Abstract
BackgroundThe native pre-fusion structure of gp120/gp41 complex of human immunodeficiency virus type 1 was recently revealed. In the model, the helices of gp41 (α6, α7, α8, and α9) form a four-helix collar underneath trimeric gp120. Gp41 is a class I fusion protein and mediates membrane fusion by forming a post-fusion structure called the six-helix bundle (6HB). The comparison of the pre- and post-fusion structures revealed the large conformational changes in gp41 during the antiparallel packing of the N- and C-terminal heptad repeats (NHRs and CHRs) in membrane fusion. Several mutagenesis studies of gp41 performed in the past were interpreted based on 6HB, the only available structure at that time. To obtain an insight about the current pre-fusion structural model and conformational changes during membrane fusion, alanine insertion mutagenesis of the NHR, CHR and connecting loop regions of HXB2 gp41 was performed. The effects of mutations on biosynthesis and membrane fusion were analyzed by immunoblotting and fusion assays, respectively. The extent of membrane fusion was evaluated by split luciferase-based pore formation and syncytia formation assays, respectively.ResultsConsistent with the current structural model, drastic negative effects of mutations on biosynthesis and membrane fusion were observed for NHR, loop, and proximal regions of CHR (up to amino acid position 643). The insertions in α9 after it leaves the four-helix collar were tolerable for biosynthesis. These CHR mutants showed varying effects on membrane fusion. Insertion at position 644 or 645 resulted in poor pore and syncytia formation. Efficient pore and syncytia formation almost similar to that of the wild type was observed for insertion at position 647, 648 or 649. However, recovery of virus infectivity was only observed for the insertions beyond position 648.ConclusionsThe mutagenesis data for HXB2 gp41 is in agreement with the recent pre-fusion structure model. The virus infection data suggested that fusion pores sufficiently large enough for the release of the virus genome complex are formed after the completion of 6HB beyond position 648.
Highlights
The native pre-fusion structure of gp120/gp41 complex of human immunodeficiency virus type 1 was recently revealed
The virus infection data suggested that fusion pores sufficiently large enough for the release of the virus genome complex are formed after the completion of six-helix bundle (6HB) beyond position 648
Insertion of an alanine residue in the N-terminal heptad repeat (NHR) and loop portions more negatively affected the biosynthesis of envelope glycoprotein (Env) than insertions in C-terminal heptad repeat (CHR) To probe the structure–function relationship of gp41, we generated alanine insertion mutants of gp41 by introducing one alanine residue at a time in the coding sequences of NHR, CHR, and the connecting loop between them (Fig. 1b)
Summary
The native pre-fusion structure of gp120/gp complex of human immunodeficiency virus type 1 was recently revealed. Gp41 is a class I fusion protein and mediates membrane fusion by forming a post-fusion structure called the six-helix bundle (6HB). To obtain an insight about the current pre-fusion structural model and conformational changes during membrane fusion, alanine insertion mutagenesis of the NHR, CHR and connecting loop regions of HXB2 gp was performed. The comparison of the pre-fusion structure with that of post-fusion gp, called the six-helix bundle (6HB), revealed the presence of dynamic conformational rearrangements during membrane fusion such as loop to helix conversions, similar to those of influenza HA2 [7,8,9,10,11,12]. The details of the conformational changes remain elusive because the intermediate forms of gp remain unknown
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
