Genetic Analysis of Papillomavirus Virion Morphogenesis

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DESCRIPTION (provided by applicant): Capsid proteins of papillomaviruses (PV) have evolved functional domains for the proper interaction between themselves, with PV viral nonstructural proteins, with PV genomic DNA, and with host cell factors, allowing for morphogenesis of infectious viral particles. Analyses performed using virus-like particles (VLPs) and pseudoviruses as an endpoint for viral morphogenesis may or may not equate to the authentic virus propagated in differentiating host tissue. Our organotypic virus culture system, capable of supporting the complete viral life cycle, coupled with our expertise in using MAbs to study capsid conformation, place us in the unique position to initiate the first studies to directly compare VLP self-assembly with authentic PV synthesis. These studies will directly investigate a functional role for L2 protein in authentic virus morphogenesis and include investigating the role of L2 in the process of VLP self-assembly. Our studies will focus on 2 main genetic approaches. One will use a chimeric genetic approach allowing investigations to begin by looking for type-specific differences between large domains of the interacting proteins of 2 viral types. The other is to specifically mutate domains that have been alluded to by other laboratories as having a function related to virion morphogenesis. To accomplish our research goals we have developed four specific aims: (1) Investigate papillomavirus type-specific structural gene interactions; (2) Compare the genetic requirements for the assembly of virus-like particles (VLPs) to authentic virion morphogenesis; (3) Genetically analyze the role of capsid protein cysteine residues in virion morphogenesis; and (4) Investigate the role of the N-terminus of the L1 capsid protein in virion morphogenesis by mutational analyses. When completed the studies proposed will for the first time provide new insight into the molecular mechanisms of virion morphogenesis in a system capable of reproducing the natural complex processes of PV morphogenesis and infection in a differentiating environment.