Binding Mechanism Study Between Specific Peptides Of Human Papillomavirus Capsid And Cells Receptors

Human papillomaviruses (HPVs), a family of non-enveloped double-stranded DNA viruses that infect epithelial cells in the skin and mucosa, have been extensively studied. To date, more than 100 different types have been identified based on DNA sequence relationships. Epidemiologic and biochemical studies have indicated that infection with low-risk HPVs as HPV-6,-11 genarally causes genital warts; while infection with certain high-risk types such as HPV-16,-18,-31,-33,-45, and-58 is an important factor associated with anogenital malignancies, particularly cervical cancer.The life cycle of HPV viruses starts with its adsorption and attachment to host cells by binding to receptor molecules on the cell surface, being followed by amplification of the viral genome and production of progeny virus. Therefore, the prerequisite for exploring the viral life cycle should be the investigation on how the viruses adsorb to cells. Because efficiently propagating HPVs is nearly impossible to obtain in vitro, VLPs generated by synthesizing L1 and L2 proteins have been used as mode in exploring interaction between the cell surface and the viral capsid. These studies implicated cell surface heparan sulfate (HS) proteoglycans (HSPGs) as the primary attachment factors for most HPV types and an involvement of electrostatic interactions between the basic amino acid residues of the capsid protein with the negatively charged sulfate groups.Firstly, we examined the interaction of five synthetic peptides of HPV-16 capsid protein with heparin, a substitute for cell surface HS, comparing the different sequential peptides of HPV-16 capsid proteins. The positively charged sequences at the C-terminus of the L1 protein and the N- and C-termini of the L2 protein of HPV-16 can efficiently bind to heparin receptors, which were characterized in the present study by quantitative isothermal titration calorimetry experiments primarily, fluorescence spectroscopy, and static right-angle light scattering. The binding constant, K, was at an order-of-magnitude of 107 M-1 for the two peptides at the N-and C-termini of HPV-16 L2 and segment b at the C-terminus of HPV-16 L1; while that for other L1 analogues were of a smaller order, illustrating the heparin binding is a typical sequence-specific and-dependent phenomenon. These results suggest that, in addition to L1, the L2 protein may participate in cell surface attachment during HPV infection and provide a solid physical-chemical foundation for the involvement of proteoglycans during HPV infection. Furthermore, the calorimetry results demonstrated that hydrophobic interactions and hydrogen bonding are involved in peptide binding to heparin in addition to the essential electrostatic interactions. Meanwhile, circular dichroism spectroscopy revealed that binding to heparin does not induce obvious secondary structural changes in the peptides.The high-risk types of human papillomaviruses (HPV) HPV-16 and HPV-18 are the predominant types associated with cervical cancer. HPV-16 and HPV-18 account for about 50% and 20%, respectively, of cervical cancers worldwide. While the reason and molecular mechanism of the distinct prevalence and distributions between them remain poorly understood, the binding affinity of cell surface receptor with capsid proteins, especially L1, may be involved. The previous studies also revealed that the C-terminus of the L1 protein can efficiently bind to heparin receptors. We examined heparin binding with two synthetic peptides corresponding to the 14 amino acid C-terminal peptides of HPV-16 and HPV-18 L1 with the goal of comparing the equivalent residues in different HPV types. Using isothermal titration calorimetry (ITC) and static right-angle light scattering (SLS), we determined the binding constant K, reaction enthalpy AH, and other thermodynamic parameters in the interaction. Especially, we assessed the role of specific residues in binding with heparin by comparing the NMR spectra of free and heparin-bounded peptides. We showed that the C-terminal of HPV-18 L1 is probably a potential candidate for the attachment factors owing to its higher binding affinity to heparin as compared to H16Ctb. Several driving forces contribute to the interaction between heparin and L1 peptides, but the H18Ctb peptide undergoes less hydrogen bonding and more hydrophobic interactions in the binding than H16Ctb. Meanwhile, in contrast to the cluster of six basic residues in the structure of H16Ctb, the unconnected basic residues in H18Ctb contributed more to the ionic interactions and lead to aggregations. Furthermore, we propose a new approach of using the C-terminal of HPV L1 as an agent to compare the binding affinity with heparin between HPV-16 and-18, then provide a possible explanation for the distinct prevalences owing to the significance of the virus adsorption and attachment to host cells.Among the 100 different types of HPV, sevaral types HPV such as HPV-5, HPV-1, and HPV-4 are not found in genital and about 40 HPV types can infect the genital tract. Genital HPV types have been subdivided into low-risk types, such as HPV-6, HPV-11, HPV-44, and HPV-55, which are found mainly in genital warts, and high-risk types, such as HPV-16, HPV-18, HPV-31, HPV-33, HPV-45, and HPV-58, which are frequently associated with invasive cervical cancer. We examined heparin binding with eight synthetic peptides corresponding to the 14 amino acid C-terminal peptides of non-genital types HPV-5, HPV-1a, HPV-4, low-risk types HPV-11, HPV-44, HPV-55 and high-risk types HPV-16, HPV-18 with the goal of comparing the equivalent residues in different HPV types. Using isothermal titration calorimetry (ITC) and static right-angle light scattering (SLS), we investigated the interactions involved in peptide binding to the HS portion of the cell surface at the molecular or sub-molecular level, compared the binding affinity with heparin between different HPV types, then provided a possible explanation for the distinct prevalences owing to the significance of the virus adsorption and attachment to host cells.In summary, we studied the interactions between specific peptides of HPV capsid peptides and heparin using physical-chemical experiments for the first time. Our study quantitatively supplied solid evidences of strong interactions and confirmed the binding mechanism between several basic-sequence-rich L1 and L2 peptides and the negatively charged polyelectrolyte, which show a potential guideline in further research of the binding mechanism in HPV infected cells.