Identification of cellular proteins that bind to the human immunodeficiency virus type 1 nef gene product in vitro: a role for myristylation.
The human immunodeficiency virus (HIV) type 1 nef gene product was expressed as an N-terminal fusion protein with glutathione-S-transferase (GST) in the baculovirus system. The resulting nefGST fusion protein was found to be authentically myristylated at the N terminus and could be purified to homogeneity by one-step affinity chromatography on immobilized glutathione. The high affinity of nefGST for glutathione was exploited to develop an assay to identify cellular proteins capable of interacting with nef. Several such proteins were identified in extracts from the Jurkat human T cell line. The interaction between nef-binding proteins and immobilized nefGST could be specifically competed by the addition of soluble nef. Cell fractionation showed that nef-binding proteins were present in both cytosolic and membrane-associated fractions. A non-myristylated derivative failed to bind to the membrane-associated proteins but was able to bind to the cytosolic group, albeit with reduced affinity. In addition, a single protein present in both soluble and membrane-associated fractions exhibited myristylation-independent binding to nef. By analogy with other myristylated proteins such as MARCKS (myristylated alanine-rich C kinase substrate) and the Rous sarcoma virus transforming protein, src, the membrane-associated proteins that bind only to myristylated nef may represent a specific membrane target for nef. The cytosolic proteins that interact with nef may constitute soluble components of an as yet unidentified signal transduction pathway which is the target of nef action in the HIV-1-infected cell.
Harris
M
M
Department of Veterinary Pathology, University of Glasgow, U.K.
Coates
K
K
D000215:Acylation; D000595:Amino Acid Sequence; D001483:Base Sequence; D002999:Clone Cells; D015870:Gene Expression; D015693:Gene Products, nef / Q000235:genetics / Q000378:metabolism*; D005982:Glutathione Transferase / Q000235:genetics; D015497:HIV-1 / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D011485:Protein Binding / Q000502:physiology; D011506:Proteins / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013329:Structure-Activity Relationship; D013601:T-Lymphocytes / Q000378:metabolism; D054311:nef Gene Products, Human Immunodeficiency Virus
D015693:Gene Products, nef; D011506:Proteins; D011993:Recombinant Fusion Proteins; D054311:nef Gene Products, Human Immunodeficiency Virus; D005982:Glutathione Transferase