Key words research : Retroviruses and HIV, Retroelements and Line, Replication and RNA chaperones, Nucleocapsid and new anti-HIV drugs.
Key words education : Master and PhD’s, post-doctoral positions ; training in molecular cloning, in monitoring HIV/HCV replication, RT-PCR, qPCR, cell culture, confocal microscopy, BSL3.

Funding research (past and present): CNRS, ENS, INSERM, ANRS, GIS Prions, Europe (FP 4-6), Région Rhône-Alpes ; Lyon I ; NIH (USA) ; Sidaction, FRM, ARC, Ligue ; Transgene, BioMérieux.
Funding education : ENS, French ministery of education, Europe (FP 4-6)

To make a long story short

From GENEVA, SWITZERLAND, TO TOULOUSE, FRANCE. The LABORETRO started in late 1985, when Caroline Gabus and Jluc Darlix (see pictures 1 & 2), moved from Geneva (Sciences II, Switzerland) to Toulouse (CNRS and University, France).
The goal was to found a laboratory dedicated to the study of the structure and replication of ubiquitous retroviruses such as MuLV (murine leukemia virus) and ASLV (avian sarcoma leukemia viruses), and of the recently discovered human AIDS virus, HIV-1. The other goal was to set up courses and a training program on Retroviruses. Based on the identification of the nucleocapsid protein (NC) as the specific partner of the genomic RNA in ASLV and MuLV virions, we wanted to study the role of NC in viral replication, firstly on an ALV clone isolated in Geneva and the Moloney MuLV, and secondly on HIV-1. Although resources were limited, we have been able to show that NC was an obligatory partner of the retroviral RNA in the course of virus assembly - specific binding to the 5’ UTR and genomic RNA dimerization – and during the reverse transcription process whereby NC chaperones the synthesis of the proviral DNA by the viral enzyme called reverse transcriptase (RT) (see Prats et al., EMBO J 1988 ; Barat et al., EMBO J 1989 ; Bieth et al., NAR 1990 ; Darlix et al., JMB 1990). At the same time, courses and a PhD training program on retroviruses have been set up.
In 1989, Philippe Lazar and Jean-Paul Lévy (INSERM and ANRS, Paris) proposed to JL Darlix to found a human virology department at ENS Lyon. This is why Caroline and JLUC moved in the middle of 1990 to the recently established ENS Sciences at Lyon Gerland.
The two original goals remained the same with an additional one on the characterization of molecules targeting the HIV-1 nucleocapsid, called NCp7.
Coming to Lyon was another uneasy start. But a combination of our energy and the enthusiasm of Guy Aubert and Jacques Daillie (ENS), of Philippe Lazar and Jean-Paul Lévy (Paris) and large funding resources effectively kick-started the LABORETRO at ENS.

The Human Virology Department at ENS-Lyon

One of MAJOR OBJECTIVE made clear by Philippe Lazar was to found a human virology Department at ENS, from a gravel parking to a fully equipped building dedicated, at least in part, to the study of human viruses at the structural, molecular and biological levels. This was reached in a time period of 3 years with the full energy of Caroline, Alain Sergeant, Marc Castellazzi and JL Darlix, for the human virology side. All of us moved to that building LR5 in October 1993 while the human virology Dpt was officially founded on the 1st of January 1994, as INSERM-ENS #412 with JL Darlix as director until December 2005.
The other goal was to install a high security laboratory called BSL3 in order to be able to study at the molecular and cellular levels the replication of human pathogenic viruses such as HIV, the human AIDS virus. This again was rendered possible by the full energy and enthusiasm of Caroline, Bernard Bigot and JLUC Darlix, and the financial supports of ANRS, Sidaction, ENS and BioMérieux.
The overall scientific context thus created favoured advanced research on human viruses and the pathogenic consequences of their replication. Indeed, this has led to the publication of several hundreds articles in high impact factor journals such as Nucleic acid research (NAR, a leader in RNA dicoveries), Journal of virology (leader in virology), Embo Journal, Nature, Science, Journal of Molecular Biology and more (for detailed informations consult the web sites ‘ncbi.nlm.nih.gov’ and ‘scopus.com’).

