Cervical cancer associated with infection by human papillomavirus (HPV) is the second leading cause of cancer deaths among women worldwide, resulting in one in six deaths. Understanding the assembly of HPV and the immune response to it offers multiple opportunities for pharmacologic and immunologic intervention to prevent disease or alter its course. Effective design of these interventions would be aided by knowledge of the structural basis of these phenomena.. Investigations to elucidate the structure of the HPV capsid and its antigenic epitopes are proposed. Because HPV does not readily replicate in cultured cells, it has been extremely difficult to study by the traditional techniques of virology and virus crystallography. To circumvent these limitations, a strategy that combines EM image reconstruction of HPV particles and HPV: antibody complexes with crystal structures of individual protein components is currently being executed. Information from image reconstruction will be combined with structures from crystallography by pseudo-atomic modeling. as applied to other system by the PPG group. With co PI Baker, we have completed a high resolution EM image reconstruction of HPV6b, and determined that it is quite reliable to at least 16 angstroms. Extension to higher accuracy and resolution and similar studies on HPV: antibody complexes will provide the data required for part of our strategy. For the other part, we have expressed both major and minor capsid proteins of HPV6b in a soluble form suitable for crystallization trials. While assembly of HPV is completely """"""""self-directed""""""""; assembly of many other biological structures occurs only with the aid of chaperone molecules. One system that employs chaperones is the assembly of the side tail fibers of phage lambda. Side tail fibers are very long, thin, jointed fibers found on wild-type lambda. These long fibers, trimmers of the Stf gene product, are analogous to similar fibers in other phages, and are formed only with the aim of the pTfa chaperone. The fibers increase efficiency of phage absorption to cells, presumably engaging a second receptor. Some studies suggest that, unlike any other chaperone, pTfa can act """"""""instructively"""""""" to alter the structure of the assembled fiber. To investigate this unusual behavior, pTfa has been the object of several attempts to crystallize the intact molecule. While all of these have failed to yield crystals suitable for x-ray diffraction, we have very recently grown crystals of a tryptic fragment of pTfa that diffract to beyond 2A. Determination of this structure and the structure of complexes of pTfa with suitable fragments of the very long, flexible Stf protein is proposed.
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