We have made tremendous progress on understanding the RNA encapsidation by the nucleocapsid, access to the RNA sequence inside the nucleocapsid, and the assembly of the bullet-shaped full virion of vesicular stomatitis virus (VSV). The focus for the next period is as following: Aim 1. Mechanism of initiation of transcription and replication. We have developed methodologies to solve the structure of specific RNA sequences inside the nucleocapsid-like partilc (NLP), as well as NLPs formed by mutant N proteins. We plan to solve a series of novel structures to address the question how the N protein helps RdRp to recognize the specific RNA sequence sequestered in the nucleocapsid. Interactions of the N protein with P or the large polymerase protein, L, will also be studied in order to define their effects on RNA sequence recognition. Aim 2. Structure and function of the L protein. We will combine X-ray crystallography, cryoEM/electron tomography, low-angle X-ray scattering and all available biological methods to study the large protein L of the viral polymerase and its complexes with other proteins. Crystals of fragments of the VSV L protein have been grown. Complexes of L-N-P proteins have been purified. The structure of the L and P proteins bound to the nucleocapsid will also be determined by electron microscopy. The ultimate goal is to show how the L protein carries out transcription and replication at separate sites, perhaps by different mechanisms. Aim 3. Assembly of VSV virion. We will work on the structure of the glycoprotein on VSV particle by electron tomography. We will also locate the L and P protein inside the virus particle. By use of all structural information, we will be able to build a complete pseudoatomic model of VSV virion and further delineate the mechanism of VSV assembly at budding. Aim 4. Structural studies of mumps virus (MuV) NLP and interactions between the N and P proteins. There are substantial mechanistic differences in RNA synthesis of paramyxoviruses, including the [unreadable]rule of six[unreadable] and polymerase stuttering during transcription, when compared to rhabdoviruses. The nucleocapsid of paramyxoviruses is likely to contain unique structural features that dictate these mechanisms. Parallel to our studies of VSV proteins, we like to solve the 3D structure of the MuV NLP by X-ray crystallography or cryoEM. Structures of P fragments and their complexes with the N protein will also be solved. Crystals of P fragments have been grown. By comparing the differences of the two classes of viruses, we can gain insight on the mechanisms of RNA transcription and replication of both.