On the forefront of EDUCATION in virology and in the relationships between pathogenic viruses and their host, master students can attend and participate to the excellent courses and training program called ‘Virus & Immunity’, since the beginning of 2005.

LaboRetro at present

Firstly, our studies focused on HIV-1, the AIDS virus (Figure 4), on the murine leukemia virus MoMuLV and next on ancient endogenous retroelements of yeast cells such as Ty1 and Ty3, that could be at the origin of the ubiquitous retroviruses found in all Eukaryotes.
Out of more than 120 published articles, one major data is that Retroviruses and Retroelements encode a protein with RNA CHAPERONE PROPERTIES. This protein called NC, for nucleocapsid, is found in the inner part of the viral particle, where 1500 NC molecules coat the viral genome in HIV-1 (reviewed in Darlix et al., 1995 ; 2000 ; 2007). NC was found to be an obligatory partner of the genomic RNA throughout the replication cycle of the virus, because it chaperones formation of the virus in infected cells, and the conversion of the genomic into DNA by the viral RNA-dependent DNA polymerase, called reverse transcriptase (RT), soon after infection of target cells. NC is also involved in the genetic variability of the virus by actively promoting recombination reactions in the course of viral DNA synthesis by RT (reviewed in Darlix et al., 1995, 2000, 2007) (Figures 5-6).
Secondly, we investigated the mechanism of viral protein synthesis in cells infected by MoMuLV and HIV-1. One important result shows that the retroviral genomic RNA can recruit in a very active way cellular ribosomes, by an IRES driven mechanism, whereby translating ribosomes gain a direct access to the start AUG codon, or even a CUG codon in the murine virus MoMuLV (Berlioz et al. ; Lopez Lastra et al., Ohlmann et al.,reviewed in Balvay et al., ).
Thridly, a major achievement of LABORETRO was the development and use of safe high titer Retroviral vectors for the genetic modification of primary blood and nerve cells. Two classes of such vectors are available on request, MLV-VL30 retrovectors and SIV-based lentiviral vectors (LV) (Lopez lastra et al. ; Goujon et al.,).

With respect to education and the PhD training program in VIROLOGY, the following persons brilliantly defended their PhD diploma during the past 30 months : Cécile Herbreteau, Carole Bampi, Caroline Goujon, Boyan Grigorov, Roland Ivanyi-Nagy.

HIV, HCV and Retroelements : Research projects since 2006

At the present time, several lines of research are being pursued on HIV, HCV and Retroelements as listed below :

• Infection of primary human blood cells by HIV-1 : Andrea Cimarelli.
• HIV-1 assembly  in cell lines and primary cells : Delphine Muriaux.
• Interactions between HIV-1 and the Prion protein : Pascal Leblanc.
• Viral RNA chaperones: Jean-Luc Darlix and Roland Ivanyi-Nagy.
• New anti-HIV drugs targeting NC : Jean-Luc Darlix and Boyan Grigorov.
• Impact of Retrotransposons and Endogenous Retroviruses on the human genome : Gael Cristofari.
  

Figure legends

Figure 4. The HIV-1 viral particle.

LEFT: Electron-microscopy pictures representing the HIV-1 particle in a mature form with a cone-like core, bottom, and where the core has an immature circular form (top). Mean diameter is 125 nm. Note that the outer envelope appears to be thicker when the particle is immature.
RIGHT: Simple scheme illustrating the architecture of the mature HIV-1 particle. From the outside to the interior of the particle:

•  The constituents of the particle envelope are the surface (SUgp120; blue) and transmembrane (TMgp41, wight) glycosylated proteins, in the form of 7-9 trimersthat are anchored in a phospholipid bilayer (grey) similar to and coming from the cell membrane.
•  The matrix protein (MAp17) molecules (1500) (light grey)  are N-myristilated and anchored into the lipid bilayer.
•  The constituents of the  core structure are the Capsid protein (CAp24; 1500 molecules), the nucleocapsid protein (NCp7; 1500 molecules) in the interior where it entirely coats the genomic RNA (9730 nt) in a dimeric form (2), cellular tRNA molecules (100 mol with a major fraction represented by tRNALys,3), viral Vpr protein (100 molecules), and the viral enzymes (80-100 mol each), reverse transcriptase (RTp66-p51), integrase (INp32) and protease (PRp12). Minute amounts of the viral proteins Vif and Nef can also be found in viral particles produced by HIV-1 infected cells.

