Inhibition of Hepatitis C Virus by the Cyanobacterial Protein Microcystis viridis Lectin: Mechanistic Differences between the High-Mannose Specific Lectins MVL, CV-N, and GNA

Abstract

Plant or microbial lectins are known to exhibit potent antiviral activities against viruses with glycosylated surface proteins, yet the mechanism(s) by which these carbohydrate-binding proteins exert their antiviral activities is not fully understood. Hepatitis C virus (HCV) is known to possess glycosylated envelope proteins (gpE1E2), and to be potently inhibited by lectins. Here, we tested in detail the antiviral properties of the newly discovered Microcystis viridis lectin (MVL) along with cyanovirin N (CV-N) and Galanthus nivalis agglutinin (GNA) against cell culture HCV, as well as their binding properties towards viral particles, target cells, and recombinant HCV glycoproteins. Using infectivity assays, CV-N, MVL and GNA inhibited HCV with IC50 values of 0.6 nM, 30.4 nM, and 11.1 nM, respectively. Biolayer interferometry analysis demonstrated a higher affinity of GNA to immobilized recombinant HCV glycoproteins compared to CV–N and MVL. Complementary studies, including FACS analysis, confocal microscopy and pre and post virus binding assays, showed a complex mechanism of inhibition for CV-N and MVL that includes both viral and cell association; while GNA functions by binding directly to the viral particle. Combinations of GNA with CV-N or MVL in HCV infection studies revealed synergistic inhibitory effects, which can be explained by different glycan recognition profiles of the mainly high-mannoside specific lectins, and supports the hypothesis that these lectins inhibit through different and complex modes of action. Our findings provide important insights into the mechanisms by which lectins inhibit HCV infection. Overall, the data suggest MVL and CV-N have the potential for toxicity due to interactions with cellular proteins while GNA may be a better therapeutic agent due to specificity for the HCV gpE1E2.

Antiviral lectins: Selective inhibitors of viral entry

“…This mechanism often requires multivalency of CRDs in the lectins themselves for adequate potency (Liu et al, 2009;Moulaei et al, 2010;Takahashi et al, 2010) and can often also rely on the cross-linking of oligosaccharides on Env by oligomeric lectins (Barrientos et al, 2006). The biochemical basis of lectin antiviral activity therefore appears Yes (Boyd et al, 1997;Gustafson et al, 1997;Balzarini et al, 2006;Witvrouw et al, 2005;Alexandre et al, 2010;Barrientos et al, 2003;O'Keefe et al, 2003;Kachko et al, 2013;Helle et al, 2006;Takebe et al, 2013;Tsai et al, 2004;Tsai et al, 2003 (260) No (Bokesch et al, 2003;Alexandre et al, 2010;Xiong et al, 2006a;Xiong et al, 2006b;Jensen et al, 2014;Garrison et al, 2014 Unk -Unknown. Botos and Wlodawer, 2003;Boyd et al, 1997;Gustafson et al, 1997;Yang et al, 1999;Kachko et al, 2013;Helle et al, 2006;Bolmstedt et al, 2001;Shenoy et al, 2001;Bewley and Otero-Quintero, 2001;Bewley, 2001 (Koshte et al, 1990;Swanson et al, 2010;Swanson et al, 2015;Gavrovic-Jankulovic et al, 2008;Dimitrijevic et al, 2012)…”

Cyanobacterial lectins characteristics and their role as antiviral agents

“…The ability of CVN and MVL to inhibit HCV by interacting with recombinant HCV glycoproteins E1E2 was confirmed by rE1E2-biolayer interferometry and confocal staining of infected cells [96]. CVN and MVN both bind to cellular proteins and HCV envelope and their binding to cell surface glycans contribute to the inhibition process [96]. SVN also targets HCV envelope glycoproteins E1 and E2 and inhibits HCV in both cell culture and pseudoparticle assays at picomolar and nanomolar concentrations [133].…”

“…These molecules are involved in HCV entry, thus CV-N exerts its inhibitory action by interacting with N-linked glycans [132]. The ability of CVN and MVL to inhibit HCV by interacting with recombinant HCV glycoproteins E1E2 was confirmed by rE1E2-biolayer interferometry and confocal staining of infected cells [96]. CVN and MVN both bind to cellular proteins and HCV envelope and their binding to cell surface glycans contribute to the inhibition process [96].…”

“…However, in contrast to CVN, no cross linking or aggregation occurs upon interaction of Cyt-CVNH with Man-9 [93]. M. viridis lectin (MVL) exhibits specificity towards chitobiose core containing oligomannosides exemplified by Man 3 GalNAc 2 and Man 6 GalNAc 2 , thus it recognizes 4-5 units of oligomannoside core [94][95][96]. Scytovirin (SVN) recognizes D3 arm of Man9GlcNAc2 and exhibits high affinity towards tetrasaccharide structure of Man␣(1-2)Man␣(1-6)Man␣(1-6)Man, thus specified as Man4 [97].…”

“…Such as, OAA recognizes the core structure of the triantennary Man-9, whereas most of other cyanobacterial lectins (CVN, MVL & SVN) recognize reducing/nonreducing end mannoses of Man-9 [100]. In terms of mammalian glycoprotein binding, MVL recognizes any oligomannoside from Man-3 to Man-9, whereas CVN binds only Man-8 or Man-9 [96]. Also, CVN specifically recognizes terminal Man␣(1-2)Man␣ disaccharides on branch arms of oligo Man-9 and Man-9 [101], whereas MVL requires occurrence of both mannose and glucosamine residues for carbohydrate recognition [95].…”