PRELIMARY STUDIES OF THE N AND P PROTEIN COMPLEX FROM VESICULAR STOMATITIS VIRUS. Todd J. Green, Ming Luo, Department of Microbiology, Center for Macromolecular Crystallography,University of Alabama at Birmingham, Birmingham, AL USA

Purification and preliminary crystallization trials of the N and P protein complex from Vesicular Stomatitis Virus (VSV) are being done. VSV is a member of Rhabdoviridae family of viruses which are characterized by their rod-like shaped morphology. VSV is responsible for causing infections in a variety of hosts ranging from cattle to humans and as a result is a virus that has undergone much research. It has 5 genes that are encoded by the positive complement to its negative-sense RNA genome. The VSV ribonucleoprotein complex is composed of the genomic RNA in association with the N, P, and L proteins and constitutes the infectious core of the virus. N is the nucleoprotein that enwraps the 11 kilobase genomic RNA. P is a phosphoprotein and L is believed to be a part of the RNA-dependent RNA polymerase. Both are needed, along with the nucleocapsid template, for VSV transcriptase activity.

We are coexpressing the N and P proteins in an Escherichia coli expression system from a single plasmid. Coexpression aids in their abilty to exist in a soluble form. N is expressed with a poly-His fusion tag; while, P is expressed as the native protein. Purification is done over a Ni column and because of the association of P with N both proteins co-purify in a single step. An addition size exclusion column is used to obtain a high purity product. This purified complex is the focus of preliminary crystallization and crystallographic studies. We have obtained crystals and are optimizing these conditions.