Negative stranded RNA viruses form a group that has a unique feature that the RNA genome is always encapsidated by a nucleoprotein. The assembly of the RNA genome with the nucleocapsid protein is required for recognition by the packaging proteins, such as the matrix protein and the nuclear export protein, and the virus specific RNA polymerase, which is also co-assembled with the nucleoprotein encapsidated RNA genome in the virion. We have developed a system to express the RNA-nucleoprotein complex of VSV in E.coli, and other important viral proteins. Using this expression system, we like to carry out the following experiments;
Aim 1. Structure of N-RNA complex of VSV. We have crystallized the N-RNA complex purified from our E.coli expression system. By combining methods of heavy atom isomorphous replacement, MAD, and density averaging, we will determine the structure of the N-RNA complex to 3.2 A resolution, with the aide of cryo-EM image construction. The 3D structure will provide a clear picture of N-RNA interactions.
Aim 2. Structure of P-N-RNA complex of VSV. In our initial attempts, we crystallized the complex of P-N-RNA purified from E.coli expression. We later found out that P was digested to a smaller fragment during crystallization. We anticipate that it was Pf that was crystallized with the N-RNA complex. A systematic screen will be conducted to crystallize the Pf-N-RNA complex and its structure will be determined. Cryo-EM will be used for determination of the lower resolution structure initially.
Aim 3. 3D structure of NS2.We have established a system to purify a large quantity of NS2 from E.coli expression. NS2 is a flexible protein that may have multiple domains linked by flexible peptides. In collaboration with Dr. XiaoLian Gao of University of Houston, we set up to solve the structure of NS2 by NMR. We will also express soluble fragments of NS2 for structural studies. The long term goal of this effort is to establish a model for RNP assembly of the negative strand RNA virus and to provide the molecular structural details of all the relevant proteins. With this system, we could look into the molecular assembly of this important class of negative strand RNA viruses.
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