Quantification of HPV16-sperm interaction.

Freshly ejaculated sperm cells were washed and incubated with Alexa488 labeled HPV16 capsids or the control protein Alexa488 MBP. Sperm-washing and the virus-sperm binding reactions were carried out as indicated at pH 8.6 or pH 7.4 (for buffer composition see Methods). The virus-sperm mixture was incubated at 37°C for 10 min and subsequently analyzed by live microscopy. The number of sperm with fluorescent staining at one or both foci on the sperm head was determined. Only live sperm, i.e. visibly moving cells, were considered. The numbers represent averages drawn from three independent experiments. In each experiment, 50 sperm cells were evaluated. Note that the association of HPV16 to sperm increases in efficiency at neutral conditions. To assess the effect of carrageenan and heparin on the HPV16-sperm interaction, HPV16 capsids were pre-incubated with carrageenan (0.1 mg/ml) or heparin (0.1 mg/ml) at RT for 10 min to allow association of the molecules to the capsids. Thereafter, the capsid-inhibitor mixture was added to the washed sperm in buffer at pH 7.4. Both heparin and carrageenan significantly reduced binding of HPV16 to sperm.