Transport Cilengitide in vitro of 6-LP VLPs depends on E protein It is known that E protein interacts with viral receptors on the host cells [22–28] resulting in the induction of receptor mediated endocytosis [25, 29, 30]. To examine whether E protein is involved in the transport of VLPs, we generated chimeric VLPs using 6-LP and Eg VLPs. 6-LP

CM Eg E VLPs have C and M/prM proteins derived from 6-LP strain and E protein from Eg strain. Eg CM 6-LP E VLPs have C and M/prM protein from Eg strain and E protein from 6-LP strain. HUVEC were exposed to wild type or chimeric VLPs and transported VLPs were detected by IFU assay at 24 h p.i (Fig. 3). The transport of Eg CM 6-LP E VLPs was similar to that of wild type 6-LP VLPs and was MDV3100 cost significantly higher than those of 6-LP CM Eg E VLPs and wild type Eg VLPs (p < 0.01). 6-LP CM Eg E VLPs www.selleckchem.com/products/gsk1120212-jtp-74057.html were rarely transported across HUVEC as well as wild type Eg VLPs. These results suggest that the transport of VLPs across HUVEC is strongly affected by E protein. 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). Multiple amino acid residues of E protein influence the transport of 6-LP VLPs The E proteins of the 6-LP and Eg strain differ at 4 amino acid residues. To determine

the residues that enhance the transport of 6-LP VLPs, we produced mutant VLPs (Table 1). 6-LP S156P VLPs and 6-LP V159I VLPs had significantly reduced transport compared to wild type 6-LP VLPs (p < 0.01) although the amount of transported VLPs was much higher than that of Eg VLPs (p < 0.01; Fig. 4A). As shown in Fig. 4B, Eg K93R VLPs and Eg T126I VLPs showed increased transport compared to wild type Eg VLPs (p < 0.05). The FER transport of Eg I159V was significantly increased (p < 0.01), although it was much lower than 6-LP VLPs. Previous studies reported that Ser 156 is involved in the N-linked glycosylation at 154, which is important for virulence and neuroinvasion [31–34]. Therefore, we expected that the transport of Eg P156 S would be increased. However, the transport of Eg P156 S VLPs was significantly lower than that of WT Eg VLPs (p < 0.01). These results suggest that multiple residues of E protein can influence the transport of VLPs. Table 1 Single and double mutant VLPs Name Wild type Position1 Substitution2 6-LP R93K 6-LP 93 R→K 6-LP I126T 6-LP 126 I→T 6-LP S156P 6-LP 156 S→P 6-LP V159I 6-LP 159 V→I Eg K93R Eg 93 K→R Eg T126I Eg 126 T→I Eg P156S Eg 156 P→S Eg I159V Eg 159 I→V 6-LP S156P V159I 6-LP 156, 159 S→P, V→I Eg P156 S I159V Eg 156, 159 P→S, I→V 1 Amino acid position of E protein.