Infectivity enhancement by human immunodeficiency virus type 1 Nef is independent of its association with a cellular serine/threonine kinase.
Nef proteins from human immunodeficiency virus type 1 isolate SF2 (HIV-1SF2) and simian immunodeficiency virus isolate mac239 (SIVmac239) have been found to associate with a cellular serine/threonine kinase designated NAK. We have recently shown that the association of Nef with NAK is isolate dependent. To identify the structural basis for Nef-kinase association, several chimeric molecules were constructed between SF2 Nef (binding NAK) and 233 Nef (a primary isolate not binding NAK) and stably expressed in HuT-78 human T cells via retrovirus-mediated gene transfer. The Nef 233/SF2/SF2 chimera in which the N-terminal 37 amino acids of SF2 Nef were replaced by those of 233 Nef showed the same ability as SF2 Nef to bind NAK. The Nef 233/SF2/233 chimera in which the N-terminal 37 amino acids and the C-terminal 72 amino acids of SF2 Nef were replaced by corresponding sequences from 233 Nef completely lost the ability to associate with the kinase activity. Furthermore, replacement of the C-terminal 72 amino acids of 233 Nef with the equivalent SF2 sequence (chimera 233/233/SF2) fully restored kinase association to 233 Nef. These results suggest that (i) the core of Nef is not sufficient for NAK binding, (ii) the C terminus of SF2 Nef contains structural determinants important for association with NAK, and (iii) the failure of 233 Nef to bind NAK is due to a defect in its C terminus. Taking advantage of the C terminus of 233 Nef being nonfunctional and using an infectious clone of HIV-1SF2, we show that association with NAK is not required for Nef-mediated infectivity enhancement. While the strong and reproducible association of some Nef isolates with NAK has been clearly established, the role of NAK in Nef function remains to be fully elucidated.
Luo
T
T
Department of Virology and Molecular Biology, St. Jude Children's Research Hospital, Memphis, Tennessee 38105, USA.
Livingston
R A
RA
Garcia
J V
JV
D002461:Cell Line, Transformed; D015693:Gene Products, nef / Q000378:metabolism*; D015497:HIV-1 / Q000302:isolation & purification / Q000378:metabolism* / Q000472:pathogenicity; D006367:HeLa Cells; D006801:Humans; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D013329:Structure-Activity Relationship; D054311:nef Gene Products, Human Immunodeficiency Virus
D015693:Gene Products, nef; D054311:nef Gene Products, Human Immunodeficiency Virus; D017346:Protein Serine-Threonine Kinases