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

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.

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From the Research Center for Asian Infectious Diseases.
the Division of Cellular and Molecular Biology, and.
the Laboratory of Structural Virology and Immunology, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100864, 100101 China, and.
the Department of Immunology, Graduate School of Medicine, University of the Ryukyus, Okinawa 903-0215, Japan.
the Division of Molecular Virology, Department of Microbiology and Immunology, Institute of Medical Science, University of Tokyo, Tokyo 108-8639, Japan.
From the Research Center for Asian Infectious Diseases,
From the Research Center for Asian Infectious Diseases,


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.


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

[Available on 2020-04-05]

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