Email: Jean-Luc.Darlix[at]ens-lyon.fr
Tel: +33 (0)4 72 72 81 69
Fax: +33 (0)4 72 72 81 37

Key words : Retroviridae, Flaviviridae, HIV NC, HCV Core, RNA chaperone, virus structure and replication.

RNA Chaperones in the living world

RNA chaperones are ubiquitous in all forms of life, from simple viruses such as HDV to sophisticated higher organisms like apes and the human kind. RNA chaperones bind a very broad range of nucleic acids and help RNA and DNA molecules adopt a stable functional conformation, likely to be unique in a DNA/RNA energy landscape. RNA chaperones are mostly unstructured proteins (defined as IUP for ‘intrinsically unstructured protein’), that are engaged in multiple interactions required to fulfill different key functions in DNA replication and maintenance, RNA splicing, trafficking and translation (Cristofari & Darlix ; review ; Tompa P, Csermely P. The role of structural disorder in the function of RNA and protein chaperones. FASEB J. 2004 Aug;18(11):1169-75. Review).
In retroviridae and flaviviridae, there is a unique abundant RNA chaperone called nucleocapsid (NC) and core protein, respectively. In the case of HIV-1, NC has multiple functions in virus structure, genome replication, genetic variability and virus assembly in infected cells (Darlix et al., 2007). Other essential RNA chaperones have recently been discovered in HIV, such as the Tat transcritional activator and the virus infectivity factor Vif.

The ongoing research projects focus on:

• the HCV core protein and its function in genomic RNA replication and virus dissemination in cell culture, and
• NC protein of the lentivirus HIV and gamma-retrovirus MLV and the identification of anti-NC molecules, that can efficiently inhibit their chaperoning activities in vitro, and virus replication in cells.

The Core protein of HCV and of other Flaviviridae

Context

To further study the properties and functions of viral RNA chaperones, we investigated the properties of the HCV Core protein (Figure 7) and of the core of other Flaviviridae, in collaboration with F Penin and JP Lavergne (CNRS, Lyon).
Our results clearly show that the HCV core as well as that from different Flaviviridae genera, namely GBV-B (GB virus B), WNV (West Nile virus) and BVDV (bovine viral diarrhoea virus) are very active RNA chaperones that can drive profound structural rearrangements of model nucleic acids and of viral RNA (Cristofari et al., Ivanyi-Nagy, et al ;, OPEN ACCESS ARTICLES PUBLISHED BY NAR). Furthermore, heat resistance of core proteins and far-UV circular dichroism spectroscopy indicated that a well-defined 3D protein structure is not a necessary prerequisite for the activities of these viral structural proteins. These data provide evidence that RNA chaperoning is possibly mediated by intrinsically disordered protein domains, and is highly conserved in Flaviviridae core proteins, despite sequence differences (Ivanyi-Nagy et al., 2008). Thus, these viral core proteins probably chaperone important RNA structural rearrangements taking place during virus replication, notably in the course of genomic RNA replication, translation and packaging.

Research projet : impact of Core mutations on HCV replication

This is presently being evaluated by targeted mutations and deletions in the basic domains of the core protein (Figure 7) within the HCV JFH infectious molecular clone.
To that end, HCV genomic RNA carrying specific mutations in the basic domains of core is transfected into the hepatocytic cell line Huh 7.5, and its replication monitored during one month, and assessed by means RT of the minus strand RNA and qPCR. Replication of 17 core mutated viruses is being monitored by virus passaging in Huh 7.5 cells, level of plus strand RNA replication and virus infectivity in cell culture.
The preliminary data indicate that the basic domains are required for HCV replication in Huh7.5 cells. More importantly specific basic residues appear to be critical determinants in HCV RNA replication ex vivo (see figure 8).

Retroviral chaperone proteins

Context

The retroviral NC is a multifunctional viral protein, involved in virus structure, replication, dissemination and variability (reviewed in Darlix et al., 2007). In fact, point mutations in the NC zinc fingers of HIV-1 (Figure 6 ; H23>C and H44>C) have a pleiotropic effect on virus structure, reverse transcription of the genomic RNA and infectivity.

Research project on HIV-1 NC : identification of molecules targeting NC

One of the original goal was to identify and characterize molecules targeting the RNA chaperoning properties of HIV-1 NC in order to inhibit its functions and therefore virus replication.

In collaboration with M Gottikh (Moscow, Russia), Gilles Divita (CNRS Montpellier) and Myriam Witvrouw (KU Leuven, Belgia), we recently identified such molecules, which block virus replication soon after cell infection by HIV-1 (Figure 5). These molecules exhibit an IC50 of about 0.3 nM and a CC50 of 8 microM. The ongoing investigations address the key questions on drug specificity with respect to other viruses and cellular chaperones, emergence of anti-NC resistance and toxicity in the animal.

