Background:  The majority of new cases of HIV in women are sexually transmitted, however, controversy remains concerning the mode of viral transmission across the cervical mucosa. Most proposed models have utilized a cervical epithelial monolayer to support their data, however, this does not mimic the natural environment. Therefore, we have combined 2 novel approaches to address female sexual transmission, fluorescently tagged HIV-1 visualization and a cervical explant model, to determine the events of penetration.

Methods:  By tethering the green fluorescent protein to Vpr, we can detect individual virions and intracellular complexes utilizing fluorescent microscopy. To investigate HIV-1 penetration we utilized the cervical explant model as previously described. The 6-mm biopsies were inoculated with either CXCR4 or CCR5 fluorescently tagged HIV-1 or photo-activatable GFP for varying time points. When harvested, samples were washed with PBS, placed in OCT and maintained at –80°C. Cryosections of 10 mm were antibody stained for p17/p24 using a Cy5 secondary antibody. Sections were then analyzed using deconvolution microscopy. To observe photo-activatable GFP labeled virions, tissue was Z scanned in FITC, then photo-activated, and then scanned again in FITC, which revealed the photo-activated virions. A scan of Cy5 provided a second confirmation of virion identification. Depth of virion penetration from the edge of the tissue was measured.

Results:  Initially, we visualized fluorescently labeled virions within the ectocervical epithelium with confirmation in p17/p24 Cy5 staining. However, tissue autofluorescence confounded our data interpretation. Therefore, the use of photo-activatable GFP-labeled virions was optimal, providing the ability to view the tissue before and after activation, thus, controlling for background. These virions were also confirmed in Cy5 staining for p17/p24. We were able to detect HIV-1 virion penetration within the ectocervical epithelium at early time points, with the deepest virion penetrating 8 mm at 2 hours. Observed virion presence within sections was heterogeneous, some showed many while others were completely void of particles.

Conclusions:  We have established a novel methodology to visualize individual HIV-1 particles within cervical tissue using photo-activatable GFP virions. This model now allows us to begin to address the question of how HIV-1 is transmitted through the genital mucosa.

Keywords: cervix; heterosexual transmission; gential tract