Controlled Self-assembly Of Human Papillomavirus Capsid Protein L1 In Vitro And Intrinsic Mechanism Study

Human papillomaviruses?HPVs?are members of non-enveloped,icosahedral,double-stranded DN A viruses.They usually infect human genitals and skin mucosa.High-risk HPV types have been shown to be the major etiological agents of cervical cancer in women.In addition,high-risk HPV can also induce a variety of malignant tumors such as head and neck squa mous cell carcinoma,oropharyngeal carcinoma,colon cancer,rectal cancer and breast cancer.However,there are still some defects in the treatment for related diseases and there is a lack of effective specific therapeutic drugs.Current PV vaccines have shown effective protection against infection with a limited number of phylogenetically related HPV types,but not against more distantly related subtypes and these vaccines have many shortcomings,such as cost expensive,poor stability,limitations of preventions,and so on.In particular,the prophylactic vaccines provide no prevention or therapeutic benefit.Therefore,urgent needs exist to develop new agents that are cost-effective and/or provide more broad-spectrum protection.HPV VLPs are composed of 72 L1 pentamers,and each L1 pentamer is made up of five L1 monomers.For the above two assembly processes,the main works of our research are summarized as follows:Firstly,a new 14 peptide,originaling essentially from the helix 5 of HPV 16L1,illustrates an IC₅₀ of 19.38 nM for the inhibition of HPV 16 L1 pentamer formation,which is highly efficient for targeting a specific protein segment.In addition,we modified the length,sequence of the peptide and mutated the hydrophilic/hydrophobic amino acids.The mechanism studies reveal that the length,sequence,and the folding of the peptide are critical factors for its inhibition.Particularly,the peptide shows similar inhibition against the pentamer formation of HPV 58L1,although it is designed specially for HPV 16 L1.This study opens a way for the development of high-efficiency,broad-spectrum inhibitors as a new class of anti-HPV agents,which could be extended to the treatment of other virus types.Secondly,thewater-solublecarboxylatopillar[5]arene?CP5A?and p-sulfonatocalix[4]arene?C4A?have been used to inhibit the HPV L1-p formation and finally prevent the assembly of virus particles.This opened a way to develop a new class of anti-HPV agents in targeting the key sites at the protein-protein interface.In the present study L-and D-proline modified calix[4]arenes?Pro-C4A?were investigated to determine any differences in their effect on the assembly of HPV 16L1 pentamer?L1-p?.The mechanism of action using model peptides was investigated by MALDI-TOF-MS and NMR and revealed that the binding was targeting the basic residues at L1 interface.This was also well supported by the trypsin digestion experiments and molecular simulations performed on the full-length L1.The large energy and morphology differences revealed by molecular simulations explain the binding disparity of L-and D-pro-C4A to L1,and consequently the selective inhibition of them on L1-p formation.The present study opens a way to develop enantioselective and cost-effective inhibitors for L1-p formation,which might be used as a new kind of anti-HPV agent and could be extended to other virus based on similar mechanisms.Thirdly,the new hybrid-assembly systems of HPV capsid protein and a europium-containing POM(EuW₁₀)have been constructed,for the first time,via the electrostatic interactions between them.By the way,both the stability of VLPs and medical activity of POMs have been improved.The co-assembly of EuW₁₀ and HPV16 L1-pentamer in buffer solution resulted in the encaps ulation of POMs in the cavity of VLPs.The sample was further purified by CsCl gradient ultracentrifugation,analysis the components of the target band by SDS-PAGE,detected the particle size by dynamic light-scattering?DLS?and the size was similar as the empty VLPs.The morphology and the composition of the particle was investigated by transmission electron microscopy?TEM?and the energy dispersive X-ray?EDX?,respectively.While the post-assembly of EuW₁₀ with the as-prepared VLPs lead only the adsorption of POMs on the external surface of particles,and both cases improved the thermal and storage stability of VLPs obviously.Particularly,the encapsulation of POMs in VLPs largely improved the antibacterial activity of EuW₁₀,and thereby,the present study will be significance both for the improvement of protein vaccines and the development of POMs medicine.In conclusion,the inhibition of HPV L1 pentamer formation has been achieved by using peptides or chiral proline modified calixarenes as inhibitors respectively.The new hybrid-assembly systems of HPV capsid protein and EuW₁₀ have been constructed,for the first time,via the electrostatic interactions between them.It lays the foundation for the development of more efficient and broad-spectrum anti-HPV agents,and provides a new method for improving the stability of protein vaccines and reducing its cost.At the same time,it provides a theoretical basis for the development of POMs drugs in the future.