.. Based on these preliminary experiments, interactions of segments 40-130 and 130-261 fused to CFP or YFP were investigated Sorafenib Tosylate CAS by FRET. As shown in Fig. Fig.2B,2B, these N- and C-terminal fragments were found to interact both with themselves and with each other. These results demonstrate that several determinants contribute to the oligomerization of NS4B, through homotypic (i.e., occurring between the same protein segment) and heterotypic (i.e., occurring between different protein segments) interactions. Whether the heterotypic interaction between the N- and C-terminal fragments occurs as an intermolecular or intramolecular association in the context of the full-length protein remains unknown. Confirmation of FRET results by coimmunoprecipitation. To corroborate the results obtained by FRET, we performed coimmunoprecipitation analyses.

To this end, the HA or FLAG tag was fused to the C terminus of full-length HCV NS4B or DV NS4B, as well as to fragments 40-130 and 130-261 from HCV NS4B. Lysates from cells coexpressing different combinations of these constructs were subjected to immunoprecipitation and Western blot analyses using anti-HA and anti-FLAG antibodies. As shown in Fig. Fig.3A,3A, these analyses revealed a specific self-interaction of HCV NS4B, while as expected, no interaction was observed between the NS4B proteins from HCV and DV. In addition, coimmunoprecipitation analyses confirmed the specificity of the homotypic and heterotypic interactions observed between the N- and C-terminal NS4B fragments 40-130 and 130-261 (Fig. (Fig.3B).3B).

Taken together, the results from the coimmunoprecipitation analyses confirm the oligomerization of HCV NS4B through several determinants. FIG. 3. Confirmation of FRET results by coimmunoprecipitation. (A) Constructs pCMVNS4B-HA, pCMVNS4B-FLAG, pCMVDV4B-HA, and pCMVDV4B-FLAG were transfected into U-2 OS cells as indicated, followed by immunoprecipitation (IP) of cell lysates with anti-FLAG M2 agarose … Oligomerization of NS4B proteins from different HCV genotypes. In order to assess whether our observations can be extended to other HCV strains and to gain insights into the genotype specificity of the identified interactions, we investigated FRET between NS4B sequences derived from the HCV H77 consensus (genotype 1a) and JFH-1 (genotype 2a) clones.

The overall amino acid sequence identity Brefeldin_A between these two NS4B sequences is 72%, while the 40-130 and 130-261 fragments display 70% and 82% identity, respectively (see Fig. S1 in the supplemental material). A schematic representation of the percent amino acid identity along the NS4B sequence, derived from the sequence alignment shown in Fig. S1, highlights the regions that are conserved between these relatively distant HCV strains (Fig. (Fig.4A4A). FIG. 4. Oligomerization of NS4B proteins from different HCV genotypes.