Entry of hepatitis C virus and human immunodeficiency virus is selectively inhibited by carbohydrate-binding agents but not by polyanions

Virology 366 (2007) 40 – 50 www.elsevier.com/locate/yviro

Entry of hepatitis C virus and human immunodeficiency virus is selectively inhibited by carbohydrate-binding agents but not by polyanions Claire Bertaux a , Dirk Daelemans b , Laurent Meertens a , Emmanuel G. Cormier a , John F. Reinus c , Willy J. Peumans d , Els J.M. Van Damme d , Yasuhiro Igarashi e , Toshikazu Oki f , Dominique Schols b , Tatjana Dragic a , Jan Balzarini b,⁎ a

Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, New York, USA b Rega Institute for Medical Research, K.U.Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium c Division of Gastroenterology and Hepatology, Montefiore Medical Center, Bronx, New York, USA d Department of Molecular Biology, UGent, Gent, Belgium e Biotechnology Research Center, Toyama Prefectural University, Toyama, Japan f Keck School of Medicine, University of Southern California, Los Angeles, USA Received 28 December 2006; returned to author for revision 24 January 2007; accepted 5 April 2007 Available online 11 May 2007

Abstract We studied the antiviral activity of carbohydrate-binding agents (CBAs), including several plant lectins and the non-peptidic small-molecularweight antibiotic pradimicin A (PRM-A). These agents efficiently prevented hepatitis C virus (HCV) and human immunodeficiency virus type 1 (HIV-1) infection of target cells by inhibiting the viral entry. CBAs were also shown to prevent HIV and HCV capture by DC-SIGN-expressing cells. Surprisingly, infection by other enveloped viruses such as herpes simplex viruses, respiratory syncytial virus and parainfluenza-3 virus was not inhibited by these agents pointing to a high degree of specificity. Mannan reversed the antiviral activity of CBAs, confirming their association with viral envelope-associated glycans. In contrast, polyanions such as dextran sulfate-5000 and sulfated polyvinylalcohol inhibited HIV entry but were devoid of any activity against HCV infection, indicating that they act through a different mechanism. CBAs could be considered as prime drug leads for the treatment of chronic viral infections such as HCV by preventing viral entry into target cells. They may represent an attractive new option for therapy of HCV/HIV coinfections. CBAs may also have the potential to prevent HCV/HIV transmission. © 2007 Elsevier Inc. All rights reserved. Keywords: Carbohydrate-binding agents (CBA); Transmission; DC-SIGN; Lectins; HCV; HIV

Introduction The hepatitis C virus (HCV) and the human immunodeficiency virus (HIV) are major human pathogens for which there is no curative treatment and no vaccine. HCV and HIV are transmitted by blood exchange and, particularly for HIV, also by sexual contact (Clarke and Kulasegaram, 2006; Lashley, 2006). Both viruses establish persistent infections, characterized by variable viremia and escape from immune surveillance through antigenic variation (Tan et al., 2006). HCV-infected individuals may remain asymptomatic or develop chronic hepatitis and ⁎ Corresponding author. Fax: +32 16 337340. E-mail address: [email protected] (J. Balzarini). 0042-6822/$ - see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.virol.2007.04.008

cirrhosis, the latter often leading to hepatocellular carcinoma (Trépo et al., 1999). HIV-infected individuals suffer progressive loss of CD4+ lymphocytes, which leads to degeneration of immune function and opportunistic infections. Because their routes of transmission are similar, the two viruses often coinfect hosts (Tan et al., 2006; Soriano et al., 2006). Most studies indicate that HIV exacerbates HCV-induced disease (Soriano et al., 2006). Even though HCV does not impact progression of acquired immunodeficiency syndrome (AIDS), it can lead to increased liver toxicity of antiviral agents targeting HIV. HCV and HIV are enveloped viruses that comprise extensively glycosylated envelope glycoproteins. The E1 envelope glycoprotein of HCV contains 4 to 5 N-linked glycans and the E2 envelope glycoprotein has 11 N-glycosylation sites (Meunier

