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 argin...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.展开更多
Foamy viruses(FVs)have broad cellular tropism infecting vertebrates from fish to human being,which indicates that Env protein has a high capability for membrane fusion.Conservative features in all FV transmembrane(TM)...Foamy viruses(FVs)have broad cellular tropism infecting vertebrates from fish to human being,which indicates that Env protein has a high capability for membrane fusion.Conservative features in all FV transmembrane(TM)proteins include a region of hydrophobic domain called membrane-spanning domain(MSD),which contains several stretches of hydrophobic amino acids.To investigate whether these features were associated with the cytotoxicity effect of TM on Escherichia coli,a series of mutants were constructed and expressed in the E.coli BL21(DE3)using pET-32a(+)as expressing vector.The results showed that only TM3 without MSD was expressed in E.coli,whereas the other two containing full or part of the MSD(TM1 and TM2)could not be expressed.Furthermore,the bacterial amount and living bacteria analysis revealed that the cytotoxicity of TM was dependent on its MSD,especially on the stretches of hydrophobic amino acids.Western blotting analysis showed that TM3 protein was purified with affinity purification.展开更多
基金supported by the Ministry of Education,Culture,Sports,Science and Technology of Japan,Japan Initiative for Global Research Network on Infectious Diseases(J-GRID).
文摘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.
基金This work was supported by National Natural Science Foundation for Youth(No.30100003).
文摘Foamy viruses(FVs)have broad cellular tropism infecting vertebrates from fish to human being,which indicates that Env protein has a high capability for membrane fusion.Conservative features in all FV transmembrane(TM)proteins include a region of hydrophobic domain called membrane-spanning domain(MSD),which contains several stretches of hydrophobic amino acids.To investigate whether these features were associated with the cytotoxicity effect of TM on Escherichia coli,a series of mutants were constructed and expressed in the E.coli BL21(DE3)using pET-32a(+)as expressing vector.The results showed that only TM3 without MSD was expressed in E.coli,whereas the other two containing full or part of the MSD(TM1 and TM2)could not be expressed.Furthermore,the bacterial amount and living bacteria analysis revealed that the cytotoxicity of TM was dependent on its MSD,especially on the stretches of hydrophobic amino acids.Western blotting analysis showed that TM3 protein was purified with affinity purification.
