Paramyxoviruses include many important human and animal pathogens. Our studies using mumps virus (MuV) will unveil the mechanism by which the viral RdRp recognizes the nucleocapsid and gains access to the viral genomic RNA sequestered inside the nucleocapsid.
Aim 1. The molecular mechanism for P functions. The P protein is essential for viral RNA synthesis and is a multi-domain protein. Our preliminary studies have shown that MuV P forms a tetramer with a pair of two parallel subunits, and another pair in the opposite orientation. Our data also showed that both N- and C-terminal regions are involved in binding specifically to the nucleocapsid, unlike P proteins of other negative strand RNA viruses (NSV) that requires only the C-terminal region.
In aim 1 a, we will determine the crystal structure of the N-terminal domains and the C-terminal domains of MuV P.
In aim 1 b, interactions of the mutant MuV P with the nucleocapsid, monomeric N protein, or the L protein, will be examined, and their effects on viral transcription and replication will be examined using a minigenome system and a reverse genetics system.
In aim 1 c, specific mutations 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.
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. 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 2 a, the three dimensional structure of the NLP or its truncated version (N379) will solved by X-ray crystallography. Crystals of NLP have been grown. How the nucleocapsid is assembled and what features may be involved in interactions with other viral proteins may be derived from the structure. How the viral RNA is encapsidated will also be revealed.
In aim 2 b, 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.
In aim 2 c, mutations will be generated based on the structure predictions, and their effects on NLP assembly and interactions with other viral proteins will be examined. Effects of mutations on viral transcriptio and replication will also be examined in a minigenome system and a reverse genetics system.
Paramyxoviruses are important human pathogens. The mechanism of their replications are different from the rest of viruses, such as the rule of six that stipulates the genome sequence to be read in blocks of six nucleotides. The experiments in this proposal are designed to unveil the unique mechanism of paramyxovirus replication.