C. Bertaux et al. / Virology 366 (2007) 40–50

et al., 1999; Drummer et al., 2003; Voisset and Dubuisson, 2004; Goffard et al., 2005). Interestingly, the glycosylation sites on E1 and E2 are highly conserved and appear to comprise a mixture of complex and high-mannose side-chains (Goffard and Dubuisson, 2003; Voisset and Dubuisson, 2004; Zhang et al., 2004a). Depending on the isolate, the HIV-1 envelope gp41 subunit carries 5 to 7 N-glycans whereas envelope gp120 carries an average of 20 to 30 N-glycans (Leonard et al., 1990; Gallaher et al., 1995). Approximately 50% of HIV-1 envelope-associated glycans are in shifting positions whereas the rest are in fixed positions (Zhang et al., 2004a). It is notable that shifting glycosylation sites are associated with complex carbohydrates whereas all high-mannose or mixed carbohydrates are in fixed positions (Zhang et al., 2004a). HCVand HIV glycans have been implicated in numerous roles including envelope glycoprotein folding and formation of the HCV E1E2 complexes (Meunier et al., 1999; Slater-Handshy et al., 2004; Goffard et al., 2005), receptor interactions and virus entry (Goffard et al., 2005), capture by C-type lectins (Geijtenbeek et al., 2000; Lai et al., 2006) and antigenic variation (Slater-Handshy et al., 2004). This last property can affect both antibody recognition as well as T cell responses. Alteration of glycosylation sites can therefore have profound consequences for the replication and transmission of these viruses. Both HCV and HIV-1 interact with two closely related membrane-associated C-type lectins, DC-SIGN and DCSIGNR (L-SIGN) (Geijtenbeek et al., 2000; Lai et al., 2006). DC-SIGN is expressed on some dendritic cells, while DC-SIGNR expression is associated with certain endothelial cell populations, including liver sinusoidal endothelial cells (Pöhlmann et al., 2003; Lai et al., 2006). Moreover, capture of HCV or HIV particles by SIGN-positive cells facilitates virus transmission to proximal lymphocytes or hepatocytes, respectively (Geijtenbeek et al., 2000; Lozach et al., 2004; Wang et al., 2004). It has been suggested that this mechanism is involved in viral dissemination within the host and the establishment of persistent viral infection (Pöhlmann et al., 2003; Lozach et al., 2004; Cormier et al., 2004). DC-SIGN and DC-SIGNR efficiently bind HCV E2 as well as HIV gp120 glycoprotein and binding is entirely dependent on the presence of high-mannose glycans (Lozach et al., 2003, 2004). The observation that high-mannose glycans are favored in fixed positions on gp120 could mean that their conservation is crucial for transmission of the virus via Ctype lectin interactions. Whereas capture and transmission of HCV and HIV may occur through common SIGN-positive carriers, the entry receptors for HCV and HIV are entirely different. HCV entry requires the CD81 tetraspanin (Wunschmann et al., 2000; Flint et al., 2001) as well as other hepatocytes-specific molecules. HCV entry cofactors include the scavenger receptor type B1 (SR-BI), the low density lipoprotein receptor (LDL-R) and glycosaminoglycans (Barth et al., 2003; Bartosch et al., 2003a, 2003b). Although these receptor molecules were shown to play a role in HCV entry, other cellular receptors involved in HCV entry remain to be identified (Voisset and Dubuisson, 2004). HIV uses CD4 and subsequently CXCR4 or CCR5 in order to

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enter lymphocytes or macrophages (Berger et al., 1999; Pöhlmann and Doms, 2002). Gp120-associated glycans adjacent to and within the variable loops 1, 2 and 3 have been shown to regulate HIV-1 coreceptor interactions (Ogert et al., 2001). We and others have previously demonstrated that carbohydrate-binding agents (CBAs) such as mannose- and GlcNAcspecific plant lectins efficiently inhibit HIV infection and prevent virus entry into its target cells (Lifson et al., 1986; Hansen et al., 1989; Balzarini et al., 1991, 1992, 2004a). The plant lectins strongly interact with the glycans present on the HIV gp120 envelope and prevent a step in the HIV entry process subsequent to CD4 receptor binding (Balzarini et al., 1991, 1992). Moreover, we have shown that the presence of CBAs in cell culture forces HIV-1 to progressively lose its glycosylation sites on gp120, thereby uncovering immunogenic epitopes on this protein (Balzarini et al., 2004b, 2005a, 2005b). Based on these findings, we proposed that CBA might exert a dual antiviral effect (Balzarini, 2005) by inhibiting entry and triggering neutralizing antibody production against cryptic epitopes. Although data supporting this concept have been indirectly provided for HIV (Reitter et al., 1998), we believe that it might extend to other chronic infections by viruses that comprise heavily glycosylated envelope glycoproteins, such as HCV. Here, we studied the ability of several plant lectins and a small molecule carbohydrate-binding agent (CBA) designated pradimicin A (Balzarini et al., 2007b) to inhibit infection of target cells by HCV as well as several other enveloped (control) viruses. We found that molecules with mannose and GlcNAc specificity markedly inhibit HCV and HIV entry into their target cells, presumably by binding to the glycans present on the viral envelope glycoproteins. CBA also prevented virus capture mediated by DC-SIGN-expressing cells. Therefore, these agents may also be considered as a novel tool to prevent virus transmission and infection (entry). The discovery that the nonpeptidic small-molecular weight CBA pradimicin A is effective against HCV and HIV in cell culture proves that development of CBAs as therapeutic agents is a realistic and achievable goal in the clinical setting. Results CBA and polyanions inhibit HCV and HIV replication in target cells We evaluated the ability of carbohydrate-binding agents and polyanions to inhibit replication of HCV (HCVcc, subtype 2a) in human hepatocellular carcinoma cells (Huh7) as well as replication of HIV-1 (strain IIIB) and HIV-2 (strain ROD) in human T lymphocyte cells (CEM) (Table 1). The mannosespecific plant lectins GNA (Galanthus nivalis agglutinin), HHA (Hippeastrum hybrid agglutinin) and CA (Cymbidium agglutinin), as well as the mannose-specific non-peptidic antibiotic pradimicin A (PRM-A) inhibited infection by both types of viruses. EC50s varied over an order of magnitude in the submicromolar range, between 0.003 and 0.030 μM. In contrast, EC50s for PRM-A were in the low micromolar range (1.80 to

