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Journal of Virology, October 2001, p. 9925-9938, Vol. 75, No. 20
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.20.9925-9938.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Cellular Membrane-Binding Ability of the C-Terminal
Cytoplasmic Domain of Human Immunodeficiency Virus Type 1 Envelope
Transmembrane Protein gp41
Steve S.-L.
Chen,1,*
Sheau-Fen
Lee,1 and
Chin-Tien
Wang2
Division of Infectious Diseases, Institute of
Biomedical Sciences, Academia Sinica, Taipei
11529,1 and Institute of Clinical
Medicine, National Yang-Ming University School of Medicine, and
Department of Medical Research and Education, Taipei Veterans General
Hospital, Taipei 11217,2 Taiwan, Republic of
China
Received 7 March 2001/Accepted 18 July 2001
The amphipathic
-helices located in the cytoplasmic tail of the
envelope (Env) transmembrane glycoprotein gp41 of human
immunodeficiency virus type 1 have been implicated in membrane
association and cytopathicity. Deletion of the last 12 amino acids in
the C terminus of this domain severely impairs infectivity. However,
the nature of the involvement of the cytoplasmic tail in Env-membrane
interactions in cells and the molecular basis for the defect in
infectivity of this mutant virus are still poorly understood. In this
study we examined the interaction of the cytoplasmic tail with
membranes in living mammalian cells by expressing a recombinant
cytoplasmic tail fragment and an Escherichia
coli
-galactosidase/cytoplasmic tail fusion protein, both of
them lacking gp120, the gp41 ectodomain, and the transmembrane region.
We found through cell fractionation, in vivo membrane flotation, and
confocal immunofluorescence studies that the cytoplasmic tail contained
determinants to be routed to a perinuclear membrane region in cells.
Further mapping showed that each of the three lentivirus lytic peptide
(LLP-1, LLP-2, and LLP-3) sequences conferred this cellular
membrane-targeting ability. Deletion of the last 12 amino acids from
the C terminus abolished the ability of the LLP-1 motif to bind to
membranes. High salt extraction, in vitro transcription and
translation, and posttranslational membrane binding analyses indicated
that the
-galactosidase/LLP fusion proteins were inserted into
membranes via the LLP sequences. Subcellular fractionation and confocal microscopy studies revealed that each of the LLP motifs, acting in a
position-independent manner, targeted non-endoplasmic reticulum (ER)-associated
-galactosidase and enhanced green fluorescence protein to the ER. Our study provides a basis for the involvement of
the gp41 cytoplasmic tail during Env maturation and also supports the
notion that the membrane apposition of the C-terminal cytoplasmic tail
plays a crucial role in virus-host interaction.
*
Corresponding author. Mailing address: Division of
Infectious Diseases, Institute of Biomedical Sciences, Academia Sinica, 128, Section 2, Yen-Chiu-Yuan Rd., Taipei 11529, Taiwan, Republic of
China. Phone: 886-2-2652-3933. Fax: 886-2-2785-8847. E-mail: schen{at}ibms.sinica.edu.tw.
Journal of Virology, October 2001, p. 9925-9938, Vol. 75, No. 20
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.20.9925-9938.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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