Production of a non-functional nef protein in human immunodeficiency virus type 1-infected CEM cells

The nef gene product of the human immunodeficiency virus (HIV) is suggested to be a negative factor involved in down-regulating viral expression by a mechanism in which the correct conformation of the nef protein is essential. The nef protein expressed by vaccinia virus recombinants is phosphorylated by protein kinase C. We investigated the synthesis of the nef protein and its state of phosphorylation during HIV-1 infection of a T4 cell line (CEM cells). Maximum synthesis of viral proteins occurred 3 days after infection, when more than 90% of cells were producing viral proteins. The synthesis of the nef protein was detected in parallel with the env and gag proteins. As expected, the nef protein was myristylated but not phosphorylated, and its half-life was less than 1 h. By the use of the polymerase chain reaction technique, we isolated and sequenced the nef gene of this HIV-1 stock. Two significant mutations were observed. Firstly, threonine, at amino acid number 15, the site of phosphorylation by protein kinase C, was mutated into an alanine, and secondly aspartic acid of the tetrapeptide WRFD, which is probably involved in GTP binding, was mutated into an asparagine. The mutated nef gene was expressed in a vaccinia virus system, in which it was not phosphorylated and its half-life was dramatically reduced compared to the wild-type nef gene product. Furthermore, down-regulation of CD4 cell surface expression was no longer affected by the mutated nef gene. These results emphasize that phosphorylation of the nef protein provides an efficient test to monitor its biological activity.