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Table 1 Antiviral activity of CBA and polyanions in cell cultures EC50 a (μM)

GNA HHA CA UDA PRM-A DS-5000 c PVAS c

CC50 b (μM) or MIC

CC50 b

HIV-1 (IIIB) (CEM)

HIV-2 (ROD) (CEM)

HCV (2a) (Huh7)

HSV-1 (KOS) (HEL)

HSV-2 (G) (HEL)

VSV (HeLa)

RSV (HeLa)

Parainfluenza-3 virus (Vero)

CEM, HEL, HeLa

Huh7

0.018 ± 0.0 0.006 ± 0.001 0.030 ± 0.010 0.140 ± 0.040 3.36 ± 1.2 0.40 ± 0.15 0.57 ± 0.25

0.011 ± 0.007 0.016 ± 0.0 0.009 ± 0.004 0.391 ± 0.106 1.80 ± 0.0 0.16 ± 0.0 0.48 ± 0.11

0.007 ± 0.003 0.003 ± 0.001 0.01 ± 0.009 0.176 ± 0.029 3.61 ± 0.78 N50 N50

N1.0 N1.0 ≥2.0 ≥5.7 N60 0.95 ± 0.007 0.7 ± 0.007

N1.0 N1.0 2.0 5.7 N60 0.65 ± 0.4 0.50 ± 0.1

N2.0 N0.4 4.0 N2.3 120 0.80 ± 0.20 0.75 ± 0.20

N2.0 N0.4 0.80 N2.3 120 0.80 ± 0.30 0.80 ± 0.30

N2.0 2.0 N4.0 N2.3 N120 N100 N100

N2 N2 N4 N5 N120 N100 N100

N10 N10 N10 N50 27 ± 0.84 N100 N100

a

50% effective concentration, or compound concentration required to inhibit virus-induced cytopathicity in HIV, HSV, VSV, RSV and parainfluenza virus-3infected cell cultures or luciferase activity in HCV-infected Huh7 cell cultures by 50%. Data are the means of at least two to three independent experiments (± SD). b Cytostatic/cytotoxic concentration, or compound concentration, required to inhibit CEM or Huh7 cell proliferation by 50% or to cause a microscopically visible altered morphology of HEL and HeLa cell cultures. c Data expressed in μg/ml.

3.61 μM). The GlcNAc-specific plant lectin UDA also markedly inhibited HCVcc and HIV infection with potencies similar to the mannose-binding lectins. Note that the potency of each CBA against HCV and HIV was within the same order of magnitude and there was a strong correlation between the inhibitory activity of the different CBAs against these two viruses (r = 0.934 (p b 0.05), Table 1 and Fig. 1, panel A). In other words, the more inhibitory a CBA was against HCV, the more inhibitory it also was against HIV. The inhibitory activity of the mannose-specific lectins and PRM-A against HCVcc and HIV-1 was efficiently diminished by the presence of mannan, but the antiviral activity of the

GlcNAc-specific UDA was decreased only by 3-fold (Table 2). Other enveloped viruses such as vesicular stomatitis virus (VSV), respiratory syncytial virus (RSV), parainfluenza virus-3 and herpes simplex virus (HSV) type 1 and type 2 were not markedly sensitive to the inhibitory activity of the CBA (Table 1). We included polyanions in these experiments as positive controls that would inhibit viral replication by binding to positively charged areas of receptors and/or envelope glycoproteins. Surprisingly however, DS-5000 and PVAS potently inhibited HIV-1 entry at 0.40 to 0.57 μg/ml and also affected replication of HSV, VSV and RSV, but were completely inactive against HCV up to a concentration of 50 μg/ml. The

Fig. 1. Correlation of the antiviral activities of CBA against HCV and HIV. (A) Correlation between the antiviral activity (EC50) of CBA against infectious HCVcc in Huh7 cells and HIV-1 in CEM cells. Correlation between the antiviral activity (EC50) of CBA against infectious HCV in Huh7 cells and HCVpp subtype 1a (B), 1b (C) and 2b (D).

