Cell-cell and virus-cell fusion assay-based analyses of alanine insertion mutants in the distal α9 portion of the JRFL gp41 subunit from HIV-1

J Biol Chem. 2019 Apr 5;294(14):5677-5687. doi: 10.1074/jbc.RA118.004579. Epub 2019 Feb 8.


Membrane fusion is the first essential step in HIV-1 replication. The gp41 subunit of HIV-1 envelope protein (Env), a class I fusion protein, achieves membrane fusion by forming a structure called a six-helix bundle composed of N- and C-terminal heptad repeats. We have recently shown that the distal portion of the α9 helix in the C-terminal heptad repeat of X4-tropic HXB2 Env plays a critical role in the late-stage membrane fusion and viral infection. Here, we used R5-tropic JRFL Env and constructed six alanine insertion mutants, 641+A to 646+A, in the further distal portion of α9 where several glutamine residues are conserved (the number corresponds to the position of the inserted alanine in JRFL Env). 644+A showed the most severe defect in syncytia formation. Decreased fusion pore formation activity, revealed by a dual split protein assay, was observed in all mutants except 641+A. Sequence analysis and substitution of inserted 644A with Gln revealed that the glutamine residue at position 644 that forms complex hydrogen-bond networks with other polar residues on the surface of the six-helix bundle is critical for cell-cell fusion. We also developed a split NanoLuc® (Nluc) reporter-based assay specific to the virus-cell membrane fusion step to analyze several of the mutants. Interestingly syncytia-competent mutants failed to display Nluc activities. In addition to defective fusion activity, a reduction of Env incorporation into virions may further contribute to differences in cell-cell and virus-cell fusions.

Keywords: NanoLuc; Vpr; envelope protein; gp41; human immunodeficiency virus (HIV); membrane fusion; syncytia formation; viral protein; virology; virus entry.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alanine / genetics
  • Alanine / metabolism
  • Biological Assay*
  • Cell Line
  • HIV Envelope Protein gp41 / genetics
  • HIV Envelope Protein gp41 / metabolism*
  • HIV-1 / genetics
  • HIV-1 / metabolism*
  • Humans
  • Membrane Fusion*
  • Mutagenesis, Insertional*
  • Protein Structure, Secondary
  • Virus Internalization*


  • HIV Envelope Protein gp41
  • gp41 protein, Human immunodeficiency virus 1
  • Alanine

Associated data

  • PDB/5FUU
  • PDB/1ENV