Role of EXT1 and Glycosaminoglycans in the Early Stage of Filovirus Entry

Abstract

Filoviruses, including both Ebola virus (EBOV) and Marburg virus (MARV), can infect humans and other animals, causing hemorrhagic fever with a high mortality rate. Entry of these viruses into the host is mediated by a single filoviral glycoprotein (GP). GP is composed of two subunits: GP1, which is responsible for attachment and binding to receptor(s) on susceptible cells, and GP2, which mediates viral and cell membrane fusion. Although numerous host factors have been implicated in the entry process, the initial attachment receptor(s) has not been well defined. In this report, we demonstrate that exostosin 1 (EXT1), which is involved in biosynthesis of heparan sulfate (HS), plays a role in filovirus entry. Expression knockdown of EXT1 by small interfering RNAs (siRNAs) impairs GP-mediated pseudoviral entry and that of infectious EBOV and MARV in tissue cultured cells. Furthermore, HS, heparin, and other related glycosaminoglycans (GAGs), to different extents, can bind to and block GP-mediated viral entry and that of infectious filoviruses. These results strongly suggest that HS and other related GAGs are attachment receptors that are utilized by filoviruses for entry and infection. These GAGs may have therapeutic potential in treating EBOV- and MARV-infected patients.

Importance: Infection by Ebola virus and Marburg virus can cause severe illness in humans, with a high mortality rate, and currently there is no FDA-approved vaccine or therapeutic treatment available. The ongoing 2014 outbreak in West Africa underscores a lack of our understanding in the infection and pathogenesis of these viruses and the urgency of drug discovery and development. In this study, we provide several pieces of evidence that demonstrate that heparan sulfate and other closely related glycosaminoglycans are the molecules that are used by filoviruses for initial attachment. Furthermore, we demonstrate that these glycosaminoglycans can block entry of and infection by filoviruses. Thus, this work provides mechanistic insights on the early step of filoviral infection and suggests a possible therapeutic option for diseases caused by filovirus infection.

FIG 1

Reductions in EXT1 mRNA and protein levels correlate with reduced infection of MARV and EBOV pseudotyped particles. (a) Introduction of siRNAs of EXT1 resulted in reduced infection by MARVpv and EBOVpv. The siRNAs of EXT1 (labeled as siRNA #1 and #2) were transfected to A549 cells, and their effects on pseudotyped Marburg virus (MARVpv), Ebola virus (EBOVpv), and influenza virus (AIVpv) were evaluated as described in Materials and Methods. A siRNA of firefly luciferase was used as a control in the experiment, and the data were normalized to nontargeting siRNA (NT). Error bars represent standard deviations. (b) Real-time PCR on cells transfected with EXT1 siRNAs showed mRNA knockdown of EXT1 at 48 and 96 h posttransfection, using NT siRNA as a control. Error bars represent standard deviations. (c) The siRNAs of EXT1 reduced the protein level of EXT1 in the target cells. An EXT1-C-Myc plasmid and EXT1 siRNAs were cotransfected to A549 cells, and the EXT1-C-Myc level in the cells was evaluated by Western analysis. In this experiment, different amounts of EXT1-C-Myc plasmid DNA (0.2 to 0.8 μg) were used. β-Actin was used as a control.

FIG 2

Heparan sulfate (HS) biosynthesis-deficient cell lines were resistant to MARVpv infection. The parental mouse L cells (L) and two lines of HS biosynthesis-deficient cells (Sog9 and Gro2C, which were derived from L cells) were challenged with MARVpv, and the infection was measured by the luciferase level of the infected cells. Error bars represent standard deviations.

FIG 3

GAGs blocked infection by MARVpv and EBOVpv in A549 cells. The pseudotyped viruses MARVpv, EBOVpv, and AIVpv were mixed with different GAGs, HS (a), heparin (b), chondroitin sulfate A (c), and chondroitin sulfate B (d), at various concentrations; the mixtures were used to challenge A549 cells, and the effects of different GAGs were evaluated as described in Materials and Methods. Error bars represent standard deviations.

FIG 4

GAGs blocked infection of MARVpv and EBOVpv in primary human pulmonary artery endothelial cells (HPAECs). The pseudotyped viruses MARVpv, EBOVpv, and AIVpv were mixed with different GAGs, HS (a), heparin (b), chondroitin sulfate A (c), and chondroitin sulfate B (d) at various concentrations; the mixtures were used to challenge HPAECs, and the effects of different GAGs were evaluated as described in Materials and Methods. Error bars represent standard deviations.

FIG 5

MARVpv and EBOVpv were able to bind to heparin and HS. (a) An ELISA (described in Materials and Methods) was used to demonstrate specific binding of MARVpv and EBOVpv to heparin and HS. AIVpv was used here as a specificity control. Error bars represent standard deviations. (b) An ELISA-based competition experiment was used to demonstrate that “cold” heparin and HS can competitively block binding of MARVpv and EBOVpv to the heparin used to coat the plates. Error bars represent standard deviations.

FIG 6

Only high-molecular-weight heparin (HMWH) could block MARVpv infection. Porcine heparin (HMWH, ∼17 to 19 kDa) and heparin hexasaccharide (LMWH, ∼1.5 kDa), at the concentrations indicated, were first mixed with MARVpv and then added to the target cells (A549), and infection by MARVpv was determined 48 h postinfection. Error bars represent standard deviations.

FIG 7

EXT1 and GAGs are involved in infection by infectious MARV and EBOV. (a) Knockdown expression of EXT1 by siRNAs reduced levels of infection by infectious MARV and EBOV. The EXT1 siRNAs were introduced to A549 cells, and their effects on infection of infectious MARV and EBOV were determined following a protocol as described in Materials and Methods. An NT siRNA was used as the control in this experiment. Error bars represent standard deviations. (b) Effects of different GAGs on blocking infectious MARV infection. Different GAGs, at different concentrations, were mixed with infectious MARV, and their effects on blocking MARV infection on A549 cells were evaluated. Error bars represent standard deviations. (c) Effects of different GAGs on blocking infectious EBOV infection. Error bars represent standard deviations.