C. Bertaux et al. / Virology 366 (2007) 40–50 Table 2 Effect of mannan on the antiviral activity of CBA and polyanions Compound

HHA GNA CA UDA PRM-A DS-5000 b PVAS b

HIV-1 EC50 a (μM)

HCV (2a) EC50 a (μM)

As such

+Mannan (2.5 mg/ml)

As such

+Mannan (2.5 mg/ml)

0.008 ± 0.005 0.013 ± 0.003 0.030 ± 0.010 0.149 ± 0.040 5.3 ± 0.78 0.45 ± 0.07 0.60 ± 0.28

0.50 ± 0.17 0.54 ± 0.22 – 0.459 ± 0.0 40 ± 13 0.30 ± 0.0 0.35 ± 0.07

0.003 ± 0.001 0.007 ± 0.003 0.012 ± 0.009 0.176 ± 0.029 3.61 ± 0.78 N50 N50

≫0.50 0.147 ± 0.039 0.047 ± 0.027 0.560 ± 0.108 ≫30 – –

a 50% effective concentration required to inhibit HIV-1-induced cytopathicity in CEM cell cultures or luciferase activity in HCV-infected Huh7 cell cultures by 50%. Data are the mean (±SD) of 2 to 3 independent experiments. b Data expressed in μg/ml.

polyanions also were not affected in their anti-HIV potential by the presence of mannan (Table 2). CBA and polyanions act at the level of viral entry We evaluated the ability of CBAs to inhibit entry of HCV pseudoparticles (HCVpp) into Huh7 cells. Fig. 2 shows that the CBAs inhibit HCVpp (subtype 1a) infection of Huh7 cells in a dose-dependent fashion. Similar to observations made with HCVcc, the mannose-specific CBAs – GNA, HHA and CA – most potently inhibited HCVpp (1a) infection, followed by GlcNAc-specific UDA and finally the mannose-specific nonpeptidic PRM-A (Fig. 2, Table 3). A similar dose-dependent inhibition by CBAs was observed with pseudoparticles bearing the envelope glycoproteins of HCV subtypes 1b and 2b (Table 3). In general, the inhibitory potential of the CBAs was even more pronounced against the HCVpp than against HCVcc, probably because the latter comprise a higher concentration of

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Table 3 Inhibition of viral entry by CBA in cell cultures using pseudotyped retroviral particles as the infectious agents Compound

GNA HHA CA UDA PRM-A DS-5000 b PVAS b

EC50 a (μM) HCVpp (1a) (Huh7)

HCVpp (1b) (Huh7)

HCVpp (2b) (Huh7)

VSVpp (C8166)

0.0009 ± 0.0002 0.0006 ± 0.0002 0.0026 ± 0.0007 0.032 ± 0.015 1.17 ± 0.31 N50 N50

0.001 ± 0.0003 0.0009 ± 0.0001 0.012 ± 0.005 0.019 ± 0.022 1.67 ± 0.06 N50 N50

0.0026 ± 0.0001 0.0011 ± 0.0004 0.0156 ± 0.0068 0.050 ± 0.054 0.924 ± 0.156 N50 N50

N2 – N4 N11 N60 – –

a

50% effective concentration, or compound concentration required to inhibit luciferase activity in HCVpp-infected Huh7 cell cultures or GFP-related fluorescence in VSVpp-infected C8166 cell cultures by 50%. Data are the means (±SD) of at least three independent experiments. b Data expressed in μg/ml.

envelope glycoproteins. However, there was a close correlation between the EC50 values measured for HCVcc and HCVpp 1a, HCVpp 1b and HCVpp 2b (r = 0.990, 0.813 and 0.840, respectively) pointing to the relevance of the HCVpp assay compared with the infectious HCVcc assay (Fig. 1, panels B, C and D). Finally, the polyanions DS-5000 and PVAS were not effective against any of the HCVpp subtypes (Table 3). In order to confirm that CBAs also inhibit HIV entry, we used an envelope glycoprotein-deficient HIV-1 (NL4.3) pseudotyped with different envelope glycoproteins that mediate a single cycle of replication. Vesicular stomatitis virus pseudoparticles (VSVpp) were used to infect C8166 cells in the presence of CBA or polyanions at different concentrations. As evidenced by Fig. 3, none of the compounds prevented infection of target cells, even at the highest test concentrations (EC50: N2 to 60 μM). These data confirm earlier reports that inhibition of HIV-1 infection by CBA and polyanions is contingent upon the presence of the HIV-1 envelope glycoproteins and that it occurs at the level of viral entry (Baba et al., 1988, 1990; Schols et al., 1990; Balzarini et al., 1991, 1992). Prevention of virus capture by Raji/DC-SIGN cells

