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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.

Author information

1
From the Research Center for Asian Infectious Diseases.
2
the Division of Cellular and Molecular Biology, and.
3
the Laboratory of Structural Virology and Immunology, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100864, 100101 China, and.
4
the Department of Immunology, Graduate School of Medicine, University of the Ryukyus, Okinawa 903-0215, Japan.
5
the Division of Molecular Virology, Department of Microbiology and Immunology, Institute of Medical Science, University of Tokyo, Tokyo 108-8639, Japan.
6
From the Research Center for Asian Infectious Diseases, jun-i@ims.u-tokyo.ac.jp.
7
From the Research Center for Asian Infectious Diseases, zmatsuda@ims.u-tokyo.ac.jp.

Abstract

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

PMID:
30737278
PMCID:
PMC6462516
[Available on 2020-04-05]
DOI:
10.1074/jbc.RA118.004579

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