Structural, mechanistic and inhibitor design studies of the Bunya- and Arenavirus L-proteins – ArenaBunya-L

We plan to apply a battery of state-of the-art structural biology techniques, to derive structural information (notably by X-ray crystallography and electron microscopy) on arena- and bunyavirus L-protein at the level of functional domains as well as that of the full-length L-protein and its complexes with vRNA. Mutagenesis based on structural results will be used to drive in vitro biochemical and in cell viral replication studies to test functional hypotheses.

Preliminary results on domain identification and expression, purification and EM of full-length bunyavirus L-protein suggest that this system may prove more tractable than influenza virus polymerase.

The long-term, ambitious goal of this project is to derive a structure-based, mechanistic model to explain how segmented, negative-strand RNA viral RdRPs, in the context of complexes with nucleocapsid (N) and vRNA, function in both transcription by cap-snatching (giving rise to translation competent viral mRNAs) and replication (giving rise to full-length copies of the genome) and how the switch between the two modes of operation is mechanistically achieved and regulated. Structural information on the cap-snatching endonuclease of arena and bunyaviruses, and possibly on other functional domains, will be used to optimize inhibitors as a first step towards antiviral drug design,

New structural results on the Lassa virus endonuclease and the nucleoprotein of LACV are very promising. We have succeded in solving the crystal structure of LACV nucleoprotein tetramer in complex with ssRNA. This has enabled us to propose a model for the viral ribonucleoprotein (RNP) complex compatible with electron micrsoscoe images of RNPs purified from virions. Publication: Structural basis for encapsidation of genomic RNA by La Crosse Orthobunyavirus nucleoprotein. Reguera J, Malet H, Weber F, Cusack S. Proc Natl Acad Sci U S A. 2013.

Arenaviridae, Bunyaviridae and Orthomyxoviridae are the principal families of segmented negative strand single-stranded RNA viruses and include many serious and/or emerging human pathogens (e.g. respectively Lassa Fever, Crimean-Congo Hemorrhagic Fever and Influenza viruses). For arena- and bunyaviruses, transcription and replication of the viral genome segments is performed by the single chain viral RNA-dependent RNA polymerase or L-protein, which is functionally similar to the hetero-trimeric influenza polymerase complex. Two distinctive features of all these polymerases is that they bind to the conserved, quasi-complementary, non-translated, 3’ and 5’ ends of each vRNA segment and that they perform transcription by ‘cap-snatching’, unlike the polymerases of non-segmented negative strand RNA viruses which possess 5’ capping activity. Despite years of study, particularly on influenza polymerase, a detailed understanding of the mechanisms of transcription and replication is lacking, largely due to the absence of high resolution structural information. In recent years, several structures have been solved of functional domains of influenza polymerase whereas the L-proteins of arena- and bunyaviruses are almost ‘virgin territory’, apart from the cap-snatching endonuclease which has recently been identified by the partners of this proposal to be at the extreme N-terminus of L and is very similar to that of influenza polymerase. This discovery has reinforced the hypothesis that the polymerase of all segmented, negative strand, single-stranded RNA viruses probably have a similar architecture. To explore this hypothesis further we plan to apply a battery of state-of the-art structural biology techniques, to derive structural information (notably by X-ray and electron microscopy) on arena- and bunyavirus L-protein at the level of functional domains as well as that of the full-length L-protein and its complexes with vRNA. Mutagenesis based on the structural results will be used to drive in vitro biochemical and in cell viral replication studies to test functional hypotheses. The long-term, ambitious goal of this project is to derive a structure-based, mechanistic model to explain how segmented, negative-strand RNA viral RdRps, in the context of complexes with nucleocapsid (N) and vRNA, function in both transcription by cap-snatching (giving rise to translation competent viral mRNAs) and replication (giving rise to full-length copies of the genome) and how the switch between the two modes of operation is mechanistically achieved and regulated. Preliminary results on domain identification and expression, purification and EM of full-length bunyavirus L-protein suggest that this system may prove more tractable than influenza virus polymerase. Furthermore it is part of the strategy of the project that each partner focuses on one of the two evolutionary diverged, but related, Arena- or Bunyavirus systems, thus increasing the chance of success as well as giving the opportunity of cross-fertilisation from one system to the other. Finally, the structural information already available on the cap-snatching endonuclease of arena and bunyaviruses, and possibly on other functional domains, will be used to optimize inhibitors as a first step towards antiviral drug design, using as a starting point the several families of inhibitors that are known to target the similar influenza virus endonuclease. In this respect it is important to remember that Arena- and Bunyavirus are largely rodent, bat or insect borne and in a world of environmental change, newly emerging threats from these families of viruses might be expected.