Fig. 2. Dose-dependent inhibition of HCVpp subtype 1a entry into Huh7 cells. Huh7 cells were infected with HCVpp (1a) in the presence of several CBA and polyanion concentrations. At 48 h post-infection, luciferase activity was measured in the cell lysates. Percent entry was calculated relative to untreated controls. Results are from at least three independent experiments ± SD.

Raji B-lymphocyte cells were modified to express DC-SIGN at their cell surface (Geijtenbeek et al., 2000; Wu et al., 2004). When Raji/DC-SIGN were exposed to cell-free HIV-1 (IIIB) particles or HCVpp, they were able to efficiently capture the virus particles as evidenced by retention of p24 antigen (∼1200 pg HIV-1 p24 or ∼ 1820 pg HCVpp p24/106 cells). Wild-type Raji/0 cells did not retain HIV-1 and HCVpp p24 (below detection limit of the assay, data not shown). When HIV-1 was exposed to different concentrations of CBAs and polyanions prior to addition to Raji/DC-SIGN cells, we observed a dose-dependent inhibition of capture (Fig. 4A). The CBAs prevented ≥ 90% of HIV-1 capture at concentrations N1 μM for GNA and HHA and N 0.1 μM for CA; ≥ 12 μM for UDA and ≥ 12 μM for PRM-A. In contrast, none of the polyanions prevented virus capture by Raji/DCSIGN cells even at concentrations as high as 250 μg/ml (Fig.

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Fig. 3. CBA do not inhibit VSVpp entry into C8166 cells. Human T-lymphocyte C8166 cells were infected with GFP-encoding HIV-1 pseudotyped with VSV-G and treated with the CBA. The degree of infection was assayed by measuring GFP expression at 48 h post-infection by flow cytometric analysis. Closed symbols (♦) represent percent of infected cells; open symbols (□) represent percent of viable uninfected cells. Results are from at least three independent experiments ± SD.

4A). Also, a pronounced concentration-dependent inhibition of DC-SIGN-directed HCVpp capture by CBAs (i.e. HHA, UDA, PRM-A) could be demonstrated (Fig. 4B). Discussion Viral entry is an attractive target for chemotherapeutic intervention. Enfuvirtide (T-20), which targets membrane fusion mediated by HIV gp41, is the only approved entry inhibitor for treatment of HIV infection (Chantry, 2004). However, a variety of other entry inhibitors directed against the coreceptors CXCR4 and CCR5 are in preclinical or clinical development (Siegert et al., 2006; Matthews et al., 2004). Carbohydrate-binding agents and polyanions have been shown to qualify as potential microbicides for prevention of HIV transmission by blocking virus entry (Balzarini and Van Damme, 2007). Here, we used the recently developed HCV cell culture system (Lindenbach et al., 2005; Wakita et al., 2005; Zhong et al., 2005) as well as HCV pseudoparticles (Hsu et al., 2003; Flint et al., 2004; Bertaux and

Dragic, 2006) to evaluate CBAs and polyanions as potential anti-HCV agents. Using both assay systems, we showed that CBAs are highly efficient in inhibiting HCV infection of Huh7 cell cultures and that their anti-HCV activity is of the same order of magnitude as their anti-HIV activity. Indeed, there was a strikingly strong correlation between the inhibitory activity of the CBAs against HCV and HIV (r = 0.934; p b 0.05). Moreover, we demonstrated that CBAs efficiently inhibited capture of virus (HIVand HCVpp) by DC-SIGN, a C-type lectin believed to play a role in viral transmission and dissemination within the host (Geijtenbeek et al., 2000). In another study, we recently demonstrated that the CBA not only dose-dependently inhibits HIV-1 capture by DC-SIGN, but also efficiently prevents subsequent virus transmission to human T-lymphocytes (Balzarini et al., 2007a). Since HCV and HIV may have, at least partially, similar modes of transmission to their target cells (Geijtenbeek et al., 2000; Lozach et al., 2004; Wang et al., 2004), it can be reasonably postulated that the CBA may also affect DCSIGN (and L-SIGN)-directed HCV capture and transmission.

