cis Expression of the F12 human immunodeficiency virus (HIV) nef allele transforms the highly productive NL4-3 HIV type 1 to a replication-defective strain: Involvement of both Env gp41 and CD4 intracytoplasmic tails

F12 human immunodeficiency virus type 1 (HIV-1) nef is a naturally occurrin g nef mutant cloned from the provirus of a nonproductive, nondefective, and interfering HIV-1 variant (F12-HIV). We have already shown that cells stab ly transfected with a vector expressing the F12-HIV nef allele do not downr egulate CD4 receptors and, more peculiarly, become resistant to the replica tion of wild type (wt) HIV. In order to investigate the mechanism of action of such an HIV inhibition, the F12-HIV nef gene was expressed in the conte xt of the NL4-3 HIV-1 infectious molecular clone by replacing the wt nef ge ne (NL4-3/chi). Through this experimental approach we established the follo wing. First, NL4-3/chi and nef-defective (Delta nef) NL4-3 viral particles behave very similarly in terms of viral entry and HIV protein production du ring the first replicative cycle. Second, no viral particles were produced from cells infected with NL4-3/chi virions, whatever the multiplicity of in fection used. The viral inhibition apparently occurs at level of viral asse mbling and/or release. Third, this block could not be relieved by in-trans expression of wt nef. Finally, NL4-3/chi reverts to a producer HIV strain w hen F12-HIV Nef is deprived of its myristoyl residue. Through a CD4 downreg ulation competition assay, we demonstrated that F12-HIV Nef protein potentl y inhibits the CD4 downregulatiom induced by wt Nef. Moreover, we observed a redistribution of CD4 receptors at the cell margin induced by F12-HIV Nef . These observations strongly suggest that F12-HIV Nef maintains the abilit y to interact with the intracytoplasmic tail of the CD4 receptor molecule. Remarkably, we distinguished the intracytoplasmic tails of Fm gp41 and CD4 as, respectively, viral and cellular targets of the F12-HIV Nef-induced vir al retention. For the first time, the inhibition of the viral life cycle by means of in-cis expression of a Nef mutant is here reported. Delineation o f the F12-HIV Nef mechanism of action may offer additional approaches to in terference with the propagation of HIV infection.