Research project on MLV : specificity of NCp10 of MoMuLV

Christelle Daudé and Caroline Gabus
To address the question of NC specificity, we used a retroviral vector system whereby Gag of MoMuLV and the VSV-G envelope are provided in trans and a MLV-based vector encoding the eGFP marker serves to monitor virus replication.
Since the NC zinc finger (ZF) is unique in MLV’s and appears to fulfill all the functions of the two zinc fingers in lentiviruses, we swapped the ZF for that of an equivalent chaperoning domain of the yeast Ty3 retrotransposon, or from the yeast Ty1 retrotransposon, the drosophila Gypsy virus, or the HCV Core.
Preliminary data indicate that all ZF recombinants were non-infectious, even when the change concerned the closely related zinc finger of Ty3. The reasons for this are presently under investigation at the molecular level.

Research project on HIV-1 Tat : mechanism of mRNA Tat translation

Nicolas Charnay in collaboration with T. Ohlmann and M. Lopez Lastra
Tat is another basic protein with RNA chaperoning activities encoded by HIV-1 (Kuciack et al  NAR open access). Tat is essential for HIV-1 replication at the level of proviral DNA transcription by the host cell machinery. However, Tat molecules are not present in the incoming infectious virions (Chertova et al., JV 2006). This raises an important question on how is Tat synthesized by the ribosomes soon after proviral DNA integration, because viral transcription is set up at an extremely low level in the absence of the transcriptional activator Tat.
To address this question the ongoing research project uses in vitro and ex vivo molecular approaches. Present results show that the Tat mRNA can very actively recruit translating ribosomes and that translation operates is by an IRES-driven mechanim as for the genomic RNA of HIV-1 and gamma-retroviruses (Brasey et al., JV ; reviewed in Darlix et al. ; Balvay et al.).

Recent Selected Publications

• Kuciak M, Gabus C, Ivanyi-Nagy R, Semrad K, Storchak R, Chaloin O, Muller S, Mély Y, Darlix JL. (2008) The HIV-1 transcriptional activator Tat has potent nucleic acid chaperoning activities in vitro. Nucleic Acids Res. doi:10.1093/nar/gkn177. [Abstract] [Full Text] [PDF] [PubMed]
  
• Berkhout B, Gorelick R, Summers MF, Mély Y, Darlix JL. (2008) 6th international symposium on retroviral nucleocapsid. Retrovirology. 5:21. [Abstract] [Full Text] [PDF] [PubMed]
  
• Houzet L, Morichaud Z, Didierlaurent L, Muriaux D, Darlix JL, Mougel M. (2008) Nucleocapsid mutations turn HIV-1 into a DNA-containing virus. Nucleic Acids Res. 36(7):2311-9. [Abstract] [Full Text] [PDF] [PubMed]
• Ivanyi-Nagy R, Lavergne JP, Gabus C, Ficheux D, Darlix JL. (2008) RNA chaperoning and intrinsic disorder in the core proteins of Flaviviridae. Nucleic Acids Res. 36(3):712-25. [Abstract] [Full Text] [PDF] [PubMed]
  
• Grigorov B, Décimo D, Smagulova F, Péchoux C, Mougel M, Muriaux D, Darlix JL. (2007) Intracellular HIV-1 Gag localization is impaired by mutations in the nucleocapsid zinc fingers. Retrovirology. 4:54. [Abstract] [Full Text] [PDF] [PubMed]

PubMed List of all Darlix Publications

Reviews

• Darlix JL, Garrido JL, Morellet N, Mély Y, de Rocquigny H. (2007) Properties, functions, and drug targeting of the multifunctional nucleocapsid protein of the human immunodeficiency virus. Adv Pharmacol. 55:299-346. [Abstract] [PDF] [PubMed]
• Ivanyi-Nagy R, Darlix JL. (2007) Ancient Retrotransposons as possible Remnants of the Primitive RNP World. In "Retrotransposition, Diversity and the Brain". Series: Research and Perspectives in Neurosciences. Gage, Fred; Christen, Yves (Eds.) [PDF]
• Ivanyi-Nagy R, Davidovic L, Khandjian EW, Darlix JL. (2005) Disordered RNA chaperone proteins: from functions to disease. Cell Mol Life Sci. 62(13):1409-17. [Abstract] [PDF] [PubMed]
• Cristofari G, Darlix JL. (2002) The ubiquitous nature of RNA chaperone proteins. Prog Nucleic Acid Res. Mol. Biol. 72:223-68. [Abstract] [PDF] [PubMed]
• Darlix JL, Lastra ML, Mely Y, and Roques B (2002). Nucleocapsid Protein Chaperoning of Nucleic Acids at the Heart of HIV Structure, Assembly and cDNA Synthesis. pp. 69-88 in HIV Sequence Compendium 2002. Edited by: Kuiken C, Foley B, Freed E, Hahn B, Korber B, Marx PA, McCutchan F, Mellors, JW, and Wolinksy S. Published by: Theoretical Biology and Biophysics Group, Los Alamos National Laboratory, Los Alamos, NM. LA-UR 03-3564 [Full Text] [PDF]