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Journal of Virology, September 1998, p. 7125-7136, Vol. 72, No. 9
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

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

Benoit Barbeau, Jean-François Fortin, Nicolas Genois, and Michel J. Tremblay*

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

Received 17 February 1998/Accepted 26 May 1998

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.

* Corresponding author. Mailing address: Unité d'ImmunoRétrovirologie Humaine, Centre de Recherche en Infectiologie, RC709, Centre Hospitalier Universitaire de Québec, Pavillon CHUL, 2705 blvd. Laurier, Ste-Foy, Québec, Canada G1V 4G2. Phone: (418) 654-2705. Fax: (418) 654-2715. E-mail: michel.j.tremblay{at}crchul.ulaval.ca.

Journal of Virology, September 1998, p. 7125-7136, Vol. 72, No. 9
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


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