The International Journal of Biochemistry & Cell Biology 44 (2012) 556–562 Contents lists available at SciVerse ScienceDirect The International Journal of Biochemistry & Cell Biology jo ur nal homep ag e: www.elsevier.com/locate/biocel Thioredoxin-1 and protein disulfide isomerase catalyze the reduction of similar disulfides in HIV gp120 Kathrin Reiser a , Katrien O. Franc ¸ ois b , Dominique Schols b , Tomas Bergman c , Hans Jörnvall c , Jan Balzarini b , Anna Karlsson a , Mathias Lundberg a,∗ a Department of Laboratory Medicine, Division of Clinical Microbiology, F68, Karolinska Institutet, SE-14186 Huddinge, Sweden b Rega Institute for Medical Research, K.U. Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium c Department of Medical Biochemistry and Biophysics, Karolinska Institutet, SE-17177 Stockholm, Sweden a r t i c l e i n f o Article history: Received 26 September 2011 Received in revised form 13 December 2011 Accepted 22 December 2011 Available online 3 January 2012 Keywords: HIV gp120 Viral entry Protein disulfide isomerase Thioredoxin-1 Antiviral agents a b s t r a c t HIV-1 enters cells via interaction of the viral glycoprotein gp120, the host cell surface receptor CD4 and the co-receptors CCR5 or CXCR4. For entry, gp120 undergoes conformational changes that depend on the reduction of one or more disulfides. Previous studies indicate that protein disulfide isomerase (PDI), thioredoxin-1 (Trx1), and glutaredoxin-1 (Grx1) catalyze gp120 reduction, but their specific disulfide targets are not known. Here, it was demonstrated that PDI and Trx1 have similar gp120 disulfide targets as determined by labeling after reduction, but with some pattern differences, including overall stronger labeling with Trx1 than with PDI. Furthermore, uneven labeling of the residues of a disulfide may reflect altered accessibility by conformational changes upon the reduction process. Since both PDI and Trx1 may be involved in viral entry, compounds that target the host redox system or the viral gp120 were tested in vitro to investigate whether redox regulation is a target for anti-HIV ther- apy. Carbohydrate binding agents (CBAs), previously shown to bind gp120 and inhibit HIV entry, were now demonstrated to inhibit gp120 disulfide reduction. Auranofin, an inhibitor of thioredoxin reductase 1 (TrxR1), also showed inhibitory activity towards HIV infection, although close to its cytotoxic concen- tration. Our results demonstrate that both the host redox system and the viral surface glycoproteins are of interest for the development of new generations of anti-HIV therapeutics. © 2012 Elsevier Ltd. All rights reserved. 1. Introduction HIV entry is initiated by the binding of the viral glycoprotein gp120 to the CD4 receptor of the target cell. gp120 subsequently undergoes a conformational change and interacts with the cellular chemokine co-receptors CXCR4 or CCR5. This leads to the inser- tion of gp41 into the cellular membrane, which mediates fusion of the viral envelope with the host cell (Ryser and Fluckiger, 2005). It is believed that redox active enzymes are responsible for these conformational changes by the reduction of important disulfides (Barbouche et al., 2003, 2005; Fenouillet et al., 2001; Gallina et al., 2002; Markovic et al., 2004; Ryser and Fluckiger, 2005). Abbreviations: aPAO, p-aminophenylarsenoxide; CAM, carbamidomethyl; CBA, carbohydrate binding agent; DTNB, 5,5-dithiobis(2-nitrobenzoic acid); DTT, dithio- threitol; GR, glutathione reductase; Grx1, glutaredoxin-1; GSH, glutathione; NEM, N-ethylmaleimide; PBMC, peripheral blood mononuclear cells; PDI, protein disul- fide isomerase; Trx1, thioredoxin-1; TrxR1, thioredoxin reductase 1. ∗ Corresponding author. Tel.: +46 8 52483616; fax: +46 8 58587933. E-mail address: Mathias.Lundberg@ki.se (M. Lundberg). There are nine highly conserved disulfide bonds in gp120 (Louwagie et al., 1995), which are important for folding and func- tion (van Anken et al., 2008). Three of these disulfide bonds are located within the V1/V2 loop, one at the base of the V3 loop and two within an area important for CD4 binding (V4 loop) (Kwong et al., 1998). Based on structural data of complexed gp120–CD4, all of these six disulfide bonds are in proximity to at least one amino acid of gp120 that makes contact to the cellular CD4 receptor (Gallina et al., 2002; Kwong et al., 1998). Studies indicate that protein disulfide isomerase (PDI) catalyzes the reduction of the disulfide bonds required for viral entry in vitro (Fenouillet et al., 2007; Ryser and Fluckiger, 2005; Ryser et al., 1994). PDI belongs to the thioredoxin superfamily of redox pro- teins and its reducing activity is dependent on electron transfer from either reduced glutathione (GSH) or thioredoxin reductase 1 (TrxR1). PDI has been detected at the cell surface, but its main function is in the endoplasmic reticulum, where it acts as an iso- merase and chaperone facilitating protein folding (Noiva, 1999; Wang, 1998). Inhibition of PDI activity led to a decrease in HIV-1 replication by inhibition of viral entry in cultured cells (Barbouche et al., 2003; Fenouillet et al., 2001; Gallina et al., 2002; Markovic et al., 2004; Ryser et al., 1994). This suggests that PDI-dependent 1357-2725/$ – see front matter © 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.biocel.2011.12.015