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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 alpha -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 beta -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 beta -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 beta -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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