| Background and objective:Defensins, a group of endogenous cationic peptides found in mammals and plants, haveabilities to actively defend against pathogen attack. To date, defensins have been consideredas a new type of antibiotic due to their broad-spectrum antibacterial property without drugresistance. Besides antibacterial activity, increasing evidences indicated that defensins showedremarkable anti-viral ability against some kind of virus. Humanα-defensin5(HD-5), whichwas found in the granules of enteric Paneth cells and expressed in the epithelial cells offemale genital tract, is presumed to have stronger antimicrobial abilities endowed byevolution upon environmental pressures.The anti-viral activity of defensin against human immunodeficiency virus (HIV) andherpes simplex virus (HSV) has been well defined. However, its anti-infection activity againstnonenveloped virus is still unclear. Human papillomavirus (HPV), a kind of nonenvelopedvirus, is a ubiquitous human pathogen that causes genital infection in female population. It isreported that approximately75%to80%of sexually active individuals will be infected byHPV in their lifetime. A cohort study indicated high-risk HPV infection was associated withthe pathogenesis of neoplasmata genitalis. Furthermore, long-term HPV16infection was theleading cause for cancer of cervix. Therefore, to further investigate the anti-HPV properties ofHD-5will provide helpful information for the prevention and treatment of HPV infection.As HPV showed strict host specificity and histodifferentiation properties, it is difficult toachieve proliferation through tissue culture. Furthermore, it is still not suitable to isolate virusfor the efficacy evaluation for the anti-virus drugs. To date, production of recombination HPVpseudovirus through amplification, expression and assemble can be accomplished bycotransfecting plasmids which expressed modified versions of the papillomavirus togetherwith a fluoresent reporter gene. Based on this, direct observation and quantitative analysis ofviral infection was available. According to our knowledge, it is very difficult to produce large amounts of defensinsthrough chemosynthesis due to the presence of three disulfide bonds, which may limit itsresearch and development. Moreover, the effects of disulfide bonds on the property ofdefensins are still controversial. Some studies demonstrated that disulfide bonds affected theantimicrobial activity of defensins, while other studies indicated that it was the positivecharges of defensins that played a pivotal role in the antimicrobial activity. Recent literaturesindicated that the absence of disulfide bonds had no effects on the antibacterial activity ofhuman beta defensin-3. Further studies were needed to investigate the effects of disulfidebonds and cysteine residues on the anti-viral activity of HD-5.Methods:In this study, HPV16pseudovirus integrated with green fluorescent protein (GFP)reporter plasmid was prepared, based on which a cellular model of human cervical cancerinfected by HPV16pseurovirus was established. The293FT cells were co-transfected withHPV pseudovirus recombinant plasmid and a GFP reporter plasmid. After isolation andpurification, virus titer was represented as the expression rate of GFP determined using flowcytometry. Three different cervical cancer cell lines were infected with HPV16pseurovirus tochoose the suitable cellular model according to the sensitivity to infection. A series of HD-5polypeptides including HD-5/N (the natural configuration of human defensin-5),HD-5/N-FAM natural configuration of human defensin-5labeled with FAM), HD-5/Acm(linear synthesis, cysteine residues were modified by acetamidomethyl) and HD-5/Abu (linearsynthesis, the cysteine residues were substituted by alpha-aminobutanol) were synthesized.The interaction between HD-5/N-FAM and cancer cell lines were determined using laserscanning confocal microscope. The expression of GFP reporter gene in the cells infected withHPV16pseudovirus was observed using fluorescence microscope and flow cytometry at48hafter incubated with HD-5polypeptides, based on which the inhibition ratio of defensins onthe viral infection was calculated. For the evaluation of indirect chemotaxis of HD-5polypeptides on Caski cells, the secretion of interleukin-8in the supernatant was determinedby ELISA. At the same time, the migration of neutrophil cell was determined using Transwellchemotaxis migration assay.Results:(1) HPV16pseudovirus containing GFP reporter gene was finally obtained, and the virus titer was up to2×10~8TU.(2) Among the three cervical cancer cell lines cultured in vitro, the highest infection ratewas detected in C-33a cell line after HPV16pseudovirus infection.(3) HD-5/N inhibited HPV16pseudovirus infection of C-33a cells in a dose-dependentmanner. At the concentration of50μg/ml, the inhibition ratio of HD-5on the viral infectionwas nearly100%.(4) HD-5could penetrate into cytoplasm and concentrate mainly in the endosome andlysosome after the cell lines were exposed to HD-5/N-FAM for2to3hours.(5) The inhibition ratios of HD-5/N, HD-5/Acm and HD-5/Abu at the concentration of20μg/ml on the HPV16infection of C-33a cells were96.48±5.67%,69.02±7.88%and2.71±1.53%, respectively. Compared with HD-5/N, significant decrease was noted in theanti-HPV activity of HD-5/Acm. Additionally, HD-5/Abu lost its anti-HPV activity comparedwith HD-5/N.(6) The secretion of IL-8in the Caski cells after HD-5stimulation ranked in an order ofHD-5/N>HD-5/Acm>HD-5/Abu. For the chemotaxis of neutrophilic granulocyte inperipheral blood, HD-5/N showed the highest property, followed by HD-5/Acm andHD-5/Abu.Conclusion:(1) Cervix cancer cell lines were infected with HPV16pseudovirus integrated with GFPreporter gene. A quantitative method for the infection of HPV virus was developed based onthe flow cytometry. All these laid the foundation for the analysis of anti-viral activity ofHD-5against HPV.(2) HD-5/N displayed high anti-viral ability against HPV. HD-5/N could penetrate intothe cytoplasm, which may lead to the speculation that HD-5/N could inhibit the viraltransportation, uncoating and replication.(3) Significant attenuation was noted in the anti-HPV16activity of HD-5/Acmcompared with HD-5/N, demonstrating disulfide bonds played an important role in theanti-viral activity of HD-5. Sharp decrease was noted in the anti-HPV16activity ofHD-5/Abu compared with HD-5/N and HD-5/Acm, indicating the presence of cysteineresidues may affect the anti-viral activity of HD-5/N.(4) Remarkable decrease was noted in the chemotaxis of HD-5/Acm and HD-5/Abu, which indicated that the secondary structure of HD-5might be associated with the chemotaxisof HD-5. Additionally, we speculated that the neutrophil recruitment directed by HD-5chemotaxis may be associated with its anti-viral activity. |
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