Relationship between human immunodeficiency virus type 1 Gag multimerization and membrane binding.
The human immunodeficiency virus type 1 (HIV-1) Gag precursor, Pr55(Gag), is necessary and sufficient for the assembly and release of viruslike particles. Binding of Gag to membrane and Gag multimerization are both essential steps in virus assembly, yet the domains responsible for these events have not been fully defined. In addition, the relationship between membrane binding and Gag-Gag interaction remains to be elucidated. To investigate these issues, we analyzed, in vivo, the membrane-binding and assembly properties of a series of C-terminally truncated Gag mutants. Pr55(Gag) was truncated at the C terminus of matrix (MAstop), between the N- and C-terminal domains of capsid (CA146stop), at the C terminus of capsid (p41stop), at the C terminus of p2 (p43stop), and after the N-terminal 35 amino acids of nucleocapsid (NC35stop). The ability of these truncated Gag molecules to assemble and release viruslike particles and their capacity to copackage into particles when coexpressed with full-length Gag were determined. We demonstrate that the amount of truncated Gag incorporated into particles is incrementally increased by extension from CA146 to NC35, suggesting that multiple sites in this region are involved in Gag multimerization. Using membrane flotation centrifugation, we observe that MA shows significantly reduced membrane binding relative to full-length Gag but that CA146 displays steady-state membrane-binding properties comparable to those of Pr55(Gag). The finding that the CA146 mutant, which contains only matrix and the N-terminal domain of capsid, exhibits levels of steady-state membrane binding equivalent to those of full-length Gag indicates that strong Gag-Gag interaction domains are not required for the efficient binding of HIV-1 Gag to membrane.
Ono
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Laboratory of Molecular Microbiology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892-0460, USA.
Demirov
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Freed
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D002462:Cell Membrane / Q000378:metabolism; D015683:Gene Products, gag / Q000235:genetics / Q000378:metabolism*; D015497:HIV-1 / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D006367:HeLa Cells; D006801:Humans; D016296:Mutagenesis; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism*; D014771:Virion / Q000502:physiology; D019065:Virus Assembly / Q000502:physiology; D054301:gag Gene Products, Human Immunodeficiency Virus
D015683:Gene Products, gag; D010446:Peptide Fragments; D011498:Protein Precursors; D054301:gag Gene Products, Human Immunodeficiency Virus; C109809:p2 gag peptide, Human immunodeficiency virus 1; C059767:p55 gag precursor protein, Human immunodeficiency virus 1