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Prot Cell    2011, Vol. 2 Issue (5) : 369-376    https://doi.org/10.1007/s13238-011-1051-0      PMID: 21667332
RESEARCH ARTICLE
Conserved arginine residue in the membrane-spanning domain of HIV-1 gp41 is required for efficient membrane fusion
Yufei Long1,2, Fanxia Meng1, Naoyuki Kondo1,3, Aikichi Iwamoto4, Zene Matsuda1,5()
1. China-Japan Joint Laboratory of Structural Virology and Immunology, Institute of Biophysics, Chinese Academy of Sciences, 15 Datun Road, Beijing 100101, China; 2. Graduate University of the Chinese Academy of Sciences, Beijing 100101, China; 3. Department of Pediatrics, Emory University School of Medicine, 2015 Uppergate Dr. Atlanta, GA 30322, USA; 4. Division of Infectious Diseases, Advanced Clinical Research Center, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan; 5. Research Center for Asian Infectious Diseases, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan
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Abstract

Despite the high mutation rate of HIV-1, the amino acid sequences of the membrane-spanning domain (MSD) of HIV-1 gp41 are well conserved. Arginine residues are rarely found in single membrane-spanning domains, yet an arginine residue, R696 (the numbering is based on that of HXB2), is highly conserved in HIV-1 gp41. To examine the role of R696, it was mutated to K, A, I, L, D, E, N, and Q. Most of these substitutions did not affect the expression, processing or surface distribution of the envelope protein (Env). However, a syncytia formation assay showed that the substitution of R696 with amino acid residues other than K, a naturally observed mutation in the gp41 MSD, decreased fusion activity. Substitution with hydrophobic amino acid residues (A, I, and L) resulted in a modest decrease, while substitution with D or E, potentially negatively-charged residues, almost abolished the syncytia formation. All the fusion-defective mutants showed slower kinetics with the cell-based dual split protein (DSP) assay that scores the degree of membrane fusion based on pore formation between fusing cells. Interestingly, the D and E substitutions did show some fusion activity in the DSP assays, suggesting that proteins containing D or E substitutions retained some fusion pore-forming capability. However, nascent pores failed to develop, due probably to impaired activity in the pore enlargement process. Our data show the importance of this conserved arginine residue for efficient membrane fusion.
Keywords human immunodeficiency virus      type-1 (HIV-1)      gp41      membrane-spanning domain (MSD)      arginine      membrane fusion     
Corresponding Author(s): Matsuda Zene,Email:zmatsuda@ims.u-tokyo.ac.jp   
Issue Date: 01 May 2011
 Cite this article:   
Naoyuki Kondo,Aikichi Iwamoto,Zene Matsuda, et al. Conserved arginine residue in the membrane-spanning domain of HIV-1 gp41 is required for efficient membrane fusion[J]. Prot Cell, 2011, 2(5): 369-376.
 URL:  
https://academic.hep.com.cn/pac/EN/10.1007/s13238-011-1051-0
https://academic.hep.com.cn/pac/EN/Y2011/V2/I5/369
Fig.1  Conservation of the arginine residue in the MSD region among different clades of HIV-1 and Arg-substitution mutants used in this study.
(A) Consensus sequences of HIV-1 gp41 MSD of different clades were from the Los Alamos HIV sequence database (http://www.hiv.lanl.gov/content/index). The predicted MSD is indicated with capital letters. (B) Substitution mutations used in this study. Mutants were constructed using a QuikChange site mutation kit based on the HXB2 strain. Substitute amino acid residues are underlined. Lysine is potentially positively-charged, like arginine. Aspartic acid and glutamic acid have a negatively-charged side chain. Asparagine and glutamine share the same backbone with aspartic acid and glutamic acid, respectively, but lack their negative charge. Alanine, leucine and isoleucine were chosen to represent uncharged amino acids.
Fig.1  Conservation of the arginine residue in the MSD region among different clades of HIV-1 and Arg-substitution mutants used in this study.
