5% �� 0.9%). Thus, FRET analyses revealed a self-interaction of HCV NS4B in the membrane environment of intact cells. No FRET was observed when one or both partners harbored the EPZ-5676 leukemia fluorescent protein at the N terminus (FRETeff, <10% [comparable to the values obtained after cotransfection of unfused CFP and YFP, i.e., the negative control]). Thus, although an interaction may have occurred between the two NS4B moieties, the fluorophores may be positioned improperly for FRET when fused to the N terminus of NS4B. This observation underscores the stringency of the FRET approach and demonstrates that colocalization to the ER or ER-derived modified membranes is not sufficient to produce FRET. Accordingly, no FRET was observed between HCV NS4B and DV NS4B, despite their colocalization, thus corroborating the specificity of the result observed for HCV NS4B.

Interestingly, no FRET was observed between DV NS4B molecules in this experimental setting. This may be related to the observation that DV NS4B does not induce membrane rearrangements and may have functions distinct from those of HCV NS4B in the DV life cycle (34). In DV, NS4A is the key protein responsible for the induction of membrane rearrangements (32). In conclusion, FRET analyses revealed a specific oligomerization of HCV NS4B in the membrane environment of intact cells. Multiple determinants contribute to the oligomerization of HCV NS4B. NS4B is a 261-amino-acid integral membrane protein comprising an N-terminal part with a predicted and a structurally resolved amphipathic ��-helix (AH1 and AH2, respectively), a central part harboring four predicted transmembrane segments, and a C-terminal part comprising a predicted and a structurally resolved ��-helix (H1 and H2, respectively) (Fig.

(Fig.2A;2A; see Fig. S1 in the supplemental material) (16). To identify regions involved in the oligomerization of NS4B, we designed fragments based on these structural elements and fused GFP to their C termini to investigate their subcellular localization. A fragment comprising the N-terminal half of NS4B (amino acids 1 to 130) displayed the same subcellular localization as full-length NS4B (Fig. (Fig.2A2A and data not shown). Localization on ER and ER-derived modified membranes was preserved after deletion of AH1 (segment 40-130).

However, further truncation at the N terminus (segment 61-130) or C terminus (segment 1-116) abrogated the typical fluorescence pattern and produced coarse cytoplasmic aggregates (Fig. (Fig.2A).2A). As shown in Fig. Fig.2A,2A, a fragment comprising the C-terminal half of NS4B (amino acids 130 to 261) also displayed the same subcellular localization AV-951 as full-length NS4B, while it was shown previously that N-terminal truncation of this fragment alters the subcellular localization (15). FIG. 2. Several determinants contribute to the oligomerization of HCV NS4B. (A) Subcellular localization of NS4B fragments.