Encapsidation of RNA by VSV N Protein In Vitro

Abstract

Sequences at the 5' end of the nascent RNA are known to be important as signals for encapsidation of the genome of vesicular stomatitis virus. In order to define the specific sequences involved in this process and to develop an in vitro encapsidation system, in vitro transcription from SP64-based plasmids was used to synthesize RNA molecules corresponding to various portions of the viral 5' plus strand sequence. Some of these RNAs were tested for their ability to bind the capsid N protein in vitro. N protein in this assay was provided either from VSV mRNA programmed reticulocyte lysates or from infected cell extracts or, in collaboration with Dr. Sue Moyer (Gainesville, Florida), purified from viral nucleocapsids. This thesis describes the construction of the SP64-based plasmids and the use of their RNA transcription products in the experiments described above. I also constructed a series of plasmids that could direct the synthesis of RNA molecules which have many of the features of VSV defective particle genomes. Two of the constructs generate a defective-like RNA carrying a reporter gene capable of expressing the bacterial lac Z protein. These RNAs have the potential, after in vitro encapsidation and transfection into mammalian cells, of producing readily detectable helper-dependent virions.