(A) Consensus sequences of HIV-1 gp41 MSD of different clades were from the Los Alamos HIV sequence database (http://www.hiv.lanl.gov/content/index). The predicted MSD is indicated with capital letters. (B) Substitution mutations used in this study. Mutants were constructed using a QuikChange site mutation kit based on the HXB2 strain. Substitute amino acid residues are underlined. Lysine is potentially positively-charged, like arginine. Aspartic acid and glutamic acid have a negatively-charged side chain. Asparagine and glutamine share the same backbone with aspartic acid and glutamic acid, respectively, but lack their negative charge. Alanine, leucine and isoleucine were chosen to represent uncharged amino acids.
Fig.2  Protein profiles of the R-substitution mutants.
The envelope proteins expressed in COS-7 cells transiently transfected with the Env expression vectors for WT or mutants were detected with anti-gp120 polyclonal antibody. The gp160 and gp120 bands are indicated.
Fig.2  Protein profiles of the R-substitution mutants.
The envelope proteins expressed in COS-7 cells transiently transfected with the Env expression vectors for WT or mutants were detected with anti-gp120 polyclonal antibody. The gp160 and gp120 bands are indicated.
Fig.3  Fusion activity and fusion kinetics of R-substitution mutants in the cell-cell fusion assay.
(A) The fusion activity was measured by syncytia formation assay. 293CD4 cells were transfected with different mutants or WT, as well as Env KO as a negative control. Nuclei of cells were stained with Hoechst. Relative fusion activity was quantified by using a fusion index (fusion index= 2 + , where is the number of multinucleated cells [number of nuclei≥5 in five visual fields] and is the number of multinucleated cells [number of nuclei<5 in five visual fields]). Fusion activities for each mutant are shown after normalization to that of WT (with WT activity set normalized to 100%). Similar results were obtained from three independent experiments. Statistical significance of the difference between the RI and RL, RD and RN, RE and RQ mutants, respectively was determined using -test. (B) DSP assay was performed to monitor the kinetics of pore formation. luciferase (RL) activity was measured from 0–5 h after co-culture. Firefly luciferase (FL) activity from parallel 293FT cells was measured to normalize transfection efficiency. RL activity normalized by FL activity was shown to reflect the fusion kinetics.
Fig.3  Fusion activity and fusion kinetics of R-substitution mutants in the cell-cell fusion assay.
(A) The fusion activity was measured by syncytia formation assay. 293CD4 cells were transfected with different mutants or WT, as well as Env KO as a negative control. Nuclei of cells were stained with Hoechst. Relative fusion activity was quantified by using a fusion index (fusion index= 2 + , where is the number of multinucleated cells [number of nuclei≥5 in five visual fields] and is the number of multinucleated cells [number of nuclei<5 in five visual fields]). Fusion activities for each mutant are shown after normalization to that of WT (with WT activity set normalized to 100%). Similar results were obtained from three independent experiments. Statistical significance of the difference between the RI and RL, RD and RN, RE and RQ mutants, respectively was determined using -test. (B) DSP assay was performed to monitor the kinetics of pore formation. luciferase (RL) activity was measured from 0–5 h after co-culture. Firefly luciferase (FL) activity from parallel 293FT cells was measured to normalize transfection efficiency. RL activity normalized by FL activity was shown to reflect the fusion kinetics.
Fig.4  Level of surface expression of R-substitution mutants.
COS-7 cells were transfected with WT or mutants and subjected to immunofluorescence assays without permeabilization. Surface-expressed gp120 was detected by staining with 2G12 primary antibody and APC-conjugated secondary antibody. Internal EGFP signal was used as an indicator of transfected cells. Representative confocal microscopic images are shown.
Fig.4  Level of surface expression of R-substitution mutants.
COS-7 cells were transfected with WT or mutants and subjected to immunofluorescence assays without permeabilization. Surface-expressed gp120 was detected by staining with 2G12 primary antibody and APC-conjugated secondary antibody. Internal EGFP signal was used as an indicator of transfected cells. Representative confocal microscopic images are shown.
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