Objective        To establish a human papillomavirus16 pseudovirus (HPV16 Psv) infection model in vitro and to determine the anti-HPV effects of human α-defensin 5 (HD-5) with different conformations.       Methods        The 293FT cells were co-transfected with HPV pseudovirus recombinant plasmid P

16L1L2

and a GFP reporter plasmid P

fwb

. After isolation and p

urification

the pseudovirus particles were used to infect cervical cancer cell line C-33a. During infection

the C-33a cells were treated respectively with 3 different configurations of HD-5

including HD-5/N with natural configuration

HD-5/Acm with Cys residues blocked by acetamidomethyl (Acm) and HD-5/Abu with Cys residues substituted by α-aminobutyric acid (Abu)

at a dose of 20 μg/ml. In 48 h later

the cells were observed by fluorescent microscopy and the viral infection rate was determined by flow cytometry assay.       Results        The 293FT cells were successfully transfected with P

fwb

and P

16L1L2

and 2.5×10

8

TU/ml of HPV-16 pseudovirus particles were finally obtained. After culturing

most of C-33a cells were infected by HPV16 pseudovirus

which displayed green fluorescence under fluorescent microscope and could be detected by flow cytometry. The inhibitory rate of HD-5/N

HD-5/Acm and HD-5/Abu on HPV infection was (96.48±5.67)%

(69.02±7.88)% and (2.71±1.53)% respectively. Compared to HD-5/N

antiviral activity of HD-5/Acm and HD-5/Abu was significantly reduced (P0.01).       Conclusion      A method for HPV16 pseudovirus infection and anti-HPV analysis based on flow cytometry assay is successfully established. The natural configuration of HD-5 contributes to enhance anti-HPV ability significantly

and Cys residues play important role in the antiviral activity of HD-5.