Oral and vaginal epithelial cell lines bind and transfer cell-free infectious HIV-1 to permissive cells but are not productively infected
Authors: Stephen J Challacombe
Ayesha Islam
Chengguo Shen
David L Moyes
Julian R Naglik
Arinder Kohli
Celia Murciano
Journal: PLoS ONE (2014)
Discipline: Multidisciplinary
Keywords: HIV-1; mucosal surfaces; oral mucosa; vaginal mucosa; epithelial cell lines
Affiliations: Department of Oral Immunology, Clinical and Diagnostic Sciences, King's College London Dental Institute, King's College London, London, United Kingdom., Department of Oral Immunology, Clinical and Diagnostic Sciences, King's College London Dental Institute, King's College London, London, United Kingdom; Department of Obstetrics and Gynecology, Boston University School of Medicine, Boston, Massachusetts, United States of America., Department of Oral Immunology, Clinical and Diagnostic Sciences, King's College London Dental Institute, King's College London, London, United Kingdom; Department of Microbiology and Ecology, University of Valencia, Valencia, Spain.

Abstract:  The majority of HIV-1 infections worldwide are acquired via mucosal surfaces. However, unlike the vaginal mucosa, the issue of whether the oral mucosa can act as a portal of entry for HIV-1 infection remains controversial. To address potential differences with regard to the fate of HIV-1 after exposure to oral and vaginal epithelium, we utilized two epithelial cell lines representative of buccal (TR146) and pharyngeal (FaDu) sites of the oral cavity and compared them with a cell line derived from vaginal epithelium (A431) in order to determine (i) HIV-1 receptor gene and protein expression, (ii) whether HIV-1 genome integration into epithelial cells occurs, (iii) whether productive viral infection ensues, and (iv) whether infectious virus can be transferred to permissive cells. Using flow cytometry to measure captured virus by HIV-1 gp120 protein detection and western blot to detect HIV-1 p24 gag protein, we demonstrate that buccal, pharyngeal and vaginal epithelial cells capture CXCR4- and CCR5-utilising virus, probably via non-canonical receptors. Both oral and vaginal epithelial cells are able to transfer infectious virus to permissive cells either directly through cell-cell attachment or via transcytosis of HIV-1 across epithelial cells. However, HIV-1 integration, as measured by real-time PCR and presence of early gene mRNA transcripts and de novo protein production were not detected in either epithelial cell type. Importantly, both oral and vaginal epithelial cells were able to support integration and productive infection if HIV-1 entered via the endocytic pathway driven by VSV-G. Our data demonstrate that under normal conditions productive HIV-1 infection of epithelial cells leading to progeny virion production is unlikely, but that epithelial cells can act as mediators of systemic viral dissemination through attachment and transfer of HIV-1 to permissive cells. 

Retracted: No

PubPeer comments:
 February 2020 correction, found here: https: journals.plos.org plosone article?id=10.1371 journal.pone.0229553  PLOS ONE apologizes for our delay in posting this Correction and acknowledges that the authors responded to the concerns about this article by providing data and clarifications in 2015. After publication of this article [1], concerns were raised about the following blots in Figs 3A and 4B: * In Fig 3A, the p24 panels for A431 and Primary cell lanes appear to be the same. The authors clarified that the correct data are shown in the published figure for Primary cells, and that this panel was duplicated in error as representing A431 p24 data. The A431 p24 panel is corrected in the updated version of Fig 3 provided with this Correction, and the original images underlying the p24 results for Primary cell and cell line experiments are included in S1u2013S3 Files. * The u03b1-actin control panels in Figs 3A and 4B appear the same for TR146, FaDu, and TZM-bl cells. The authors explained that the same blot was used in generating the p24, p55, and u03b1-actin data shown in Figs 3A and 4B for TR146, FaDu, A431, and TZM-bl. Hence the same control blots apply to both figures. This is reflected in the updated figure legends provided with this notice. * In Fig 3A, the u03b1-actin control panels for A431 and Primary cell lanes appear to be the same as lanes 3, 4, and 5 of the TR146 FaDu u03b1-actin panel. In Fig 4B, the u03b1-actin control panel for A431 appears to be identical to lanes 2, 3, and 4 of the u03b1-actin panel for TR146 and FaDu cell lines. The authors clarified that the wrong u03b1-actin data were shown in the published Fig 3A for A431 and Primary cells and in Fig 4B for A431 cells. This is corrected in the updated figures and the original u03b1-actin blot images for the cell lines and Primary cell u03b1-actin panels are provided in S4 and S5 Files.  In reviewing the primary image data, it came to light that the p24 data shown in Fig 3A for TZM-bl cells were obtained using a shorter blot exposure than the data for the other three cell lines (see S2 and S3 Files). The same blot was used to obtain the p24 data for all four cell lines, and the u03b1-actin data for all cell lines reflect the same exposure of the same blot. The TZM-bl panel has been revised in the updated figure so that the p24 data for all four cell lines reflect the same exposure of the same blot (S3 File). The updated figure and supporting raw image data in S2 and S3 Files indicate that p24 was expressed at higher levels in TZM-bl than in the other three cell lines. As a result, statements comparing expression across cell lines are not supported. Specifically, the following sentences (second and fourth) from the second paragraph of the u2018HIV-1 binding to epithelial cellsu2019 subsection of the Results are not supported: u201cp24 was present in TR146, FaDu and A431 protein lysates at levels similar to that found with TZM-bl cells, indicating that both R5 and X4 virus are captured by both oral and vaginal epithelial cells (Fig 3A).u201d u201cGiven the identical HIV-1 binding data between primary and carcinoma epithelial cells, all other experiments were performed with TR146, FaDu and A431 cells.u201d In addition, the Primary cell p24 panel has been updated in the revised Fig 3A so that the aspect ratio in the figure panel aligns more closely with the original blot image (see S1 File). The authors apologize for the errors in the published article and confirm that the original data underlying other results in this article are available upon request. , 
 Here are the original concerns as reported to the journal in May 2014, plus an additional finding shown with pink boxes, that I noticed just now. * Red boxes:  In Fig 3A, the p24 panels for A431 and Primary cell lanes appear to be the same.  * Boxes in three shades of blue:  In Fig 3A, the u03b1-actin control panels for A431 and Primary cell lanes appear to be the same as lanes 3, 4, and 5 of the TR146 FaDu u03b1-actin panel. In Fig 4B, the u03b1-actin control panel for A431 appears to be identical to lanes 2, 3, and 4 of the u03b1-actin panel for TR146 and FaDu cell lines. * Pink boxes: The P55 lanes of TR146 and FaDu look remarkably similar to those of A431. ![file] Here is a closeup of Figure 4B, with enhanced contrast to show the striking similarities between the two p55 panels, marked in pink. Note how the A431 panel is supposed to show three lanes only, while six lanes appear to be visible, squeezed in the horizontal direction. ![file] One could wonder if the number of errors admitted by the authors could be adequately addressed with a correction, in particular in light of the new pink-marked concern. ,