Downloaded from www.microbiologyresearch.org by IP: 54.167.64.178 On: Mon, 18 Jul 2016 04:46:54 Complexity in human immunodeficiency virus type 1 (HIV-1) co-receptor usage: roles of CCR3 and CCR5 in HIV-1 infection of monocyte-derived macrophages and brain microglia Lokesh Agrawal, 1 Christina R. Maxwell, 1 Paul J. Peters, 2 Paul R. Clapham, 2 Sue M. Liu, 3 Charles R. Mackay 3 and David S. Strayer 1 Correspondence Lokesh Agrawal lokesh.agrawal@jefferson.edu 1 Department of Pathology and Cell Biology, Thomas Jefferson University, Philadelphia, PA 19107, USA 2 Department of Molecular Genetics and Microbiology, University of Massachusetts Medical School, Worcester, MA 01605, USA 3 Garvan Institute of Medical Research, 384 Victoria St, Darlinghurst, NSW 2010, Australia Received 1 August 2008 Accepted 24 November 2008 CCR3 has been implicated as a co-receptor for human immunodeficiency virus type 1 (HIV-1), particularly in brain microglia cells. We sought to clarify the comparative roles of CCR3 and CCR5 in the central nervous system (CNS) HIV-1 infection and the potential utility of CCR3 as a target for manipulation via gene transfer. To target CCR3, we developed a single-chain antibody (SFv) and an interfering RNA (RNAi), R3-526. Coding sequences for both were cloned into Tag- deleted SV40-dervied vectors, as these vectors transduce brain microglia and monocyte-derived macrophages (MDM) highly efficiently. These anti-CCR3 transgenes were compared to SFv- CCR5, an SFv against CCR5, and RNAi-R5, an RNAi that targets CCR5, for the ability to protect primary human brain microglia and MDM from infection with peripheral and neurotropic strains of HIV-1. Downregulation of CCR3 and CCR5 by these transgenes was independent from one another. Confocal microscopy showed that CCR3 and CCR5 co-localized at the plasma membrane with each other and with CD4. Targeting either CCR5 or CCR3 largely protected both microglia and MDM from infection by many strains of HIV-1. That is, some HIV-1 strains, isolated from either the CNS or periphery, required both CCR3 and CCR5 for optimal productive infection of microglia and MDM. Some HIV-1 strains were relatively purely CCR5-tropic. None was purely CCR3-tropic. Thus, some CNS-tropic strains of HIV-1 utilize CCR5 as a co-receptor but do not need CCR3, while for other isolates both CCR3 and CCR5 may be required. INTRODUCTION Several chemokine receptors are used, together with CD4, for human immunodeficiency virus type 1 (HIV-1) entry into cells (Deng et al., 1996; Feng et al., 1996). T-cell tropic (T-tropic) viruses use CXCR4 as a co-receptor (Feng et al., 1996), whereas macrophage-tropic (M-tropic) HIV-1 uses CCR5 (Choe et al., 1998). Some strains of HIV-1 may bind CCR3 and other chemokine receptors and use them to enter cells that express them following stable transfection (Doranz et al., 1996), but the role of CCR3 in primary cells in HIV-1 infection is unclear. CCR5 is the major co-receptor for M-tropic HIV-1. It is utilized for HIV-1 entry into macrophages in the peripheral immune system and into brain microglia cells (Albright et al., 1999; Gorry et al., 2001; Shieh et al. , 1998; Smit et al., 2001). It is mainly expressed on eosinophils, microglial cells, basophils and some T lymphocytes (Berger et al., 1999). The extent to which it functions as a co-receptor for HIV-1 entry into central nervous system (CNS) cells is conjectural, but considerable data suggest that some forms of HIV-1 envelope may bind to CCR3, so it could act as a minor co-receptor for HIV-1 entry (Alkhatib et al. , 1997; Choe et al. , 1996; Ghorpade et al., 1998; He et al., 1997; Ho et al. , 2004; Martin-Garcia et al. , 2002; Peters et al., 2004; Shieh et al., 1998). The cloned envelopes of many primary HIV-1 isolates show comparable tropism for CCR3 and CCR5 (Aasa- Chapman et al. , 2006). We approached the study of the roles of CCR3 and CCR5 in CNS HIV-1 infection from the perspective of the potential therapeutic applicability of targeting CCR3 and CCR5 to protect CNS and monocyte-derived macrophages (MDM) A supplementary table is available with the online version of this paper. Journal of General Virology (2009), 90, 710–722 DOI 10.1099/vir.0.006205-0 710 006205 G 2009 SGM Printed in Great Britain