Isolated Human Astrocytes Are Not Susceptible to Infection by M- and T- Tropic HIV-1 Strains Despite Functional Expression of the Chemokine Receptors CCR5 and CXCR4 AGNE ` S BOUTET, 1 * HASSAN SALIM, 2 YASSINE TAOUFIK, 1 PIERRE-MARIE LLEDO, 2 JEAN-DIDIER VINCENT, 2 JEAN-FRANC ¸ OIS DELFRAISSY, 1 AND MARC TARDIEU 1 1 Laboratoire d’immunologie antivirale syste ´mique et ce ´re ´brale, Inserm E 0109, Faculte ´ de Me ´decine Paris-Sud, Le Kremlin-Bice ˆtre Cedex, France 2 CNRS, Institut de Neurobiologie Alfred-Fessard, UPR 2197, Gif-sur-Yvette Cedex, France. KEY WORDS chemokine receptors; CD4; HIV; astrocytes; microglial cells; development ABSTRACT Within the brain, HIV-1 targets the microglia and astrocytes. Previous studies have reported that viral entry into astrocytes is independent of CD4, in contrast to microglia. We aimed to determine whether chemokine receptors play a role in mediating CD4-independent HIV-1 entry into astrocytes. We found that embryonic astrocytes and microglial cells express CCR5, CCR3, and CXCR4 transcripts. Intracel- lular calcium levels in astrocytes were found to increase following application of RAN- TES, MIP-1 (CCR5-agonist), SDF-1 (CXCR4-agonist), but not eotaxin (CCR3-agonist). In microglial cells, eotaxin was also able to modulate internal calcium homeostasis. CD4 was not present at the cell surface of purified astrocytes but CD4 mRNA could be detected by RT-PCR. Neither HIV-1 9533 (R5 isolate) nor HIV-1 LAI (X4 isolate) penetrated into purified astrocytes. In contrast, mixed CNS cell cultures were infected by HIV-1 9533 and this was inhibited by anti-CD4 mAb in 4/4 tested cultures and by anti-CCR5 mAb in 2/4. Thus, the HIV-1 R5 strain requires CD4 to penetrate into brain cells, suggesting that CCR5 cannot be used as the primary receptor for M-tropic HIV-1 strains in astrocytes. Moreover, inconstant inhibition of HIV-1 entry by anti-CCR5 mAb supports the existence of alternative coreceptors for penetration of M-tropic isolates into brain cells. GLIA 34:165–177, 2001. © 2001 Wiley-Liss, Inc. INTRODUCTION The cellular tropism displayed by different human im- munodeficiency virus type 1 (HIV-1) isolates is related to their use of specific chemokine receptors that mediate HIV-1 entry in conjunction with CD4. Virus strains that infect primary T cells and transformed T-cell lines (T- tropic strains) use the -chemokine receptor CXCR4 (X4 strains), while those that replicate in primary T cells and macrophages (M-tropic strains) typically use the -che- mokine receptor CCR5 (R5 strains) (Berger et al., 1998; Miller and Meucci, 1999). Other chemokine receptors such as CCR2b, CCR3, CCR8, CCR9, CX 3 CR1, and APJ in addition to some orphan receptor such as GPR1, GPR15, and STRL33 have been shown to bind HIV and SIV envelopes (for review, see Berger et al., 1999). In vivo, the contribution of these alternative receptors to infection and pathogenesis remains obscure. HIV-1 invades the Abbreviations used: AIDS, acquired immunodeficiency syndrome; FCS, fetal calf serum; FITC, fluorescein isothiocyanate; GAPDH, glyceraldehyde phospho- dehydrogenase; GFAP, glial fibrillary acidic protein; GPCR, G protein-coupled receptor; HIV-1, human immunodeficiency virus type 1; LTR, long terminal repeat; MDM, monocyte-derived macrophage; MIP, macrophage inflammatory protein; PBMC, peripheral blood mononuclear cell; PBS, phosphate-buffered saline; PTX, pertussis toxin; RANTES, regulated on activation normal T ex- pressed and secreted; SDF, stromal cell-derived factor; SIV, simian immunode- ficiency virus. Grant sponsor: Institut National de la Recherche Me ´dicale (INSERM; Grant number: 96012; Grant sponsor: Centre National de la Recherche Scientifique (CNRS); Grant sponsor: Agence Nationale de Recherche sur le SIDA (ANRS); Grant sponsor: Universite ´ Paris-Sud. *Correspondence to: Dr. Agne `s Boutet. Laboratoire d’immunologie antivirale sys- te ´mique et ce ´re ´brale, Inserm E 0109, Faculte ´ de Me ´decine Paris-Sud, 63, rue Gabriel Pe ´ri, 94276 Le Kremlin-Bice ˆtre Cedex, France. E-mail: agnes.boutet@kb.u-psud.fr Received 22 September 2000; Accepted 23 February 2001 GLIA 34:165–177 (2001) © 2001 Wiley-Liss, Inc.