Computational protocol: α-Defensin HD5 Inhibits Human Papillomavirus 16 Infection via Capsid Stabilization and Redirection to the Lysosome

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Protocol publication

[…] Serial dilutions of HPV16 PsVs in serum-free DMEM (SFM) were used to infect HeLa cells in 96-well plates for 4 h. fcHPV16 PsV infection was in the presence or absence of 20 µM furin inhibitor (decanoyl-RVKR-CMK; Calbiochem) to assess the efficacy of the furin cleavage of L2. The cells were washed and cultured in complete medium for ~44 h. Total eGFP expression was quantified with a Typhoon 9400 variable mode imager (GE Healthcare) and ImageJ software (). A standard curve of infection was constructed in Prism software (version 5.0d; GraphPad Software, Inc.). A virus concentration resulting in ~80% total signal was used in inhibition studies.To determine antiviral concentrations of HD5, increasing concentrations of HD5 were incubated with HPV16 PsV or fcHPV16 PsV on ice for 45 min in SFM. Folded HD5 was made from a synthesized 80% pure linearized peptide (CPC Scientific, Sunnyvale, CA) and purified by reverse-phase high-pressure liquid chromatography (). A 20 μM concentration of furin inhibitor was also added to the fcHPV16 PsV samples. The mixture was added to HeLa cells in a 96-well plate and incubated at 37°C for 4 h. Cells were washed and cultured with complete medium for ~44 h. Total eGFP fluorescence was quantified as described above and normalized to a control sample infected without inhibitors using ImageJ. Fifty percent inhibitory concentrations (IC50s) were determined using nonlinear regression in Prism. [...] The day before the infection, 6 × 104 HeLa or HaCaT cells were seeded on glass coverslips. Cells were infected with 1.2 × 109 to 2.4 × 109 particles of fcHPV16 EdU PsV (MOI, ≈2 × 104 particles per cell, assuming a doubling of the cells overnight) or 1.6 × 109 particles of fcHPV16 L2-FLAG. Cells that were to be treated with HD5 were infected with 3- to 6-fold less virus than untreated controls. The virus was incubated with the cells at 4°C to allow binding, unbound virus was washed off with cold medium, and the cells were further incubated at 4°C for an additional hour with medium, 10 µM HD5, or 10 µM HD5 Abu. HD5 Abu is an HD5 analogue containing l-α-aminobutyric acid in place of cysteine, which was chemically synthesized as previously described (). A 20 μM concentration of furin inhibitor was added to samples infected with fcHPV16 L2-FLAG PsV due to the presence of a minor fraction (<15%) of PsV containing uncleaved L2 in this preparation. Cells were washed with cold phosphate-buffered saline (PBS) and fixed in 2% paraformaldehyde (PFA) for the 0-h time points or shifted to 37°C for the indicated times and then fixed. Cells were permeabilized with 20 mM glycine–0.5% Triton X-100 in PBS for 20 min and then stained with antibody anti-EEA1 (1:250; Cell Signaling), anti-LAMP1 (1:250; Abcam), anti-TGN46 (1:100; Abcam), anti-FLAG (1:1,000; Sigma), or 33L1-7 (1:100; a kind gift from Martin Sapp, LSU Shreveport) diluted in 1% bovine serum albumin (BSA)-0.05% Tween 80-PBS. Secondary antibodies labeled with Alexa Fluor 555 (Life Technologies) were used at 1:1,000. EdU-labeled viral genomes were stained with the Click-iT kit according to the manufacturer’s instructions (Life Technologies). Cell nuclei were stained with TO-PRO-3 (1:1,000; Life Technologies). Coverslips were mounted in ProLong Gold (Life Technologies). z-stack images of least three fields of view for each sample were taken on a Zeiss 510 Meta laser scanning confocal microscope.Images analysis was performed using ImageJ. Individual cell borders were defined using bright-field images. Only images in the z-stack that were coplanar with the nucleus were used for all image analysis. Background levels of fluorescent stains were determined using uninfected cells. For colocalization studies, Manders coefficients were obtained using the JaCoP plugin of ImageJ for at least 40 individual cells per condition (). For nuclear colocalization, total EdU-positive pixels in the nucleus were divided by those in the entire cell. For uncoating studies, total EdU-positive and 33L1-7-positive pixels were quantified in ImageJ. […]

Pipeline specifications

Software tools ImageJ, JACoP
Applications Laser scanning microscopy, Microscopic phenotype analysis
Organisms Human papillomavirus type 16, Homo sapiens
Diseases Adenoviridae Infections, Papillomavirus Infections