C. Bertaux et al. / Virology 366 (2007) 40–50

Fig. 4. Inhibition of DC-SIGN-mediated capture of HIV-1 particles by CBA and polyanions (panel A) and HCVpp by CBA (panel B). The virus was pre-exposed to different concentrations of CBA prior to addition to Raji/DC-SIGN cells. After 2 h, the unbound virus was carefully washed-out and the remaining virus that was captured by the Raji/DC-SIGN cells was quantified by a p24 ELISA assay. Results are from at least three independent experiments ± SD (for HIV-1) and two independent experiments ± SD (for HCVpp).

In contrast, CBAs do not efficiently inhibit infection and replication of other enveloped viruses such as HSV-1 and -2, VSV, RSV and parainfluenza virus-3. This intriguing observation could be due to differences in the particular carbohydrate content or spatial arrangement of the glycans on the envelopes of these viruses. Herpes simplex virus type 1 contains at least three principal surface glycoproteins (gC, gB, gD). The gC is highly glycosylated and contains besides several N-linked glycans and numerous O-linked glycans (Biller et al., 2000). It was shown by Mardberg et al. (2004) that particularly the Oglycosylation pattern determines the attachment of the virus to permissive cells as well as viral cell-to-cell spread. The oligosaccharides present on the human parainfluenza virus-3 F and HN glycoproteins predominantly consist of complex type

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glycans (Tanaka et al., 2006) and are important for fusion activity. The attachment glycoprotein G of human RSV is known to be extensively and predominantly O-glycosylated (Wertz et al., 1989). The F (fusion) protein exists of a single F1 subunit and at least two different forms of the F2 subunit. The latter subunit forms differ in the glycosylation patterns (Rixon et al., 2002). The SH surface glycoprotein of RSV forms an oligomeric complex with the F and G glycoproteins (Feldman et al., 2001), but the exact nature of the RSV envelope glycans is unknown. Thus, it is unclear whether the envelopes of these viruses bear (a significant amount of) high-mannose type residues. Also, it would seem excluded that the CBA interacts with a cellular glycoprotein that is present on the CEM and the human hepatoma cells and not on the HEL, Vero or HeLa cells. Indeed, previous studies have shown that a short preincubation of virus (HIV) but not cells (CEM) markedly increases the antiviral activity of the CBA (Balzarini et al., 2004a). It was also previously shown that HIV-1 gp120 strongly interacts with HHA and PRM-A (Balzarini et al., 2007b). These observations point to a surprisingly marked degree of selectivity of the CBAs against some enveloped viruses and a striking similarity in this respect between HCV and HIV. It is still not entirely clear which types of glycans are present on the HCV envelope. Current literature points to a mixture of complex and high-mannose-rich side-chains (i.e. Duvet et al., 1998; Flint et al., 2004; Lozach et al., 2004; Op De Beeck et al., 2004). The positions of the sidechains are highly conserved (Goffard and Dubuisson, 2003; Zhang et al., 2004a). A similar glycan profile exists on the HIV1 envelope gp120 (Zhang et al., 2004b) and may explain the close correlation of CBAs activity against both viruses. Such a close correlation between the antiviral activity of the CBA against HIV and HCV is even more striking given the fact that different cell types in which HCVcc, HCVpp and HIV were generated may differently affect the glycosylation pattern of the viral glycoproteins. These findings point to a rather highly conserved pattern of glycan arrays present on the viral envelope glycoproteins that may be required for an efficient viral entry. Whereas the antiviral activity of the CBA closely correlated for HCV and HIV, the polyanions only inhibited HIV infection but not HCV infection (Baba et al., 1988, 1990). HIV infectivity is increased by electrostatic interactions between the HIV-1 envelope and heparan sulfate proteoglycans on the target cells (Batinic and Robey, 1992; Roderiquez et al., 1995), and it is thought that the overall basic charge on gp120 is determined by the V3 loop (Moulard et al., 2000). Moreover, the V3 loop binds to polyanions (Batinic and Robey, 1992; Moulard et al., 2000), resulting in inhibition of HIV entry into its target cells. The interaction sites of polyanions and envelopes of the other viruses that are inhibited by these agents are currently unknown. However, given the complete inactivity of polyanions against HCV, we assume that the HCV envelope glycoproteins do not contain a positively charged configuration capable of interacting with these molecules. During the preparation of this manuscript, a study appeared (Helle et al., 2006) on the anti-HCV activity of cyanovirin-N (CV-N), a prokaryotic mannose-specific lectin that had previously been shown to efficiently inhibit HIVentry at nanomolar

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(Peumans et al., 1984; Van Damme et al., 1987, 1988, 1991). Pradimicin A (PRM-A) was obtained from Prof. T. Oki and Prof. Y. Igarashi (Japan) and PVAS (sulfated polyvinylalcohol) was synthesized by Dr. S. Görög (Budapest, Hungary). Dextran sulfate-5000 was purchased from Sigma (St. Louis, MO).

