Human papillomavirus (HPV) E6 oncoprotein targets certain tumor suppressors such as MAGI-1 and SAP97/ hDlg for degradation. A short peptide at the C terminus of E6 interacts specifically with the PDZ domains of these tumor suppressors, which is a property unique to high-risk HPVs that are associated with cervical cancer. The detailed recognition mechanisms between HPV E6 and PDZ proteins are unclear. To understand the specific binding of cellular PDZ substrates by HPV E6, we have solved the crystal structures of the complexes containing a peptide from HPV18 E6 bound to three PDZ domains from MAGI-1 and SAP97/Dlg. The complex crystal structures reveal novel features of PDZ peptide recognition that explain why high-risk HPV E6 can specifically target these cellular tumor suppressors for destruction. Moreover, a new peptide-binding loop on these PDZs is identified as interacting with the E6 peptide. Furthermore, we have identified an arginine residue, unique to high-risk HPV E6 but outside the canonical core PDZ recognition motif, that plays an important role in the binding of the PDZs of both MAGI-I and SAP97/Dlg, the mutation of which abolishes E6's ability to degrade the two proteins. Finally, we have identified a dimer form of MAGI-1 PDZ domain 1 in the cocrystal structure with E6 peptide, which may have functional relevance for MAGI-1 activity. In addition to its novel insights into the biochemistry of PDZ interactions, this study is important for understanding HPV-induced oncogenesis; this could provide a basis for developing antiviral and anticancer compounds.Human papillomaviruses (HPVs) are small double-stranded DNA tumor viruses that induce hyperproliferative lesions in epithelial tissues (28). A subset of HPV types that act as the etiological agent of cervical cancers are called "high-risk" types; these include HPV16, HPV18, HPV31, and HPV45 among others (26,27,29,45). The oncogenic potential of the high-risk types is dependent on the cooperative action of the two early viral gene products, E6 and E7, which bind and alter the activity of cell cycle regulatory proteins.The targeting of p53 for degradation by E6 has extensively been studied, and this is an important feature for the oncogenic activity of the high-risk papillomaviruses that cause cancer (19,36,37). However, a large amount of evidence suggests the existence of p53-independent functions of E6 that are also necessary and important for transformation. Support for this idea comes from the observation that the transformation of cells or the induction of epithelial hyperproliferation in transgenic animals by E6 does not always correlate with its ability to degrade p53 (22,33).Numerous cellular proteins have been identified as targets of HPV E6 in cell transformation. These proteins are involved in a variety of cellular processes, such as calcium signaling (4), cell adhesion (41), transcriptional control (5), DNA synthesis (25), apoptosis (8), cell cycle control (10), DNA repair (20), and small G-protein signaling (11). One group of these targets of...

dHuman papillomavirus (HPV) entry is accompanied by multiple receptor-induced conformational changes (CCs) affecting both the major and minor capsid proteins, L1 and L2. Interaction of heparan sulfate (HS) with L1 is essential for successful HPV16 entry. Recently, cocrystallization of HPV16 with heparin revealed four distinct binding sites. Here we characterize mutant HPV16 to delineate the role of engagement with HS binding sites during infectious internalization. Site 1 (Lys278, Lys361), which mediates primary binding, is sufficient to trigger an L2 CC, exposing the amino terminus. Site 2 (Lys54, Lys356) and site 3 (Asn57, Lys59, Lys442, Lys443) are engaged following primary attachment and are required for infectious entry. Site 2 mutant particles are efficiently internalized but fail to undergo an L1 CC on the cell surface and subsequent uncoating in the endocytic compartment. After initial attachment to the cell, site 3 mutants undergo L1 and L2 CCs and then accumulate on the extracellular matrix (ECM). We conclude that the induction of CCs following site 1 and site 2 interactions results in reduced affinity for the primary HS binding site(s) on the cell surface, which allows engagement with site 3. Taken together, our findings suggest that HS binding site engagement induces CCs that prepare the virus for downstream events, such as the exposure of secondary binding sites, CCs, transfer to the uptake receptor, and uncoating.H uman papillomaviruses (HPVs) are small, nonenveloped epitheliotropic DNA viruses. HPV infection usually induces benign papillomas of the skin and mucosa. However, certain species, especially HPV16, are known as "high risk" due to their involvement in the progression to invasive carcinomas. Infection by HPV is considered necessary, though not sufficient, for the development of cervical cancer (1, 2). HPV infection is also associated with various anogenital and head and neck cancers (3). Despite the clear medical importance of preventing HPV-induced lesions, limited molecular detail regarding the attachment and entry of the virus is available. HPVs productively infect only epithelial cells in the skin and mucosa and depend on the differentiation of these cells for the completion of the viral life cycle (4). To bypass this obstacle, an in vitro surrogate system for viral propagation using a marker gene encapsidated into the viral capsid proteins was developed (5-7). This pseudovirus system overcomes the tropism and species specificity for viral propagation displayed by HPVs, allowing for study of the early events in the infection process.The papillomavirus virion is composed of the major and minor capsid proteins, L1 and L2, respectively. L1 is present in 360 copies organized into 72 pentamers, referred to as capsomeres (8-10). The L2 protein is present in an undetermined number of copies and is initially hidden inside the L1 structure prior to attachment to the cell surface (11). The outer virion shell, formed via pentavalent and hexavalent capsomere interactions between L1 molecules, medi...

