Structure and functions of Nipah virus C protein – NiPah-C

The project uses a combination of complementary approaches in proteomics, protein biochemistry, biophysics, structural biology, and molecular and cellular virology using state-of-the-art facilities at each partner's site to:
(1) Map the NiV C interactome using TAP-MS.
(1) Mapping the NiV C interactome by TAP-MS. (2) Reconstructing and characterizing complexes with host proteins.
(3) To understand the interdependence in the expression of superimposed proteins and the expression of a truncated C' protein.
(4) To understand how viral proteins created de novo by overprinting have unusual sequence properties and are often implicated in viral pathogenicity.

(1) The crystal structure of the C-terminal domain of the C protein from a homologuous virus has been solved and refined.
(2) Recombinant Nipah viruses lacking expression of C or expressing the C protein from an independent cassette have been generated

- Structural characterization of full-length TPMV C protein by SAXS.

- Comparison of the infections of cell-cultures by w.t. NiV, NiV?C and NiV TMPV-C to identify the roles of C protein and determine if TPMV C can play the same roles in the context of a NiV lacking C expression.

- Mapping of the interactome by tap-tag/mass spectrometry using full-length TPMV C as a bait and comparison with the interactome of NiV C.

- Partner 3 will validate the interactome data by repeating the tap-tag sélection using another set of tags and together with partner 1, will characterize the interactome of NiV C in NiV infected cells.

- The cloning full-length NiV C in other vectors has started for future expression in insect and mammalian cells.

- Reconstitution of complexes by mixing NiV C C-terminal moiety with the purified partners and biophysical characterization

- Optimization of the conditions for minigenome expression experiments

- On the basis of our current knowledge and of the analysis of Tupaia virus C protein structure, mutations in NiV C proteins have been designed to perturb interactions with different partners and these mutations will be tested with the minigenome system

- Growth characteristics of the viruses lacking expression of C or C' or expressing C or C' from the independent cassette are expected to be studied as soon as the BSL4 lab will be opened for operation

STRUCTURAL DESCRIPTION OF THE NIPAH VIRUS PHOSPHOPROTEIN AND ITS INTERACTION WITH STAT1. Jensen MR, Yabukarski F, Communie G, Condamine E, Mas C, Volchkova V, Tarbouriech N, Bourhis J-M, Volchkov V, Blackledge M & Jamin M. doi.org/10.1016/j.bpj.2020.04.010

The objective of the Nipah-C project is to decipher the roles of the nonstructural accessory C protein (NiV C) in Nipah virus replication and pathogenicity by structurally and functionally characterizing the interactions with different viral and host partners, by disrupting specifically some of these interactions and evaluating the effects on viral replication and pathogenicity and by investigating how a protein can be created de novo by overprinting on an existing gene, building on some of our previous works and on promising preliminary results.

NiV is a nonsegmented negative-sense RNA virus of the family Paramyxoviridae, which express three nonstructural proteins (V, W and C) from the overlapping P and C genes. These proteins are virulence factors involved in antagonizing the host defenses and in hijacking cellular machineries. Partner 2 has demonstrated that recombinant NiV deficient in expression of full-length C protein can replicate in cellula but is attenuated in vivo, and recently, new insights have been gained about some biological functions of NiV C, but the underlying molecular mechanisms and their importance for virulence and pathogenicity remain largely unknown. We are today in an optimal situation for addressing the issues.The barriers to overcome are (1) the identification of the viral and host factors that directly interact with NiV C, (2) the reconstitution of protein complexes involving NiV C and an partner and the determination of their structure and (3) the possibility to delete or mutate the NiV C protein without perturbing the expression or integrity of the cognate P, V and W proteins that are expressed from the overlapping gene.

To meet the objectives, the partners will bring complementary expertises and use state-of-the-art techniques in proteomics, molecular biophysics, structural biology and molecular and cellular virology including in the BSL-4 Merieux laboratory in Lyon. Specifically, we will (1) map the interactome of NiV C, (2) reconstitute several protein complexes from purified components and characterize their properties and structure, (3) generate recombinant Nipah viruses that either express no C protein or express C from an independent transcription cassette, evaluate their fitness for viral replication and by mutagenesis evaluate the role of the different protein-protein interactions, and (4) select artificial proteins that specifically bind to NiV C to perturb the function of NiV C in the context of cells infected with wild type NiV expressing its nonstructural proteins from natural P and C genes. Building on our earlier work and on different preliminary results, notably (1) an initial mapping of the NiV C interactome, (2) the discovery using new anti-C antibodies that a truncated form of C is also expressed in NiV infected cells, (3) the availability of new P gene constructs from which no C or no aberrant forms of P can be produced and (4) the successful production and purification of different forms of NiV C as well as the reconstitution of two protein complexes, we are very optimistic about the success of the project.

Nipah virus (NiV) is one of the most pathogenic and deadliest zoonotic viruses infecting humans and at risk for pandemics. NiV causes severe encephalitis and respiratory disease with case fatality rates that can exceed 70%, but the underlying molecular mechanisms of its pathogenicity are unknown. No vaccine or specific treatment is currently approved for human therapy while the person-to-person transmission observed in recent NiV outbreaks raises the specter of a new threat for human health and economy. We thus need a better understanding of the molecular mechanisms of NiV pathogenicity.