concentrations (Boyd et al., 1997; O'Keefe et al., 2000; Bolmstedt et al., 2001). In this study, it was shown that CV-N blocks the interaction of the HCV glycoprotein E2 and the receptor CD81 through binding of the drug to the envelope Nlinked glycans. These data clearly confirm that targeting HCV envelope glycans might be a promising approach in the development of novel antiviral therapies. Our results with plant lectins and more importantly with the non-peptidic smallmolecule pradimicin A are in full agreement with the observations and conclusions made by Helle et al. (2006). Moreover, it is notable that CBAs inhibit HCV entry of all three subtypes to the same extent. The current treatment with ribavirin and pegylated interferon-α is effective in only half of the treated HCV-infected patients and is relatively toxic (Feld and Hoofnagle, 2005). Thus, CBAs are attractive new lead drugs for HCV treatment through an entirely different mechanism of action than the currently existing therapies. Our observations imply that one single CBA may be equally and simultaneously effective against both HCV and HIV in vivo. One-third of HIV-infected individuals suffer from coinfection with HCV (Soriano et al., 2006; Tan et al., 2006). A more rapid liver disease progression is seen in these individuals, leading to end-stage liver disease complications, as well as a higher risk of developing hepatotoxicity following initiation of antiretroviral therapy (Soriano et al., 2006). Concomitant HCV and HIV therapy therefore would be highly advantageous to these patients and CBAs fit the profile of agents that would simultaneously target both pathogens. The discovery that small-size non-peptidic CBAs such as pradimicin A inhibit both HIV (Balzarini et al., 2007b; and data in this study) and HCV indicates that CBA treatment of HCV/HIVinfected individuals may become a feasible goal. We recognize that plant lectins and also cyanovirin may have the disadvantage of being proteins, expensive to produce on a large scale, subject of proteolytic cleavage, endowed with a short plasma half-life, immunogenic and not orally bioavailable (Balzarini and Van Damme, 2007). The data presented in this study demonstrate the proof-of-concept that CBAs in general have the potential to efficiently prevent HCV and HIV entry into susceptible cells. Moreover, CBAs may also prevent virus capture by C-type lectins and subsequent transmission and dissemination within the host. Importantly, we also demonstrated that pradimicin A, a non-peptidic small-molecule antibiotic (M.W. 836) with mannose specificity (Ueki et al., 1993; Hiramoto et al., 2005), behaves as a lectin and enables efficient prevention/inhibition of HCV (and HIV) entry. Screening for non-peptidic small molecule CBAs therefore is a realistic goal.

Construct FL-J6/JFH-5′C19Rluc2AUbi was used to generate HCVcc and was a generous gift of Dr. C. Rice (The Rockefeller University, New York, NY). This clone is a monocistronic, full-length HCV genome that expresses Renilla luciferase (Rluc) and was derived from the previously described infectious genotype 2a HCV genome J6/JFH1 (Lindenbach et al., 2005; Tscherne et al., 2006). HCVcc was generated as described (Lindenbach et al., 2005). HIV-1 (IIIB) was provided by Dr. R.C. Gallo (Institute of Human Virology, University of Maryland, Baltimore, MD). HIV-2 (ROD) was provided by Dr. L. Montagnier (at that time at the Pasteur Institute, Paris, France). Virus stocks were prepared in MT-4 cell cultures. When full cytopathicity was reached (5 days), the cell cultures were centrifuged and the supernatants divided in aliquots and stored at − 80 °C. Respiratory syncytial virus (RSV), vesicular stomatitis virus (VSV) and parainfluenza virus-3 were obtained from the ATCC (Rockville, MD). The origin and characterization of herpes simplex virus type 1 (HSV-1) and HSV-2 were described previously (De Clercq et al., 1980). All viruses were stored at − 80 °C until use.

Materials and methods

Production of pseudoparticles

Carbohydrate-binding compounds and polyanions

Retroviral pseudotypes were generated by cotransfection of 293T cells with replication-deficient HIV-1 vectors and envelope glycoproteins of individual viruses. Thus, the vesicular stomatitis virus glycoprotein (VSV-G) was coexpressed with pNL4.3-ΔE-GFP (Zhang et al., 2004b) (a kind gift of Dr R. Siliciano, Johns Hopkins University, Baltimore MD).

