Elucidating Virus and Host Determinants of Nipah Virus Matrix Protein Trafficking and Budding

Nipah virus is a founding member of the newly described Henipavirus genus within the Paramyxoviridae family. The henipaviruses first came to light via spillover from their fruit bat reservoir, causing high mortality and instigating great concern. The cause of this high mortality, unusual among the paramyxoviruses, is unknown. Here, we investigate the molecular mechanisms underlying the functions of the Nipah virus matrix protein (NiV-M), which has the central role in virus assembly and budding, yet remains poorly understood. First, we developed new methods to modify the virus and the host to facilitate functional investigations. For the virus, we established and improved a reverse genetics system for Nipah virus to allow efficient rescue of replication-competent viruses from cDNA, which can be modified with mutations or insertion of useful reporter genes. For the host, we used the CRISPR/Cas gene-editing technology to knock-in a destabilization domain tag on all copies of an essential gene, thus allowing inducible knockdown of the tagged protein upon removal of the stabilizing compound from cell culture. We then investigated NiV-M function with two complementary approaches: mutagenesis of conserved residues and motifs in NiV-M, some of which mediated interactions with host factors, and comprehensive identification of cellular factors and pathways interacting with NiV-M. In doing so, we identified post-translational modifications, palmitoylation and ubiquitination, that regulate NiV-M function, and specifically found that ubiquitination of a conserved lysine within a nuclear localization sequence regulated nuclear-cytoplasmic trafficking of NiV-M. The conserved interaction of NiV-M and other paramyxovirus matrix proteins with nuclear import/export factors, vesicular trafficking pathways, and factors involved in actin cytoskeletal dynamics confirmed the importance of intracellular trafficking pathways for matrix function. Finally, we extended our study of matrix function beyond matrix itself by describing the Nipah virus C protein-mediated recruitment of the cellular ESCRT complex, which was required for efficient NiV-M-driven budding of Nipah virus