In Vitro Controlled Pentamer Self-assembly Of Human Papillomavirus (HPV) Capsid Proteins L1

Human papillomavirus (HPV) is the causative agent of cervical cancer and otheranogenital and oropharyngeal cancers. HPVs are members of a group of non-enveloped DNAtumor viruses.1The outer shell of the virus particle consists of72pentamers of the majorcapsid protein L1. The L1contains all the information needed for particle assembly and alsohas the ability to self-assemble in vitro into empty virus like particles (VLPs) from thepentamer at low pH and high ionic strength. Large scale investigations have been performedto monitor the formation of VLPs from pentamers. Although the assembly of VLPs requiresthe pentamer form of L1, to the best of our knowledge, few studies have focused on theformation of pentamers and, particularly, no report has monitored the transformation of L1monomers into pentamers. Whether it is expressed in a prokaryotic or eukaryotic system, themain capsid protein L1has the intrinsic ability to form pentamers, which makes it verydifficult to obtain a stable L1monomer in vitro. Until now, the key factors that influence theformation of pentamers have been unknown, which has greatly hindered the development ofanti-viral agents.In the present study, the glutathione-S-transferase (GST) fused HPV16L1protein fromE. coli has been used as a model to monitor pentamer formation. The steric effects of theGST-tag at the N-terminal of L1hinder the strong hydrophobic interaction between monomersinside the pentamer and allow us to obtain the GST–L1monomer before GST is cleaved.After GST cleavage, the released L1readily forms the pentamer, and the slowly ongoingformer process can provide a platform to follow the formation kinetics of the pentamerassembly process. Studying the intrinsic assembly kinetics of pentamer formation is anecessary prerequisite for understanding the next step of VLP assembly and therefore it playsa key role in virus control. Time-dependent kinetic studies of the process were performed forthe first time by using static light scattering and Western Blot analysis, where the essentialfactors were revealed, offering a new biotechnical approach for virus control and/or thedevelopment of anti-viral agents.Creating disturbances in virus assembly is a particularly attractive method for antiviralintervention because viral structures are formed by multiple, relatively weak noncovalentinteractions, and essentially the viral infectivity is critically dependent on capsid formation and its stability. Therefore, novel small-molecule inhibitors of HPV, targeting specific sites ofproteins to stop further virus assembly, are needed to complement or replace currenttreatments. Although inhibitors have been widely used for controlling the assembly process ofother health-related viruses, few/no small-molecular inhibitors have been identified for HPVassembly mainly due to the insufficient target sites and unavailability of agents. Smallmolecules that recognize protein surfaces specifically are important tools for modifyingprotein interactions. Molecules that target the cationic side chains of lysine and arginine havegreat potential as generic surface binders.GroEL(chaperone) binds the disordered C-terminal domains of HPV L1and furtheraffects the formation of pentamer/VLPs during the process of coexpression and co-purifying,so it is another promising study that we should consider for vaccine development. Chaperonecoexpression is a common strategy for improving the folding quality of overexpressedproteins, whereas the late release of it from target protein in purification often a bother. Thetight association of the E. Coli chaperone GroEL with HPV capsid protein L1brought verydifficult exclusion of it in elution, the role of co-purifying GroEL on L1folding, especially onthe pentamer formation has not been investigated. Through three elaborately designedpurification routes, the contribution of co-purifying GroEL on L1folding on pentamerformation were evaluated, and essentially the mechanism of the process were compared anddiscussed in detail, explaining well the crucial importance of co-purifying GroEL in thepentamer formation of HPV L1, and the consequent VLPs assembly. The discovery andexplanations of the dependence of pentamer formation of HPV L1on the co-purifyingmolecular chaperones offers a unique biotechnical strategy for vaccine development.