Identification And Function Analysis Of Critical Residues On Minor Structural Protein L2 Of Human Papillomavrius That Involved In Its Interaction With Major Structural Protein L1

Persistent infection of high-risk human papillomavirus(HPV)is a necesssary cause of cervical cancer,one of the most common malignant tumors in the female genital system.HPV is non-enveloped,double-stranded DNA virus that consists of multiple copies of the major(L1)and minor(L2)capsid proteins.L2 protein plays a critical role in viral infection,including primary attachment,host cell entry,immune escape,vesicular escape and assembly of virus particles,etc.However,no high resolution three-dimensional structure of L2 protein are available,and the interaction mechanism between L1 and L2 has not been studied clearly.In particular,the key sites of L2 protein interaction with L1 is completely unknown.Therefore,it is of great significance to elucidate the key sites of the interaction between HPV minor structural protein L2 and major structural protein Ll and their functional mechanism.To explore key sites and functions of the interaction between HPV minor structural protein L2 and major structural protein L1,the functions of four key residues(F256,R315,Q317 and T340),locating in the lumen of L1 pentamer and identified in the previous phase of our group,which affected the virus infection and may interact with L2 protein,were further analyzed.First,the L1-F256A,L1-R315A,L1-Q317A,and L1-T340A mutant pseudoviruses were prepared on a large scale,and purified by ultracentrifugation to a higher purity.The conformational changes and amount of minor structural proteins L2 were analyzed by ELISA and Western blotting.The results showed that except for L1-F256A,the amount of L2 protein in the mutant virions was significantly lower than that of the wild type.However,the L2 expression level of the four mutant viruses was similar to that of the wild type in 293FT cells,indicating that the mutation of four sites on L1 didn't affecting L2 expression,demonstrating that mutations at three key sites,R315,Q317,and T340,induce L2 protein decreasing in particle assembly;Detecting mutations of F256 by the HPV 16 L2 antibody 14H6 revealed some conformational changes on the N terminus of L2.The immunofluorescence assay was utilized to visualize the changes in the primary cell attachment of mutant pseudoviruses.The results showed that the mutations in the four sites did not affect the viral adsorption of extracellular matrix and cell membranes,but prevented the endocytosis of the pseudoviruses.The above research suggested that L1-F256,R315,Q317 and T340 are extremely important for virus infection and are involved in the interaction of major structural and minor structural proteins of the virus.Since there is no structural information about the minor structural protein L2,we first analyzed the primary structure information of L2 protein and used software to analyze the charged state and predicted the residue exposure after protein folding.Based on the properties of the four key amino acids of L1,selected negatively charged and hydrophilic amino acids exposed on the surface of L2 for mutation analysis.The properties and functions of L2 mutant pseudoviruses were studied by similar methods of L1.Virus infection analysis showed that the infection ability of L2-D337A-D338A-D340A and L2-D329A mutant pseudoviruses was significantly reduced;the amounts and conformational changes of L1 and L2 proteins in the mutant pseudoviruses were analyzed using double antibody sandwich ELISA and Western blotting.L2-D337A-D338A-D340A caused the loss of L2 protein in the virus particles after L-D337-D338-D340 mutation,but the L2 protein expression in the cell was similar to that of the wild type.There was no significant change in L2 protein content in L2-D329A mutant pseudotyped virus,but the immunoreactivity of L2 protein was decreased,indicating that the mutation at 329 site resulted in the conformational change of L2 during particle assembly.Furthermore,it was proved that the mutation of D337,D338,D340 in the minor structural protein L2 resulted in a decrease in L2 protein incorporated into the particles or the conformational change of L2 protein.Similarly,immunofluorescence analysis of mutant pseudovirus infections revealed that loss of L2 protein in the mutant pseudovirions resulted in L2-D337A-D338A-D340A pseudovirus loss of cytosolic capacity,but did not interfere with its adsorption of extracellular matrix and cell membranes.The L2-D329A mutant pseudovirus could enter the cell normally,and its infection ability was significantly reduced,indicating that this site may be related to the subsequent infection events after the virus enters the cell.Based on the key sites of L1 and L2 protein interactions obtained in the above studies,attempts were made to introduce disulfide bonds inside the virus particles and to lock the interaction of the structural proteins L1 and L2,which laid the foundation for the analysis of the three-dimensional structure of L2.The key amino acids interacting between L1 and L2 proteins were mutated to cysteine and paired with each other to prepare pseudoviruses.The pseudovirus formed by the pairing of L1-Q317C and L2-D329C was analyzed by reducing and non-reducing Western blotting.It was found that a protein band of approximately 180 kD in size was formed,indicating that these two amino acids were successfully crosslinked to form disulfide bonds in virus particles.Double-antibody sandwich ELISA detection showed that the immunoreactivity of L2 protein was decreased,indicating that the introduction of the disulfide bond of 16L1-Q317C+L2-D329C pseudovirus caused a conformational change of L2 protein,which may result in the exposure changes of amino acids 21-30 of L2 N-terminus,and the infection rate analysis showed that their ability to infect significantly decreased;Immunofluorescence was used to further analyze the initial infection process of 16L1-Q317C+L2-D329C pseudovirus.It was found that the mutant pseudovirus could normally adsorb the extracellular matrix and cell membrane and enter into the cell normally,indicating that the introduction of disulfide bonds affects the pseudovirus adsorption cells.In summary,this research further studied the properties and functions of the four key amino acids in L1 involved in capsid protein interaction.For the first time,the key sites that interact with L1 proteins in L2,a minor structural protein,were identified,and the roles of these key sites in viral particle assembly and infection were analyzed.Based on these key sites involved in L1 and L2 interactions,disulfide bonds were successfully introduced into the viral capsid,laying the foundation for the solutions of high-resolution L2 protein structure and elucidation of HPV assembly and infection mechanisms.It also provides structural and functional information for the design of HPV broad-spectrum vaccines or therapeutic drugs.