Conformational changes of the HIV-1 envelope protein during membrane fusion are inhibited by the replacement of its membrane-spanning domain.
To help understand the dynamic nature of membrane fusion induced by the human immunodeficiency virus-1 (HIV-1) envelope protein, we developed a new cell-based real-time assay system employing a pair of novel reporter proteins. The reporter proteins consist of a pair of split Renilla luciferase (spRL) fused to split green fluorescent protein (spGFP). The spGFP modules were chosen not only to compensate weak self-association of spRL but also to provide visual reporter signals during membrane fusion. Use of this reporter together with a membrane permeable substrate for Renilla luciferase achieved a simple real-time monitoring of membrane fusion using live cells. We analyzed the HIV-1 envelope mutants whose membrane-spanning domains were replaced with that of glycophorin A or vesicular stomatitis virus G-protein. These mutants showed a slower kinetics of membrane fusion. The analysis of membrane fusion in the presence of fusion inhibitors, soluble CD4 and C34, revealed that these replacements prolonged the period during which the mutants were sensitive to the inhibitors, as compared with the wild type. These results suggest that the mutations within the membrane-spanning domains exerted an allosteric effect on the HIV-1 envelope protein, probably affecting the receptor-induced conformational changes of the ectodomain of the protein.
Kondo
Naoyuki
N
Research Center for Asian Infectious Diseases, Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan.
Miyauchi
Kosuke
K
Meng
Fanxia
F
Iwamoto
Aikichi
A
Matsuda
Zene
Z
D017252:CD3 Complex / Q000378:metabolism; D015704:CD4 Antigens / Q000378:metabolism; D006021:Glycophorins / Q000235:genetics; D015699:HIV Envelope Protein gp120 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015700:HIV Envelope Protein gp41 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008561:Membrane Fusion; D008562:Membrane Glycoproteins / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014759:Viral Envelope Proteins / Q000235:genetics; D014779:Virus Replication
D017252:CD3 Complex; D015704:CD4 Antigens; C028391:G protein, vesicular stomatitis virus; D006021:Glycophorins; D015699:HIV Envelope Protein gp120; D015700:HIV Envelope Protein gp41; D008562:Membrane Glycoproteins; D011993:Recombinant Fusion Proteins; D014759:Viral Envelope Proteins