A gain-of-function R620W polymorphism in the PTPN22 gene, encoding the lymphoid tyrosine phosphatase LYP, has recently emerged as an important risk factor for human autoimmunity. Here we report that another missense substitution (R263Q) within the catalytic domain of LYP leads to reduced phosphatase activity. High-resolution structural analysis revealed the molecular basis for this loss of function. Furthermore, the Q263 variant conferred protection against human systemic lupus erythematosus, reinforcing the proposal that inhibition of LYP activity could be beneficial in human autoimmunity.

High risk human papillomavirus types 16 (HPV16) and 18 (HPV18) can cause cervical cancer. Efficient infection by HPV16 and HPV18 pseudovirions requires interactions of particles with cell-surface receptor heparan sulfate oligosaccharide. To understand the virus-receptor interactions for HPV infection, we determined the crystal structures of HPV16 and HPV18 capsids bound to the oligosaccharide receptor fragment using oligomeric heparin. The HPV-heparin structures revealed multiple binding sites for the highly negatively charged oligosaccharide fragment on the capsid surface, which is different from previously reported virus-receptor interactions in which a single type of binding pocket is present for a particular receptor. We performed structure-guided mutagenesis to generate mutant viruses, and cell binding and infectivity assays demonstrated the functional role of viral residues involved in heparin binding. These results provide a basis for understanding virus-heparan sulfate receptor interactions critical for HPV infection and for the potential development of inhibitors against HPV infection. Human papillomaviruses (HPVs)4 are non-enveloped small DNA viruses of great medical importance. Among the large group of HPVs known by now, sexually transmitted genital high risk HPV types are the cause for the development of a variety of epithelial tumors, especially cervical carcinoma (1). Cervical cancer is the second leading cause of death among female cancer patients worldwide. HPV16 and HPV18 stand out, as they are causally linked to Ͼ70% of cervical cancer cases (2).HPV particles consist of 72 pentamers of the major capsid protein L1, which forms the virus outer shell and encapsidates the viral DNA (3, 4). The minor capsid protein L2 is present at up to 72 copies and is hidden inside the capsid with exception of a small N-terminal section (5, 6). Efficient infection by HPV16 and HPV18 pseudoviruses requires the interactions of the L1 protein with extracellular matrix (ECM)-and cell surface-resident heparan sulfate receptor in vitro (7-9) as well as in vivo models (10). Homologs of heparan sulfate polysaccharide or heparin, secreted by mast cells, can inhibit HPV infection (7-9).Cell-surface heparan sulfates are linear and highly negatively charged oligosaccharides that are covalently linked to proteins. They can serve as the attachment receptors for several important human virus pathogens (7,11,12). Despite considerable efforts, the interactions between HPV and the heparan sulfate oligosaccharides that initiate infection are poorly understood. Here, we determined the co-crystal structure of HPV16 and HPV18 capsids bound to oligomeric heparin. We found that the highly negatively charged heparin fragment binds to multiple locations on the capsid surface mainly through charge-charge interactions. On the basis of the structure, we generated mutant virus to disrupt the interactions with heparin. ECM and cell binding assays combined with infectivity measurements showed that substitution of key HPV residues involved in bi...

Human papillomaviruses (HPVs) are known etiologic agents of cervical cancer. Vaccines that contain virus-like particles (VLPs) made of L1 capsid protein from several high risk HPV types have proven to be effective against HPV infections. Raising high levels of neutralizing antibodies against each HPV type is believed to be the primary mechanism of protection, gained by vaccination. Antibodies elicited by a particular HPV type are highly specific to that particular HPV type and show little or no cross-reactivity between HPV types. With an intention to understand the interplay between the L1 structure of different HPV types and the type specificity of neutralizing antibodies, we have prepared the L1 pentamers of four different HPV types, HPV11, HPV16, HPV18, and HPV35. The pentamers only bind the type-specific neutralizing monoclonal antibodies (NmAbs) that are raised against the VLP of the corresponding HPV type, implying that the surface loop structures of the pentamers from each type are distinctive and functionally active as VLPs in terms of antibody binding. We have determined the crystal structures of all four L1 pentamers, and their comparisons revealed characteristic conformational differences of the surface loops that contain the known epitopes for the NmAbs. On the basis of these distinct surface loop structures, we have provided a molecular explanation for the type specificity of NmAbs against HPV infection.