Following attachment to primary receptor heparan sulfate proteoglycans (HSPG), human papillomavirus type 16 (HPV16) particles undergo conformational changes affecting the major and minor capsid proteins, L1 and L2, respectively. This results in exposure of the L2 N-terminus, transfer to uptake receptors, and infectious internalization. Here, we report that target cell cyclophilins, peptidyl-prolyl cis/trans isomerases, are required for efficient HPV16 infection. Cell surface cyclophilin B (CyPB) facilitates conformational changes in capsid proteins, resulting in exposure of the L2 N-terminus. Inhibition of CyPB blocked HPV16 infection by inducing noninfectious internalization. Mutation of a putative CyP binding site present in HPV16 L2 yielded exposed L2 N-terminus in the absence of active CyP and bypassed the need for cell surface CyPB. However, this mutant was still sensitive to CyP inhibition and required CyP for completion of infection, probably after internalization. Taken together, these data suggest that CyP is required during two distinct steps of HPV16 infection. Identification of cell surface CyPB will facilitate the study of the complex events preceding internalization and adds a putative drug target for prevention of HPV–induced diseases.

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 cytomegalovirus (HCMV) infection is usually benign in healthy individuals but can cause life-threatening disease in those with compromised immune systems. Approved drugs available to treat HCMV disease, including ganciclovir, cidofovir, and foscarnet, have significant toxicities that limit their use in certain patient populations. LJP538 and LJP539 are human monoclonal antibodies that are being evaluated as immunoglobulin therapeutics. The antibodies target glycoproteins gB and the gH/ gL/UL128/UL130/UL131a pentameric complex, respectively. Here we present an in vitro characterization of these antibodies. We show that LJP538 and LJP539 are more potent than a marketed immunoglobulin at inhibiting HCMV infection of various cell lines relevant to pathogenesis. We find that LJP538 and LJP539 are active against a panel of clinical isolates in vitro and demonstrate minor-to-moderate synergy in combination. Passage of HCMV in the presence of LJP538 or LJP539 alone resulted in resistance-associated mutations that mapped to the target genes. However, no loss of susceptibility to the combination of antibodies was observed for >400 days in culture. Finally, the binding regions of LJP538 and LJP539 are conserved among clinical isolates. Taken together, these data support the use of LJP538 and LJP539 in combination for clinical trials in HCMV patients.

1 The aim of this study was to establish the utility of a¯uorometric imaging plate reader (FLIPR) assay to assess human adenosine A 2B receptor function by characterizing its receptor pharmacology and comparing this pro®le to that obtained using a microphysiometer. 2 FLIPR was used, in conjunction with a Ca 2+ -sensitive dye (Fluo-3-AM), to measure rapid rises in intracellular calcium in a Chinese Hamster Ovary (CHO-K1) cell line stably transfected with both the human A 2B receptor and a promiscuous G a16 protein. Microphysiometry was used to measure rapid changes in the rate of extracellular acidi®cation in a Human Embryonic Kidney (HEK-293) cell line also stably transfected with human A 2B receptor. 3 Activation of A 2B receptors by various ligands caused a concentration-dependent increase in both the intracellular calcium concentration and the extracellular acidi®cation rate in the cells tested, with a similar rank order of potency for agonists: NECA4N 6 -Benzyl NECA4adenosine5R-PIA4CPA4S-PIA4CHA4CGS 21680. No comparable e ects were observed in the nontransfected control cell lines. 4 The rank order of potency of the agonists examined was the same in all studies, whereas absolute potency and e cacy varied. Thus, all compounds exhibited greater potency in FLIPR than the microphysiometer and the e cacies obtained with CHO-K1+G a16 +A 2B cell line and FLIPR were greater than those obtained with HEK-293+A 2B cell line in the microphysiometer. 5 ZM-241385 was the most potent of a range of adenosine antagonists tested with a pA 2 of 8.0 in both the FLIPR and microphysiometer assays. 6 In conclusion, the pro®le of the responses to both A 2B receptor agonists and antagonists in FLIPR were similar to those obtained by the microphysiometer, although both potency and e cacy values were higher in the FLIPR assay. With this caveat in mind, this study shows that FLIPR coupled with a cell line transfected with both the human A 2B receptor and a promiscuous G a16 protein provides a useful, high throughput method for the assessment of A 2B receptor function.