Chikungunya Virus Strains from Each Genetic Clade Bind Sulfated Glycosaminoglycans as Attachment Factors

Abstract

Chikungunya virus (CHIKV) is an arthritogenic alphavirus that causes debilitating musculoskeletal disease. CHIKV displays broad cell, tissue, and species tropism, which may correlate with the attachment factors and entry receptors used by the virus. Cell-surface glycosaminoglycans (GAGs) have been identified as CHIKV attachment factors. However, the specific types of GAGs and potentially other glycans to which CHIKV binds and whether there are strain-specific differences in GAG binding is not fully understood. To identify the types of glycans bound by CHIKV, we conducted glycan microarray analyses and discovered that CHIKV preferentially binds GAGs. Microarray results also indicate that sulfate groups on GAGs are essential for CHIKV binding and that CHIKV binds most strongly to longer GAG chains of heparin and heparan sulfate. To determine whether GAG-binding capacity varies among CHIKV strains, a representative strain from each genetic clade was tested. While all strains directly bound to heparin and chondroitin sulfate in ELISAs and depended on heparan sulfate for efficient cell-binding and infection, we observed some variation by strain. Enzymatic removal of cell-surface GAGs and genetic ablation that diminishes GAG expression reduced CHIKV binding and infectivity of all strains. Collectively, these data demonstrate that GAGs are the preferred glycan bound by CHIKV, enhance our understanding of the specific GAG moieties required for CHIKV binding, define strain differences in GAG engagement, and provide further evidence for a critical function of GAGs in CHIKV cell attachment and infection. IMPORTANCE Alphavirus infections are a global health threat, contributing to outbreaks of disease in many parts of the world. Recent epidemics caused by CHIKV, an arthritogenic alphavirus, resulted in more than 8.5 million cases as the virus has spread into new geographic regions, including the Western Hemisphere. CHIKV causes disease in the majority of people infected, leading to severe and debilitating arthritis. Despite the severity of CHIKV disease, there are no licensed therapeutics. Since attachment factors and receptors are determinants of viral tropism and pathogenesis, understanding these virus-host interactions can enhance our knowledge of CHIKV infection. We analyzed over 670 glycans and identified GAGs as the main glycan bound by CHIKV. We defined specific GAG components required for CHIKV binding and assessed strain-specific differences in GAG-binding capacity. These studies provide insight about cell-surface molecules that CHIKV binds, which could facilitate the development of antiviral therapeutics targeting the CHIKV attachment step.

Overview on Chikungunya Virus Infection: From Epidemiology to State-of-the-Art Experimental Models

“…The FUZ is involved in the clathrin-mediated endocytosis pathway, and TSPAN9 helps viral entry by two possible mechanisms: (i) virus orientation to the early endosome and/or (ii) modulation of the endosome membrane to be more permissive to the fusion process (Ooi et al, 2013). Nevertheless, other molecules, such as glycosaminoglycans (GAGs), T-cell immunoglobulin and mucin (TIM) family, Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin (DC-SIGN), AXL receptor tyrosine kinase, and membrane protein complex CD147, have all been described to participate in CHIKV-target cell interaction and to act as alternative cell receptors for CHIKV (Silva et al, 2014;Acharya et al, 2015;van Duijl-Richter et al, 2015;Schnierle, 2019;McAllister et al, 2020;De Caluwé et al, 2021), although binding to them might not be sufficient to trigger virus internalization (Wang et al, 1992;Klimstra et al, 2003;La Linn et al, 2005;Kielian et al, 2010). Other infection routes occur in epidermal and muscle cells.…”

Emerging chikungunya virus variants at the E1-E1 inter-glycoprotein spike interface impact virus attachment and Inflammation

“…In addition to interactions with GAGs, E1 residues 156, 211, and 226 each lie within the predicted contact zones of Mxra8 (Figure 9A, oval) [13,18]. Notably, the recent characterization of CHIKV-GAG interactions found Mxra8 expression to be inversely correlated to GAGdependency for CHIKV binding and infection [16]. Also intriguing, considering our data, is that in vivo, Mxra8 deficient mice exhibit a modest decrease in titers at the sight of injection following infection via footpad, but dramatically decreased foot swelling [17].…”

“…For instance, binding to the ubiquitous host GAGs is, in part, modulated by the type of amino acid at E2 residue 82 [11,14]. Residue 82 is a highly conserved glycine in nearly every CHIKV isolate, except for CHIKV 181/25, which has an arginine at residue 82 and altered GAG utilization [14][15][16]. Moreover, the CHIKV 181/25 E2 residue D71 has been shown to be critical for interactions with Mxra8 [17].…”

New Insights into Chikungunya Virus Infection and Pathogenesis

“…Glycosaminoglycans (GAGs), a family of negatively charged polysaccharides, interact with a structurally conserved and positively charged domain in E2. These membrane proteins were proposed to enhance infection by promoting E1/E2 dissociation (62,63). The functional importance of GAGs was recently reassessed in genome-wide loss of function screens performed in HAP1 cells that identified GAGs' biosynthesis enzymes (B3GAT3, SLC35B2, PAPSS1, NDST1) as critical factors for CHIKV infectivity (64,65).…”