Promotion of vesicular transport of endothelial cells, including pinocytosis and transcytosis, is also

affected by these cytokines [47]. Paracellular invasion by disruption of the tight junction induced by cytokines could occur in vivo, however, there is a possibility that WNV also utilizes a transcellular pathway, which might be promoted by inflammatory cytokines. The analysis of VLPs with chimeric E proteins showed that E protein determines the difference in the transport across HUVEC between the 6-LP and Eg strains. Our data also suggest that multiple amino acid residues of E protein are influential. It has been well known that the sequence NYS/T at the residues 154-156 is important EPZ5676 mw for glycosylation associated with the virulence of WNV and that strains possessing proline at the residue 156 lack

glycosylation [10, 31–33]. The BIBW2992 mouse prototype WNV strain B956 has a 4 amino acid deletion in the residues 156-159 resulting in absence of glycosylation [48]. The position of glycosylation seems to be also important, since the WNI-25 and WNI-25A strains which have N-glycosylation at the residue 155, do not show neuroinvasive phenotype [49, 50]. The present study suggests that the combination of Ser 156 and Val 159 is important for transport of VLPs across endothelial cells, which might be associated with the invasion of WNV into the target organs. The transport of Eg P156 S VLPs was lower than that of WT Eg VLPs in spite of the presence of glycosylation. The residues 156-160 form two turns of α-helix, termed αA’, although E proteins of Dengue virus serotype 2 (DENV-2) and Tick-borne encephalitis virus (TBEV) lack the amino acids 157-160 resulting in the absence of this structure[51]. The α-helix shifts the glycosylation site about 5 Å to the exterior and lateral surfaces of E protein with respect to those of E proteins of DENV-2 and TBEV.

Davis et al. [52] showed that modulation of N-glycosylation of WNV E protein modified the attachment to DC-SIGNR. As well as the existence of proline Thymidine kinase and the deletion of the amino acids between the residues 156-160, there is a possibility that the combination of amino acid residues at 156 and 159 might affect the structure of αA’ and position of glycosylation site, resulting in modulation of the binding affinity to a lectin or unknown binding molecules on HUVEC. This, in turn, could be a reason for the unsuccessful transport of Eg P156 S VLPs. Conclusion In this study, we propose a transcellular mechanism by which WNV crosses endothelial cells and enters the target organs. We also suggest that higher transendothelial migration ability could be one of the determinants of the different virulence of the NY and Eg strains, and that this depends on Ser 156 and Val 159 of E protein. Methods Cell culture HUVEC were purchased from Lonza Group Ltd.