No abstract

We previously reported the presence of two cellular serine/threonine protein kinases incorporated in human immunodeficiency virus type 1 (HIV-1) particles. One protein kinase is MAPK ERK2 (mitogen-activated protein kinase), whereas the other one, a 53-kDa protein, still needs to be identified. Furthermore, we demonstrated that the capsid protein CAp24 is phosphorylated by one of those two virion-associated protein kinases (Cartier, C., Deckert, M., Grangeasse, C., Trauger, R., Jensen, F., Bernard, A., Cozzone, A., Desgranges, C., and Boyer, V. (1997) J. Virol. 71, 4832-4837). In this study, we showed that CAp24 is not a direct substrate of MAPK ERK2. Moreover, using site-directed mutagenesis of each of the 9 serine residues of CAp24, we demonstrated the phosphorylation of 3 serine residues (Ser-109, Ser-149, and Ser-178) in the CAp24. Substitution of each serine residue did not affect viral budding, nor viral structure. By contrast, substitution of Ser-109, Ser-149, or Ser-178 affects viral infectivity by preventing the reverse transcription process to be completely achieved. Our results suggest that CAp24 serine phosphorylation is essential for viral uncoating process.

We have developed an in vitro reconstructed vaginal mucosa integrating Langerhans cells (LCs), obtained by differentiation of umbilical cord blood CD34(+) hematopoietic progenitor cells, and, in this model, we have investigated the infection of LCs by human immunodeficiency virus type 1 (HIV-1). Proviral DNA of X4 (LAI and NL4-3) and R5 (Ba-L) HIV-1 strains were detected in LCs integrated in the reconstituted mucosa. Infection of LCs could be specifically inhibited by the chemokines stromal cell-derived factor 1 (SDF-1) and RANTES (regulated on activation, normally T cell-expressed and -secreted), confirming the presence of functional coreceptors on LCs generated in vitro. A complete inhibition of LCs, by use of azidothymidine, a reverse-transcriptase inhibitor, was also observed. Moreover, HIV-1-infected LCs of the reconstructed mucosa were able to transmit R5 or X4 HIV-1 strains to activated peripheral blood mononuclear cells. Such a model could be a useful tool to study the mechanisms involved in transmission of HIV in the female genital tract.

All three-dimensional in vitro mucosal models constructed, thus far, have only been reconstituted by epithelial cells. We have developed a reconstructed oral and vaginal epithelium that integrates Langerhans' cells (LC), the dendritic cells (DC) of malpighian epithelia. The epithelium was composed of gingival or vaginal keratinocytes seeded on a de-epidermized dermis (DED) and grown in submerged culture for 2 weeks. LC precursors, obtained after differentiation of cord blood-derived CD34+ hematopoietic progenitor cells (CD34+HPC) by granulocyte macrophage-colony stimulating factor (GM-CSF), tumor necrosis factor-alpha (TNF-alpha), transforming growth factor-beta (TGF-beta) and Flt3-ligand (Flt3-L), were introduced after 6-8 days of culture into the reconstituted epithelium. The in vitro reconstituted mucosal epithelium formed a multilayered, well-differentiated epithelial structure, confirmed by the immunohistochemical expression of cytokeratins 4, 6, 10, 13, 14, 16 and involucrin. LC were identified in the basal and suprabasal epithelial layers by CD1a antigen, S100 protein and Langerin/CD207 expression, and by transmission electron microscopy. Type IV collagen was expressed at the chorio-epithelial junction, and most ultrastructural features of this junction were visualized by electron microscopy. This in vitro reconstructed gingiva or vagina integrating LC represents interesting models very similar to native tissues. Because LC play an important role in the mucosal immune system, our models could be useful for conducting studies on interactions with pathogenic agents (viruses, bacteria etc.), as well as in pharmacological, toxicological and clinical research.