Transcellular transport of West Nile virus-like particles across human endothelial cells depends on residues 156 and 159 of envelope protein

Abstract

Background: West Nile virus (WNV) causes viremia after invasion to the hosts by mosquito bite. Endothelial cells could play an important role in WNV spread from the blood stream into the central nervous system and peripheral tissues. Here, we analyzed the capacity of virus-like particles (VLPs) of the highly virulent NY99 6-LP strain (6-LP VLPs) and the low virulence Eg101 strain (Eg VLPs) to cross cultured human endothelial cells.

Results: 6-LP VLPs were transported from the apical to basolateral side of endothelial cells, whereas Eg VLPs were hardly transported. The localization of tight junction marker ZO-1 and the integrity of tight junctions were not impaired during the transport of 6-LP VLPs. The transport of 6-LP VLPs was inhibited by treatment with filipin, which prevents the formation of cholesterol-dependent membrane rafts, suggesting the involvement of raft-associated membrane transport. To determine the amino acid residues responsible for the transport of VLPs, we produced mutant VLPs, in which residues of E protein were exchanged between the 6-LP and Eg strains. Double amino acid substitution of the residues 156 and 159 greatly impaired the transport of VLPs.

Conclusion: Our results suggest that a transcellular pathway is associated with 6-LP VLPs transport. We also showed that the combination of the residues 156 and 159 plays an important role in the transport of VLPs across endothelial cells.

Figure 1

Transport of 6-LP and Eg VLPs across a monolayer of HUVEC. HUVEC were exposed to VLPs for 0, 8 or 24 h. The numbers of transferred VLPs were determined by IFU assay. Gray bars, 6-LP VLPs. White bars, Eg VLPs. The graphs show the mean of three determinations. The error bars show SD. The results are representative of 2 independent experiments. *p < 0.01.

Figure 2

Transcellular transport of 6-LP VLPs in HUVEC. (A) Distribution of tight junction marker ZO-1 in HUVEC. HUVEC were exposed to 6-LP VLPs or treated with TNF-α for 24 h. The cells were fixed and processed for immunofluorescence staining of ZO-1. Bars represent 50 μm. (B) Transfer of Dx70k into a monolayer of untreated, 6-LP VLP-exposed or TNF-α treated HUVEC. HUVEC were exposed to 6-LP VLPs or treated with TNF-α in the presence of FITC-labeled 70k Dx (FITC-70k Dx). After 24 h, media were collected from lower chambers and the fluorescence of transferred 70k Dx was measured by a fluorescent plate reader. Relative transfer of FITC-70k Dx was calculated as described in METHODS. The graphs show the mean of three determinations. The error bars show SD. The results are representative of 2 independent experiments. *p < 0.01. (C) Transport of 6-LP VLPs in HUVEC treated with endocytosis inhibitors. HUVEC were exposed to 6-LP VLPs in the presence or absence of 5 μg/ml of chlorpromazine or 1 μg/ml of filipin. The cells treated with 0.1% DMSO were used as control. After 24 h, media at the lower chamber were collected and subjected to IFU assay. *p < 0.01. (D) Transfer of FITC-70k Dx in HUVEC treated with endocytosis inhibitors. FITC-70k Dx was added to HUVEC with or without 5 μg/ml of chlorpromazine or 1 μg/ml of filipin. After 24 h, medium was collected from the lower chambers and the fluorescence was measured. Relative transfer of FITC-70k Dx was calculated as described in METHODS. The graphs show the mean of three determinations. The error bars show SD. The results are representative of 2 independent experiments.

Figure 3

Role of WNV E protein in the transport of VLPs. HUVEC were exposed to 6-LP, Eg, 6-LP CM Eg E or Eg CM 6-LP E VLPs. After 24 h, media at the lower chamber were collected and subjected to IFU assay. The graphs show the mean of three determinations. The error bars show SD. The results are representative of 2 independent experiments. * represents p < 0.01 (versus 6-LP).

Figure 4

Effect of single amino acid substitutions in E protein on the transport of VLPs. HUVEC were exposed to mutant VLPs. After 24 h, media at the lower chamber were collected and subjected to IFU assay. (A) Transport of mutant 6-LP VLPs. *represents p < 0.01 (versus 6-LP). (B) Transport of mutant Eg VLPs. * and ** represent p < 0.01 and p < 0.05, respectively (versus Eg). The graphs show the mean of three determinations. The error bars show SD. The results are representative of 2 independent experiments.

Figure 5

Effect of double amino acid substitutions of E protein on the transport of VLPs. HUVEC were exposed to 6-LP, 6-LP S156P V159I, Eg P156 S I159V or Eg VLPs. After 24 h, media at the lower chamber were collected and subjected to IFU assay. * p < 0.01 (versus 6-LP). The graphs show the mean of three determinations. The error bars show SD. The results are representative of 2 independent experiments.

Figure 6

Glycosylation of E protein in wild type and mutant VLPs. 293T cells were cotransfected with replicon RNA and plasmids encoding structural genes or empty vector for mock control. The supernatants were collected and subjected to Western blotting with anti-WNV E protein monoclonal antibody.