 Cellular proteins and enzymes can  be incorporated into the viral particle: for example APOBEC3G in the absence of Vif, UDG, Actin, several Tetraspanins etc...

Figure 5. HIV-1 REPLICATION: the major steps are illustrated here from 1 to 7.

•  (1,2) Infection is mediated by interactions between viral SU-TM and cellular CD4 and CCR5°CXCR4 membrane proteins, promoting entry of the viral core into the cell cytoplasm. Note that several modes of infection can take place: by cell-free viruses or directly by cell-to-cell transmission via the virological synapse.
• (3) Following uncoating probably corresponding to core-destabilization  with/without intervention of restriction factors such as TRIM 5a , viral DNA is synthesized by RT chaperoned by the NC molecules.
• (4) The complete viral DNA is in a poorly defined molecular complex, the pre-integration complex (PIC) within which it enters the nucleus via nucleo-pores.
• (5) Viral DNA is integrated into active regions of the host genome by the viral integrase, probably assisted by NC molecules, and by the cellular factor LEDGF/p75.
• (6) The proviral DNA is transcribed by the host cell machinery, RNA polII and factors, which also needs the trans-activator Tat. The genomic RNA undergoes a large number of splicing reactions to generate the viral mRNA of genomic length, of 4 kb and 1.8 kb. The genome and the 4 kb m RNAs need the viral REV to be exported from the nucleus. The genomic RNA and viral 4 kb and 1.8 kb mRNAs are efficiently translated in the cytoplasm probably by an original IRES mechanism. Gag-NC and NC may well regulate the translation process of the genomic RNA, via a negative feed-back at the early stage  of virus assembly.
• (7) Virus assembly is driven by Gag and requires a number ofcoordinated  interactions in  infected cells;

a- Gag-NC/genomic RNA and cellular membrane/MA-Gag that constitute the two necessary assembling platforms.

b- Gag and Gag-Pol, and cellular tRNAs.

c- Gag-Vpr.

d- Gag with cellular proteins ALIX and TSG101 for Gag trafficking and virus budding.

e- Gag with the viral Envelope constituents and cellular Tip47 to end up with complete infectious viruses produced by an infected cell. Note that reverse transcription is negatively controlled by NC at that stage.

Highly Active Anti-Retroviral Therapies (HAART) target RT (stage 3) and PR during assembly (stage 7). New anti-HIV-1 drugs targeting IN will soon be used in combination with anti-RT and/or anti-PR drugs (detailed at http://www.aidsmeds.com/list.shtml).

Figure 6. Structure and functions of HIV-1 NC protein.

Top. Representation of the HIV-1 polyprotein precursor Pr55 gag with a molecular weight of 55 kDa.
Middle. During virus formation Gag and Gag-Pol molecules assemble to form new viral particles. In addition, this causes protease activation and leads to Gag-Pol and Gag processing via the cleavage of specific sites in Pol and in Gag (shown here). Consequently, mature structural Gag proteins and Pol enzymes are found in newly formed infectious viruses. The sequence of the large form of NCp7 is shown. Note that the two zinc fingers, ZF1 and ZF2, and  flanking basic residues are highly conserved in all HIV-1 strains known to date.
Bottom. Illustration of the 3D structure of HIV-1 NCp7, kindly provided by Dr. Nelly Morellet (Paris). The N- and C-terminal domains are mostly unstructured (yellow) while the central domain corresponding to ZF1, ZF2 (both in blue) and the basic linker (in red) fold into a globular structure. This small central globular domain proved to be essential in Gag assembly, genomic RNA dimerization and packaging, virion structure and genomic RNA replication by RT. In addition, this domain was found to negatively control viral cDNA synthesis during assembly. Note that the F and W residues are essential determinants for the selective packaging of the genomic RNA.
New anti-NC drugs target the central globular domain, shown here in blue and red