Persistent infection by one of >15 oncogenic Human papillomavirus (HPV) types is a necessary cause of cervical cancer. Papillomavirus has only two capsid proteins;L1 and L2. Although L1 is known to form the capsid and mediates initial binding to cells, the functions of L2 remain to be determined. Whereas vaccination with L1 virus-like particles induces type-restricted immunity, we show that vaccination of animals with L2 provides broad immunity from viral challenge via neutralizing antibodies. Therefore, our long-term goal is the rational development of a single, inexpensive pan-oncogenic HPV preventive vaccine through dissection of mechanisms relating to L2 function in the infectious process and L2-dependent neutralization. We find that L2 is required for appropriate intracellular trafficking and binds to the cellular proteins syntaxin-18 and VAV-2. L2 neutralizing antibodies permit virus binding to cell monolayers, but their epitopes overlap the binding sites of interacting cellular proteins, suggesting: L2 antibody-dependent neutralization occurs by preventing interaction between L2 and key cellular proteins that mediate papillomavirus trafficking. To address this hypothesis we propose Specific aim 1: Characterize the effect of L2-specific neutralizing monoclonal antibodies upon the trafficking of virions and interaction with cellular proteins during infection, and Specific aim 2: Generate fine mutants within HPV L2 that prevent interaction with cellular proteins and determine their influence upon the particle to infectivity ratio of HPV pseudotype virions. Conflicting reports of L2 function in the literature likely reflect differences in the plethora of experimental models applied to study the molecular virology of papillomavirus. The organotypic raft model is the 'gold standard'that most closely replicates the entire HPV life cycle. Therefore we propose Specific aim 3: Validate the neutralizing activity of the L2 monoclonal antibodies and the phenotype of mutant HPV16 L2 in the context of the viral genome using the organotypic raft model of the complete viral life cycle. Finally we propose Specific Aim 4: Determine the three dimensional structure of HPV16 bound to neutralizing and non-neutralizing L2-specific monoclonal antibodies by cryo- electron microscopy and image reconstruction. These studies will identify conserved interactions between L2 and cellular proteins that are critical to infection and targeted by protective antibodies.
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