Modulation of human immunodeficiency virus type 1-induced syncytium formation by the conformational state of LFA-1 determined by a new luciferase-based syncytium quantitative assay.
The ICAM-1/LFA-1 interaction has been clearly demonstrated to play an active role in syncytium formation induced by human immunodeficiency virus type 1 (HIV-1). Since it is known that a high-affinity state of LFA-1 for ICAM-1 can be induced through conformational change, such a high-affinity state may also contribute to the process of syncytium formation. In this study, we have investigated the involvement of the conformational status of LFA-1 in HIV-1-dependent syncytium formation by using the anti-LFA-1 antibody NKI-L16, which is known to activate the high-affinity state. Initial visual observations by light microscopy indeed suggested that the addition of the NKI-L16 antibody led to bigger and more numerous syncytia when different cell lines were tested. To further analyze this NKI-L16-dependent increment of syncytium formation in a quantitative assay, a new luciferase-based assay was developed by using a T-cell line containing an HIV-1 long terminal repeat (LTR)-driven luciferase construct (1G5) in coincubation with an HIV-1-positive cell line (J1.1). Upon fusion, the viral Tat protein could diffuse to the 1G5 cells, leading to a transcriptional increase of the HIV-1 LTR-driven luciferase gene. Initial evaluation of this assay showed a good correlation between the level of syncytium formation determined by microscopic observation and the level of measured luciferase activity. In addition, this assay showed a greater induction of enzymatic activity correlating with syncytium formation in comparison to a similar incubation with the HeLa-CD4-LTR-beta-gal indicator cell line. By using this test, NKI-L16 treatment of 1G5/J1.1 cells led to a three- to sevenfold increase in HIV-1 LTR-driven luciferase activity. The syncytium-dependent luciferase activity in NKI-L16-treated cells could be blocked by classical syncytium inhibitors such as soluble CD4, anti-CD4, and anti-gp120 antibodies. Inhibition could also be observed with specific blocking agents for the chemokine receptor CXCR4, as well as with soluble ICAM-1, anti-LFA-1, anti-ICAM-1, and anti-ICAM-2 blocking antibodies, indicating the requirement for the LFA-1/ICAM interaction. Treatment of peripheral blood mononuclear cells with NKI-L16 resulted in a higher level of syncytium formation in the presence of the cell line J1.1. Conversely, when PBMCs were infected with two different syncytium-inducing HIV-1 primary isolates, coincubation with NKI-L16-pretreated 1G5 cells led to higher levels of luciferase activity for both virus isolates. Our results therefore show for the first time a direct role for the LFA-1 high-affinity state in virus-mediated syncytium formation. Based on the demonstration that an increase in ICAM-1 binding is induced by T-cell activation, these data suggest an in vivo involvement of the high-affinity state of LFA-1 in HIV-1-induced syncytium formation. Moreover, syncytia might preferentially occur in lymph nodes, since this microenvironment harbors a high proportion of activated T cells.
Centre de Recherche en Infectiologie, Centre Hospitalier Universitaire de Québec, Pavillon CHUL, and Département de Biologie médicale, Faculté de Médecine, Université Laval, Ste-Foy, Québec, Canada G1V 4G2.
B
B
Barbeau
J F
JF
Fortin
N
N
Genois
M J
MJ
Tremblay
D000906:Antibodies / Q000276:immunology; D015703:Antigens, CD / Q000378:metabolism; D015704:CD4 Antigens / Q000378:metabolism; D015815:Cell Adhesion Molecules / Q000378:metabolism; D015726:Giant Cells; D015497:HIV-1 / Q000502:physiology*; D006367:HeLa Cells; D006801:Humans; D018799:Intercellular Adhesion Molecule-1 / Q000378:metabolism; D019169:Jurkat Cells; D007963:Leukocytes, Mononuclear; D008156:Luciferases / Q000378:metabolism*; D016169:Lymphocyte Function-Associated Antigen-1 / Q000276:immunology / Q000378:metabolism*; D019718:Receptors, CXCR4 / Q000378:metabolism; D014407:Tumor Cells, Cultured
D000906:Antibodies; D015703:Antigens, CD; D015704:CD4 Antigens; D015815:Cell Adhesion Molecules; C065289:ICAM2 protein, human; D016169:Lymphocyte Function-Associated Antigen-1; D019718:Receptors, CXCR4; D018799:Intercellular Adhesion Molecule-1; D008156:Luciferases