1311 © Rapid Science Publishers ISSN 0269-9370 Introduction HIV-1 is able to recognize at least two distinct cellular receptors: CD4, which is expressed by CD4+ lympho- cytes and monocyte-derived macrophages [1], and galactosylceramide (GalCer), a glycosphingolipid found in the plasma membrane of oligodendrocytes [2] and intestinal epithelial cells [3]. The characterization of GalCer as an alternative receptor for HIV-1 has been done on the following basis: (i) anti-GalCer antibodies inhibit HIV-1 entry into CD4-/GalCer+ neural and colonic cell lines [2–5]; (ii) GalCer binds HIV-1 surface envelope glycoprotein gp120 with a high affinity [2,6,7]; (iii) inhibitors of gp120 binding to GalCer Co-expression of CXCR4/fusin and galactosylceramide in the human intestinal epithelial cell line HT-29 Olivier Delézay, Nathalie Koch*, Nouara Yahi*, Djilali Hammache, Christian Tourres*, Catherine Tamalet* and Jacques Fantini Objective: To detect the expression CXCR4/fusin in human intestinal epithelial cells and to assess its potential role in the pathway of HIV-1 infection mediated by the alternative gp120 receptor galactosylceramide (GalCer). Methods: GalCer+ (HT-29, HT-29/CD4+) and GalCer- (Caco-2/Cl2, Cl14 and Cl14/CD4+) human intestinal cell lines were analysed for CXCR4/fusin expression using the monoclonal antibody (MAb) 12G5. This MAb was then evaluated for its ability to inhibit HIV-1 infection in permissive cells. HIV-1 infection was measured by detection of p24 antigen, polymerase chain reaction amplification, and cocultivation with CD4+ cells. Results: CXCR4/fusin was detected on the surface of HT-29 and HT-29/CD4+, but not on Caco-2/Cl2, Cl14 and Cl14/CD4+ cells. Ninety per cent of CXCR4/fusin+ HT-29 and HT-29/CD4+ cells co-expressed GalCer. Infection of HT-29 cells by laboratory isolates of HIV-1 was inhibited by both anti-GalCer and anti-CXCR4/fusin MAbs. Expression of CD4 rendered HT-29 cells sensitive to HIV-1(89.6), a macrophage-tropic isolate that does not recognize GalCer. The 12G5 MAb blocked HIV-1 infection of HT-29/CD4+ cells. In contrast, the expression of HIV-1 receptors, i.e., CD4, GalCer or both, into CXCR4/fusin-negative intestinal cells did not confer sensitivity to HIV-1 infection. The resulting receptor-positive cell lines could, however, bind HIV-1, whereas the original cell lines could not. Conclusion: HIV-1 entry into human intestinal cells involves both GalCer and CXCR4/fusin. HIV-1 isolates such as 89.6 that are able to use CXCR4/fusin as coreceptor, but do not bind to GalCer, do not infect these cells. These data raise the possibility that CXCR4/fusin may function as a coreceptor for HIV-1 entry into CD4-/GalCer+ intestinal epithelial cells. AIDS 1997, 11:1311–1318 Keywords: fusin, galactosylceramide, coreceptor, glycolipids, CD4, fusion, intestinal cells From the Laboratoire de Biochimie et Biologie de la Nutrition, URA-CNRS 1820, Faculté des Sciences de St Jérôme, 13397 Marseille Cedex 20 and the *Laboratoire de Virologie, Unité Fonctionnelle SIDA, Hôpital de la Timone, 13005 Marseille, France. Sponsorship: This work was supported by a grant (to J.F.) and a fellowship (to O.D.) from the Fondation pour la Recherche Médicale (SIDACTION). Requests for reprints to: Dr Jacques Fantini, Laboratoire de Biochimie et Biologie de la Nutrition, Unité Propre de Recherche de l’Enseignement Supérieur associée au Centre National de la Recherche Scientifique A6033 1820, Faculté des Sciences de St Jérôme, Service 342, 13397 Marseille Cedex 20, France. Date of receipt: 30 January 1997; revised: 15th May 1997; accepted: 27 May 1997.