Mechanism of Paramyxovirus Replication

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DESCRIPTION (provided by applicant): Our studies using mumps virus (MuV), a paramyxovirus, will unveil the mechanism by which the viral RdRp recognizes the nucleocapsid and gains access to the viral genomic RNA sequestered inside the nucleocapsid. It will also address how the "rule of six" is imposed by the nucleocapsid and how mRNA editing is regulated by interactions between P and N. Aim 1. The molecular mechanism for P functions. Our preliminary studies have shown that MuV P forms a tetramer with one pair of two parallel subunits, and another pair in the opposite orientation. This orientation that places the N-terminal and C-terminal regions on both ends of the MuV P tetramer is a novel structure moiety of P. Our data also showed that both N- and C-terminal regions are involved in binding specifically to the nucleocapsid, unlike P proteins of other NSVs that requires only the C- terminal region. In aim 1a, we will determine the crystal structure of the N-terminal domains and the C-terminal domains of MuV P. Crystal hits have been observed. In aim 1b, certain regions of P may be truncated and their effects on viral transcription and replication will be examined using a mini-genome system and a reverse genetics system. In aim 1c, specific mutational analysis based on the crystal structure of the N-terminal domain, the oligomerization domain and the C-terminal domain of MuV P will be carried out. For these mutants, interactions with the N proteins will be examined and their effects on viral transcription and replication will also be examined using a mini-genome system and a reverse genetics system. Alternative approaches include H/D exchange by mass spectrometry to map protein interactions. Aim 2. The molecular mechanism for N functions. We have previously prepared a nucleocapsid-like particle (NLP) that contains 13 N subunits and a piece of random RNA. This NLP corresponds to one turn of the helical nucleocapsid of MuV. MuV P and its nucleocapsid binding domains (both at N- and C- terminal regions) were shown to bind NLP. Proteolytic removal of the C-terminal region at residue 379 did not disrupt NLP or P binding. In aim 2a, the three dimensional structure of the NLP or its truncated version (N379) will be solved by X-ray crystallography. Crystals of NLP have been grown. In aim 2b, the location of P interactions with MuV NLP will be determined. We will solve the cryoEM structure of P or P fragments in complex with NLP or truncated NLP. When possible, P fragments may be cocrystallized with NLP or truncated NLP and the respective structure will be solved by X- ray crystallography. H/D exchange by mass spectrometry will be an alternative approach. In aim 2c, mutations will be generated to alter interactions between N and P, and their effects on NLP assembly and protein binding will be examined. Effects of mutations on viral transcription and replication will also be examined in a mini- genome system and a reverse genetics system.