Controlled Assembly Of Human Papillomavirus Capsid Protein L1in Vitro

Human papillomaviruses (HPV) is DNA viruses that infect basal epithelial cells ofskin and mucosal. High-risk HPV is the major causative agent of cervical cancer andprecancerous lesions, and it has been implicated in99.7%of cervical squamous cellcancer cases worldwide. Nowadays the pre-existing HPV infection is troublesome tobe cured. However, the prophylactic HPV vaccines that based L1-VLPs could preventthe incidence of HPV-associated cervical cancer and other tumors, therefore, the studyabout the prophylactic HPV vaccines is very important for human being. Whereas, therecombinant yeast and insect cells are currently the systems of choice for prophylacticHPV vaccines, which are largely unavailable in developing countries, as vaccine’sproduction, distribution and storage costs are too high. Especially, the current VLPsvaccines are not expected to protect against the almost30%of cervical cancers thatare HPV16-independent and HPV18-independent. Therefore, the development of aneconomically advantageous broad-spectrum vaccine would be prior and urgent needfor the introduction of HPV vaccines into these developing countries.The VLPs are structurally and immunologically similar to the native HPV virionsTherefore, throughing the study and analysis of VLPs, which can grasp the capsidstructure and biological functions of HPV, and further identify the inherent law ofHPV transmission. The literatures have reported that both the cysteine residues thatform capsid-stabilizing disulfide bonds and the h4of L1were indispensable for VLPsassembly. However, the molecular mechanism and key factor on VLPs assembly hasso far not been studied in depth. And, the study about the intrinsic relationshipbetween protein structure and their functions is very important for the development ofthe HPV vaccines and anti-HPV reagents. Therefore, to reveal both of the molecularmechanism and the assembled kinetics of VLPs assembly, we reconstructed the L1protein structure to realize the study of the controlled assembly from monomer topentamer, and to VLPs.In the present study, we proposed to realize the controlled assembly ofbi-hybrid-VLPs by using the recombined HPV16L1Bi and HPV18L1Bi proteins from E. coli. For that, the h4and the related turn sequences in wild-type HPV16L1and HPV18L1(HPV16L1Wt and HPV18L1Wt) were exchanged. Firstly, therecombinant HPV16L1Bi and HPV18L1Bi were successfully constructed, expressed.And then, the L1proteins were purified from cell lysates with GST affinitychromatography and size-exclusion chromatography (SEC). In the fast protein liquidchromatography (FPLC) elution profile, both HPV16L1Bi and HPV18L1Bi proteinsshowed two inclusion peaks in the target protein with elution positions at about265and53kDa, corresponding to the molecular weights of a L1pentamer (L1-P, majorpeak) and a L1-M (very minor peak), respectively. The recent study showed that theproteins from the monomer peak could not assemble into pentamer, possibly becausethey existed as partially misfolded, metastable proteins. Afterwards, FPLC andtransmission electron microscopy (TEM) confirmed that HPV16L1Bi and HPV18L1Bi pentamer (HPV16L1Bi-P and HPV18L1Bi-P) could form soluble pentamers.These results indicated that the exchange of h4and its related turn sequences betweenHPV16L1Wt and HPV18L1Wt did not affect the pentamers formation.After assembly assay, sole HPV16L1Bi-P or HPV18L1Bi-P and their mixture ofcould assemble into VLPs, respectively. The results of time-dependent static lightscattering (SLS) showed that they had the different molecular mechanism, itsuggested that HPV16L1Bi-P and HPV18L1Bi-P probably assemble into the hybridHPV16/18L1Bi VLPs. The hybrid HPV16/18L1Bi VLPs was further confirmed byfluorescence resonance energy transfer (FRET) and complex-immunoprecipation(Co-IP) assays. And the ratio of HPV16L1Bi-P and HPV18L1Bi-P was about3:5,which was estimated by UVP Bioimaging system of labworks software. The result ofDLS confirmed the protein from Co-IP still keeping the capsid-like structure with theaverage size of52.65nm. The HPV pseudovirus neutralization assay exhibited thathybrid HPV16/18L1Bi VLPs had high immunogenicity and induced highneutralizing antibodies titres