Abstract
Filoviruses infect a wide range of cell types with the exception of lymphocytes. The intracellular proteins cathepsin B and L, two-pore channel 1 and 2, and bona fide receptor Niemann–Pick Disease C1 (NPC1) are essential for the endosomal phase of cell entry. However, earlier steps of filoviral infection remain poorly characterized. Numerous plasma membrane proteins have been implicated in attachment but it is still unclear which ones are sufficient for productive entry. To define a minimal set of host factors required for filoviral glycoprotein-driven cell entry, we screened twelve cell lines and identified the nonlymphocytic cell line SH-SY5Y to be specifically resistant to filovirus infection. Heterokaryons of SH-SY5Y cells fused to susceptible cells were susceptible to filoviruses, indicating that SH-SY5Y cells do not express a restriction factor but lack an enabling factor critical for filovirus entry. However, all tested cell lines expressed functional intracellular factors. Global gene expression profiling of known cell surface entry factors and protein expression levels of analyzed attachment factors did not reveal any correlation between susceptibility and expression of a specific host factor. Using binding assays with recombinant filovirus glycoprotein, we identified cell attachment as the step impaired in filovirus entry in SH-SY5Y cells. Individual overexpression of attachment factors T-cell immunoglobulin and mucin domain 1 (TIM-1), Axl, Mer, or dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) rendered SH-SY5Y cells susceptible to filovirus glycoprotein-driven transduction. Our study reveals that a lack of attachment factors limits filovirus entry and provides direct experimental support for a model of filoviral cell attachment where host factor usage at the cell surface is highly promiscuous.
Highlights
Ebola and marburgviruses are enveloped, negative single-strand RNA viruses of the Filoviridae family [1]
To further characterize the step of filovirus infection impaired in SH-SY5Y cells, we studied filovirus GP-specific attachment using recombinant soluble Ebola virus (EBOV) GP1 fused to human Fc or a human
We tested a panel of immortalized cell lines for EBOV and Marburg virus (MARV) GP-mediated transduction to correlate susceptibility to the presence of plasma membrane proteins or endosomal proteins required during the early phases of infection
Summary
Ebola and marburgviruses are enveloped, negative single-strand RNA viruses of the Filoviridae family [1]. The viral glycoprotein (GP), the only viral surface protein, exclusively mediates the entry and internalization of filoviruses into cells. The precursor protein GP0 is synthesized on the endoplasmic reticulum, and cleaved in the constitutive secretory pathway into the surface unit GP1, which binds to host cell factors, and the transmembrane unit GP2, which mediates fusion of viral envelopes with endosomal membranes. Almost any cell type with the notable exception of lymphocytes is susceptible to infection by authentic filoviruses in vitro [14,15], or to transduction by retrovirus particles pseudotyped with GP [16,17]. The broad cell tropism observed in infected primates, where virus can be isolated from all organs but not from lymphocytes [14,20,21], is recapitulated in vitro
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