Characterization of the Filovirus-Resistant Cell Line SH-SY5Y Reveals Redundant Role of Cell Surface Entry Factors

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.

The Phosphatidylserine Receptor TIM-1 Enhances Authentic Chikungunya Virus Cell Entry

“…At MOI of 0.01, TIM-1 WT expression increased susceptibility by 12-fold whereas Axl WT expression resulted in a twofold increase (Figure 1B,C). Notably, TIM-1 WT and Axl WT expressing HEK293T cells were more susceptible to CHIKV (33-fold and 10-fold respectively) and Ebola virus (18-fold and five-fold respectively) glycoprotein-based lentiviral pseudoparticles demonstrating that the proteins are functional as previously reported [18,[67][68][69] (Supplementary Figure S2B). Next, we analyzed the susceptibility of the cells to authentic CHIKV at different hours post infection (hpi).…”

When Dendritic Cells Go Viral: The Role of Siglec-1 in Host Defense and Dissemination of Enveloped Viruses

“…Initial EBOV attachment to the DC surface is mediated by several receptors that recognize different elements on the viral membrane and often have a redundant activity [105]. C-type lectin receptors (CLRs) such as the dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) and the liver/lymph node sinusoidal endothelial C-type lectin (LSECtin) mediate viral attachment through binding to viral glycoproteins [106,107], while receptors of the TIM/TAM families (comprising the T cell immunoglobulin and mucin domain receptor along with Tyro-Axl-Mer receptors) recognize phosphatidylserine lipids present on the viral envelope [108] ( Figure 2B).…”

Filovirus Antiviral Activity of Cationic Amphiphilic Drugs Is Associated with Lipophilicity and Ability To Induce Phospholipidosis

“…In our study, we found that DIPL strongly correlates with both antiviral activity and with hydrophobicity. However, visible DIPL itself is not required for antiviral activity of CADs since effects like EBOV VLP inhibition (32) Filoviruses are late-penetrating viruses that are taken up into endosomes following low-specificity interactions with one or more of various cell surface attachment factors (34). Within the endosome, they undergo gp priming by low-pH dependent cathepsin and interact with their endolysosomal receptor NPC1, a cholesterol transporter (50).…”

“…Experiments were performed in the endothelium/lung-hybrid cell line EA.hy926 that is easy to cultivate and gives low background. Besides monocytes, macrophages, endothelial cells, hepatocytes and fibroblasts, this cell line was shown to be susceptible to several enveloped viruses including filoviruses(11,13,34,35). -3.7 µmol/L) and C max(sertraline) = 0.352 µmol/L, which can accumulate up to 20fold in the liver…”