to HPV16and HPV18. This study may provide thesuggestions for the design of effective multivalent hybrid-VLP vaccines for theprevention of infection with multiple types of HPV.Meanwhile, we found that the HPV capsid protein L1contains a small alpha-helixnear the C-termini, helix5(h5). The accidental mutation of R466in the h5of HPV16L1to Ala (R466A) produced only monomeric peak without pentameric peak,indicating its crucial importance in pentamers formation. To understand the structural role of critical residues in h5, we further mutated each of the highly conservedresidues,464LGRKFL469, in the h5of HPV16L1to Ala (L464A, G465A, R466A,R466H, K467A, F468A, L469A). Firstly, the L1genes of the above mutants weresuccessfully constructed, expressed and purified as GST-L1fusion protein. In theFPLC elution profile, the mono-site mutations of the absolutely conserved residues,464LGR466, in h5of HPV16L1completely disrupted the pentamer formation, beingsignificant because it ceased one of the indispensable steps of virus life cycle andtherefore supplied a potential target for the development of anti-HPV reagents.However, other three mono-site mutants, K467A, F468A and L469A, produce bothL1-P and L1-M, FPLC and CD indicated that KFL did not affect the pentamersformation and secondary structure of L1proteins. In addition, the mutation of L469Ain HPV16L1Wt produced an approximately2-fold pentameric yield of HPV16L1Wt,being crucially important in lowering the cost of future HPV VLPs vaccine.Three mono-site mutants, HPV16L1-L464A, HPV16L1-G465A and HPV16L1-R466A, produce no pentamer, but produce monomer. However, the HPV16L1-R466A mutant behaved differently from the other mutants, it producedapproximately3-5times more soluble monomeric L1than any other Ala mutant. Thisunusually high yield of soluble monomeric L1for HPV16L1-R466A is surprising,because monomeric L1generally has low solubility, as observe for HPV16L1Wt andthe other mutants. Afterward, we similarly mutate R466in HPV16L1to His (R466H)and R467in HPV18L1to His (R467H), respectively. FPLC results showed that onlythe monomer peak was observed, confirming that both of mutations led to solublemonomeric L1protein. These results indicated that arginine in h5indeed played anindispensable role in the HPV L1pentamers formation.To understand further the functional and structural roles of h5for L1solubility,stability, and pentamer formation, we inspected in detail the structure of HPV16L1inconjunction with our mutational biochemical and biophysical data. We examined theh5sequences in all papillomavirus types. It indicates that the LGR sequence isabsolutely conserved in L1proteins of all papillomavirus types. This observationsuggested that the LGR sequence in h5must be important for L1structure andfunction.The mono-site mutations in the h5disrupted the L1pentamer formation, therefore,these interactions may be targeted for inhibiting pentamer formation and inhibiting viral particle assembly. With this in mind, we created a15-amino acid syntheticpeptide that contained the h5, with the sequence of PLGRKFLLQAGLKAK (referredas h5-pep), and incubated it with HPV16L1Wt protein. In the FPLC elution profile,the h5-pep effectively inhibited the pentamer formation of HPV16L1Wt. This resultsuggested that the highly conserved h5interaction site among papillomaviruses mayserve as a target in developing potential new anti-HPV therapeutics. These findingslay the groundwork for the development of peptidic inhibitors as prophylactic and/ortherapeutic agents for HPV.In summary, basing on the molecular mechanisms from pentamer to VLPs, wedesigned and constructed the recombinant HPV16L1Bi and HPV18L1Bi from E.coli. And we have successfully prepared a new form, with the type-specific biologicalfunction, hybrid HPV16/18L1Bi VLPs. Meanwhile, we found that the LGR sequencewas absolutely conserved in L1proteins of all papillomavirus types, and these threeresidues were indispensable for the pentamer formation and potential target for thedevelopment of anti-HPV reagents. In addition, the mutation of L469A in HPV16L1Wt produced an approximately2-fold pentameric yield of HPV16L1Wt. Thepresent study provides the useful structure information and new ideas for the researchof HPV vaccines and anti-HPV reagents.