregnard, C., Hue, P., Basbous, J., Yatim, A., Larroque, M., Kirchhoff, F., Constantinou, A., Bijan, S., Benkirane, M. .
Premature Activation of the SLX4 Complex by Vpr Promotes G2/M Arrest and Escape from Innate Immune Sensing
(2014), CELL : 156, 1-2, 134-145
Latreille D, Bluy L, Benkirane M, Kiernan RE..
Identification of histone 3 variant 2 interacting factors
(2014), Nucleic Acids Res. : 42,6, 3542-3550

 

All the publications of the group

Staff:


Name / First name Room Phone status

   Researcher

BENKIRANE Monsef 200 9996 DR (CNRS) E-mail    Photo  
BENNASSER Yamina 130-131 9988 CR (INSERM) E-mail    Photo  
LAGUETTE Nadine 132-133 9976 CR (CNRS) E-mail    Photo  

   Engineer & Administrative

MALIRAT Nathalie 132-133 9976 AI (CNRS) E-mail    Photo  

   Post-Doc

BREGNARD Christelle 132-133 9976 (CNRS) E-mail    Photo  
CRIBIER Alexandra 130-131 9988 (CNRS) E-mail    Photo  
DESCOURS Benjamin 130-131 9988 (CNRS) E-mail    Photo  
MORCHIKH Mehdi 132-133 9976 (CNRS) E-mail    Photo  
PETITJEAN Gael 130-131 9988 (CNRS) E-mail    Photo  
RAHM Nadia 130-131 9988 (Univ.Montp.1) E-mail    Photo  
STADELMAYER Bernd 132-133 9976 (CNRS) E-mail    Photo  CV in "ADUM"

   PhD students

LOUIS Tania 132-133 9976 (Univ.Montp.2) E-mail    Photo  

   Fixed-term contract

LATREILLE Daniel 132-133 9976 IR (CNRS) E-mail    Photo  CV in "ADUM"
RICHARD Chloé DR13 - SPV-119 83540 IE (CNRS) E-mail    Photo  

   Visitor

CHABLE-BESSIA Christine 130-131 9988 E-mail    Photo  

Laboratory of Molecular Virology. Group

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