Both epidemiologic and molecular biological studies indicate that certai human papillomavirus types, particularly HPV 16 and HPV 18 are involved in the etiology of squamous cell carcinomas of the cervix. Prevention of HPV infection with vaccines, or the use of vaccines or antiviral agents to modulate infection and tumor development is an extremely important goal. The recent development in our laboratory of a recombinant vaccinia virus expression system that allows the assembly of HPV proteins into capsids provides an opportunity to study capsid structure and assembly. This information could be used to design peptides or antibodies that interfere with the formation of infectious virions. The study will examine three HPV types: 1,6 and 16 to allow an assessment of the features that are shared among papillomaviruses and those that are unique to a particular type. The study will combine biochemical, genetic, microscopic and structural approaches to characterize HPV capsids.
The specific aims of this proposal are: (1) To characterize the structure of the L1 capsomers and capsid. Molecular modeling will be used to determine if there is structural similarity between the L1 proteins of papillomaviruses and the VP1 proteins of polyomaviruses. Image analysis of cryoelectron micrographs and mass measurements of scanning transmission electron micrographs will be used to study the L1 capsomers and the capsid at the 3.0 nm level and to attempt to locate L2. Attempts will be made to crystallize the L1 capsomer. (2) To characterize the domains of the capsid proteins that are required for self-assembly including the intracapsomer among the fiv L1 - containing subunits, the intercapsomer contacts among the L1 proteins in the capsid shell, and interactions between L1 and L2. The ability of L1 to form intra- and intercapsomer contacts will be assessed using wild type and mutated capsid proteins by coimmunoprecipitation, fractionation analysis, electron microscopy, and the two hybrid system of Fields and Song. The two hybrid system will also be used to identify contacts between L1 and L2. (3) To characterize post-translational modifications of the L1 and L2 proteins. Post-translational modifications will be determined by metabolic labeling. 2D gel analysis and mutagenesis. Modifications will be compared among the three HPV types in vaccinia and baculovirus expression systems to look for consistent changes. The ability of L2 to effect posttranslational modification of L1 will be examined. (4) To characterize the requirements for encapsidation of viral DNA. Viral DNA will be replicated to high copy number transiently by the expression of the E1 and E2 genes, or will exist as high copy episomal DNA in cell lines, and will be packaged using the recombinant vaccinia viruses to provide the capsid proteins. The requirement for the L2 protein will be determined.
