VISUALIZING INDIVIDUAL, FLUORESCENTLY TAGGED HIV-1 VIRIONS IN CERVICAL TISSUE.
This study by researchers at the Northwestern University School of Medicine demonstrated how MatTek’s EpiVaginal human cervico-vaginal tissue equivalent can be used in the development of methodologies to visualize the interaction of HIV-1 virus with cervical mucosa/tissue. 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. To gain insights into the interaction of HIV with human cervical explant cultures, researchers at the Northwestern University School of Medicine have developed a technology that allows the fluorescent identification of virions of HIV in thin sections of tissue. Methods: By tethering the green fluorescent protein to Vpr, researchers can detect individual virions utilizing fluorescent microscopy. However, the detection of GFP in tissue sections is confounded by autofluorescence making the identification of HIV labeled using standard technology suboptimal. Therefore, the researchers developed a system of virion labeling based on a photo-activatable form of GFP. To observe photo-activatable GFP labeled virions, the tissue section was initially scanned in the green channel to define the background. After photo-activation, the section is scanned again in the green channel. The new signal detected after photo-activation represents the labeled virions. Results: Analysis of the interaction of HIV with MatTek EpiVagival 3-D tissue cultures of squamous epithelium revealed that HIV binding and entry was temperature dependent because no virus was observed to enter the tissue at 4°C. In contrast the virus penetrated the raft cultures efficiently at 37°C. The mechanism of entry of virus into the deeper layers of the EpiVaginal tissues appeared to be transcytosis because it was blocked by bafilomycin A, which is known to perturb intracellular vesicle trafficking. Analysis of HIV applied to explant culture of ectocervix revealed that virus entry was a rare event. Observed virion presence within ectocervix sections was heterogeneous, some cultures showed many while most others were completely void of particles. This is consistent with the view that the squamous epithelium of the ectocervix is an efficient barrier to HIV entering the mucosa. In contrast, HIV was found to efficiently enter the columnar epithelium of the endocervix in explant cultures. Conclusions: Northwestern University School of Medicine researchers have established a novel methodology to visualize individual HIV-1 particles within cervical tissue using photo-activatable GFP virions. This model now allows the researchers to begin to address the question of how HIV-1 is transmitted through the genital mucosa.
Cervical Tissue, Cervical mucosa, Columnar epithelium, Ectocervix, EpiVaginal, Fluorescently Tagged HIV-1 Virions, Genital mucosa, Green fluorescent protein (GFP), HIV, HIV-1, HIV-binding, Photo-activatable GFP, Sexually transmitted, Squamous epithelium, Transcytosis, Viral transmission, Vpr
Bafilomycin A, HIV, HIV-1, MatTek EpiVaginal tissue
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