The human papillomavirus (HPV) major structural protein L1 composes capsomers that are linked together through interactions mediated by the L1 C terminus to constitute a T=7 icosahedral capsid. most prevalent oncogenic genotype in HPV-associated anogenital and oral cancers. Here we use cryo-EM reconstruction techniques to solve the structures of the HPV16 capsid complexes using H16.U4 fragment of antibody (Fab). Different from most other antibodies directed against surface loops, H16.U4 monoclonal antibody is unique in targeting the C-terminal arm of the L1 protein. This monoclonal antibody (MAb) is used throughout the HPV research community in HPV serological and vaccine development and to define mechanisms of HPV uptake. The unique binding mode of H16.U4 defined here shows important conformation-dependent interactions within the HPV16 capsid. By targeting an important structural and conformational epitope, H16.U4 may identify subtle conformational changes in different maturation stages of the HPV capsid and provide a key probe to analyze the mechanisms of HPV uptake during the early stages of virus contamination. Our analyses precisely define AS703026 important conformational epitopes on HPV16 capsids that are key targets for successful HPV prophylactic vaccines. INTRODUCTION Human papillomavirus (HPV) infections continue to be a significant health burden in patient populations (1, 2). Although commercial vaccines targeting the viral capsid proteins have been applied successfully to protect against high-risk HPV, the efficacy of vaccines is usually genotype specific, and vaccines provide little therapeutic benefit against existing infections (3). Understanding the antigenic nature of the HPV capsid offers an opportunity to discover structural features that are crucial to capsid integrity and conserved across species. Panels of monoclonal antibodies and mutational analyses have helped to define several antigenic epitopes (4,C10); however, determining the conformational epitopes around the capsid surface requires AS703026 structural analyses, which can be accomplished by cryo-electron microscopy (cryo-EM) technology. Since the HPV life cycle depends on the differentiation of keratinocytes, it is difficult to purify high-titer virus stocks for structural studies. Vegfa Virus-like particles (VLPs) that are devoid of viral genome (11) have been used successfully for structural studies (8, 12, 13), whereas both pseudovirus (PsV) and quasivirus (QV), which contain expression plasmid DNA (14, 15), have been used for structural studies and infectivity assays (9, 10). For the work presented here, quasivirus has been used throughout. Papillomaviruses form a nonenveloped T=7 icosahedral capsid that is 55 to 60 nm in diameter and contains a circular double-stranded DNA (dsDNA) genome of 8 kb. The capsid is usually comprised of 360 copies of the AS703026 L1 major structural protein and an uncertain number of the L2 minor structural protein (15, 16). Five copies of the L1 protein intertwine to form each capsomer, and 72 capsomers interact to constitute a capsid. Twelve capsomers lie on an icosahedral five-fold vertex and are referred to as pentavalent capsomers, whereas the remaining 60 capsomers are each surrounded by six other capsomers and referred to as hexavalent capsomers. The C terminus of each L1 protein, called the C-terminal arm, extends along the capsid floor to interact with the neighboring capsomer before returning to the original donor capsomer (9, 17, 18). Intercapsomer disulfide bonds are formed between cysteine C428 and C175, which stabilize the icosahedral structure and play an important role in computer virus maturation (18, 19). The core of the capsomer is composed of the common viral structural motif, the antiparallel -strands BIDG and CHEF (20). Nearly all known conformational epitopes are located on one or more outwardly facing surface-exposed loops that connect the -strands (21). We recently reported a cryo-EM study of four different neutralizing monoclonal antibodies (MAbs) that interact with the human papillomavirus 16 (HPV16) capsid (10). Monoclonal antibodies H16.V5, H16.1A, H16.14J, and H263.A2 examined in the previous study all target conformational epitopes located on combinations of the.