The mannose-specific plant lectins from G. nivalis (GNA), Hippeastrum hybrid (HHA) and Cymbidium hybrid (CA) and the GlcNAc-specific plant lectin from Urtica dioica (UDA) were derived and purified from these plants, as described before

Cells Human embryo kidney cells (293T) were purchased from ATCC and human hepatoma cells (Huh7 and Huh7.5) were a kind gift of Dr. C. Rice (Rockefeller University, New York, USA). These cell lines were grown in Dulbecco's Modified Essential Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin, MEM nonessential amino acids and sodium pyruvate. Human Tlymphocytic CEM cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA). The wild-type Raji/0 and DC-SIGN-expressing Raji/DC-SIGN cells (Geijtenbeek et al., 2000) were a kind gift of Dr. L. Burleigh (Institut Pasteur, Paris, France). MT-4 cells were obtained from Dr. N. Yamamoto (Tokyo University, Japan). The origin of the C8166 cells was described earlier (Salahuddin et al., 1983). These cell lines were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) (BioWhittaker Europe, Verviers, Belgium), 2 mM L-glutamine and 0.075% NaHCO3. Infectious viruses

C. Bertaux et al. / Virology 366 (2007) 40–50

47

Supernatants containing VSV-G-pseudotyped HIV-1 were collected 60 h after transfection. HCVpp were generated by cotransfection of NLluc+env− reporter vector and a vector expressing the HCV envelope glycoproteins as previously described (Bertaux and Dragic, 2006). HCV envelope glycoprotein-encoding sequences corresponding to subtypes 1b and 2b were PCR-amplified from patient sera starting from the last 60 amino acids in the Core to the end of glycoprotein E2. For all pseudotypes, supernatants were filtered (0.45 μ) or clarified by centrifugation and stored at − 80 °C until further use.

Inhibition of HIV-1 and HCVpp capture by Raji/DC-SIGN cells

Inhibition of viral infection by the test compounds

Statistical analysis

All assays were carried out with serial dilutions of compounds added to cells at the time of infection. Mannan (2.5 mg/ml; Sigma) was added to some infections. For inhibition of HCV infection, Huh7 cells (2 × 104) were plated and 24 h later infected with supernatants containing infectious HCVcc in the presence of compounds. Twenty-four hours post-infection, the mixture of virus and test compounds was replaced by fresh medium, and luciferase activity was measured in cell lysates 24 h later using the Dual Luciferase Assay System (Promega). For inhibition of HIV entry, CEM cells (5 × 105) were infected with HIV-1 (IIIB) or HIV-2 (ROD) at 100 TCID50 per ml of cell suspension in the presence of compounds. After 4 days, giant cell formation was scored visually in the CEM cell cultures. The half-maximal effective concentration (EC50) corresponds to the compound concentration required to reduce syncytium formation by 50% in the virus-infected CEM cell cultures. The halfmaximal cytostatic concentration (CC50) corresponds to the compound concentration required to inhibit the CEM or Huh7 cell proliferation by 50%. The measurement of the antiviral effects of CBAs on other viruses was scored microscopically when the cytopathic effect (CPE) had reached 100% in the control (untreated) virusinfected cell cultures. Confluent monolayers of human embryonic lung fibroblast (HEL), African green monkey kidney (Vero) or human cervix carcinoma (HeLa) cells were exposed to HSV-1, HSV-2 and VSV, RSV and parainfluenza-3, respectively, at 100 CCID50 (cell culture infective dose-50) in the presence of various dilutions of the test compounds. CPE was quantified at 3 days post-infection.

The correlation coefficients between the antiviral EC50 values were calculated according to the equation Y = aX + b in which Y is the EC50 value on the ordinate axis and X is the EC50 value on the abscissa axis; a is the slope of the correlation line and b is the intercept of this line with the X-axis. The correlation coefficient r is calculated according to the formula: X X X x: y xy
n vffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi  X 2 !  X 2 ! u u X X x y 2 2 t y
x
n n

Inhibition of virus entry by the test compounds Human T-lymphocyte C8166 cell cultures were infected with the GFP-encoding VSV-G pseudotyped HIV-1 in the presence of the test compounds at different concentrations. GFP expression was quantified by flow cytometry at 48 h postinfection. Acquisition was stopped when 10,000 events were counted and data analysis was carried out with Cell Quest software (BD Biosciences). Cell debris was excluded from the analysis by gating on forward versus side scatter dot plots. To measure inhibition of HCVpp (1a, 1b and 2b) entry into Huh7 cells by CBAs and polyanions, an essentially similar procedure was used except that luciferase activity was measured in cell lysates 48 h post-infection.

Virus particles (2.2 μg p24/ml) were exposed to serial dilutions of the test compounds for 30 min. Following this, the drug-exposed virus suspensions were mixed with Raji/DCSIGN cell suspensions (106 cells) for 60 min at 37 °C after which the cells were thoroughly washed twice with culture medium. The Raji/DC-SIGN cell suspensions were then analyzed for the amount of captured virus by HIV-1 p24 Ag ELISA.

The following formula has been used to measure the standard deviation: sX ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi ð¯x
xÞ2 ðn
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