Downloaded from www.microbiologyresearch.org by IP: 54.70.40.11 On: Sun, 25 Nov 2018 08:54:43 Journal of General Virology (1999), 80, 1975–1982. Printed in Great Britain ................................................................................................................................................................................................................................................................................... Mutations in the env gene of human immunodeficiency virus type 1 NDK isolates and the use of African green monkey CXCR4 as a co-receptor in COS-7 cells Julie Dumonceaux, 1 Chantal Chanel, 1 Susana Valente, 1 Laurence Quivet, 1 Pascale Briand 1 and Uriel Hazan 1, 2 1 INSERM Unite  380 Laboratoire de Ge  ne  tique et Pathologie Expe  rimentales, Institut Cochin de Ge  ne  tique Mole  culaire, 22 rue Me  chain, 75014 Paris, France 2 Universite  Paris 7 Denis Diderot, UFR de Biochimie, 75251 Paris Cedex 05, France A previous report from this laboratory described the isolation of the first CD4-independent human immunodeficiency virus type 1 isolate, m7NDK. This independence of CD4 is due to seven mutations located in the C2, V3 and C3 regions of the gp120 protein. The present report describes the entry features of the m5NDK virus, which contains five of the seven m7NDK mutations, located in the V3 loop and C3 region. The entry of this virus is strictly CD4-dependent but it can fuse with African green monkey (agm) COS-7 cells bearing human CD4 (h-CD4). This fusion is directly due to the five mutations in the env gene. It has also been shown that entry of m7NDK is CD4-independent in COS-7 cells. Since the wild-type NDK and m7NDK viruses use the human CXCR4 protein as co- receptor, agm-CXCR4 was cloned and used in transfection and fusion inhibition experiments to show that this receptor can be used by the m5 and m7NDK viruses. The wild-type NDK virus, which does not enter COS-7 cells, can use agm-CXCR4, but only when the receptor is transfected into target cells. Although co-receptor nature and expression levels are still major determinants of virus entry, this is the first case where a few mutations in the env gene can overcome this restriction. Introduction CD4 is the primary receptor for the human immuno- deficiency viruses HIV-1 and HIV-2, but binding to it is not sufficient for infection. Membrane fusion requires a cell-surface cofactor (or co-receptor), which belongs to the family of seven- transmembrane-domain G-coupled protein receptors (Berson et al., 1996) (for reviews see Berson & Doms, 1998; Doms & Peiper, 1997 ;Moore et al., 1997). A number of cofactors have been identified, the most common being CXCR4 (Feng et al., 1996) and CCR5 (Alkhatib et al., 1996; Deng et al., 1996; Dragic et al., 1996). CXCR4 and CCR5 allow membrane fusion with X4 and R5 strains, respectively (Berger et al., 1998). Other receptors have been characterized in vitro and allow less efficient virus entry ; they include CCR2b (Zhang et al., 1997), CCR3 (He et al., 1997 ; Rucker et al., 1997), CCR8 (Rucker et al., 1997), CX3CR1V28 (Combadiere et al., 1998 ; He et al., 1997 ; Author for correspondence : Uriel Hazan (at INSERM). Fax 33 1 40 51 64 07. e-mail hazanicgm.cochin.inserm.fr Reeves et al., 1997; Rucker et al., 1997), US28 (Pleskoff et al., 1997 b), STRL33 (or Bonzo) (Alkhatib et al., 1997 ; Deng et al., 1997 ; Li et al., 1990 ; Liao et al., 1997), GPR15 (or Bob) (Deng et al., 1997; Farzan et al., 1997), apj and CCR9 (Choe et al., 1998). A few HIV isolates can use simian (rhesus and fascicularis monkey) (Himathongkham & Luciw, 1996), murine (Bieniasz et al., 1997 ; Tachibana et al., 1997), feline (Willett et al., 1997) or rat (Pleskoff et al., 1997 a) CXCR4 when the receptor is expressed in transfected human CD4-positive, CXCR4-nega- tive cells. No HIV enters the simian cell line COS-7, derived from African green monkey (agm), under normal conditions of co-receptor expression. The nature of the co-receptor, the cell type producing the co-receptor and the presence of human (h-) CD4 seem to be the most important factors determining virus entry (Dimitrov, 1997). While CD4 is almost always required, two CD4-inde- pendent HIV isolates have been characterized after long-term culture, the HIV-2 RodB isolate (Clapham et al., 1992 ; Reeves & Schulz, 1997) and the HIV-1 m7NDK virus (Dumonceaux et 0001-6314  1999 SGM BJHF