GAG, VIF AND NEF GENES CONTRIBUTE TO THE HOMOLOGOUS VIRAL INTERFERENCE INDUCED BY A NONPRODUCER HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 (HIV-1)VARIANT - IDENTIFICATION OF NOVEL HIV-1-INHIBITING VIRAL PROTEIN MUTANTS

We previously demonstrated that expression of the nonproducer F12-huma n immunodeficiency virus type 1 (HIV-1) variant induces a block in the replication of superinfecting HIV that does not depend on the downreg ulation of CD4 HIV receptors. In order to individuate the gene(s) invo lved in F12-HIV-induced interference, vectors expressing each of the n ine F12-HIV proteins were transfected in HIV-susceptible HeLa CD4 cell s. Pools of cell clones stably producing each viral protein were infec ted with HIV-1, and virus release was measured in terms of reverse tra nscriptase activity in supernatants. We hereby demonstrate that HeLa C D4 cells expressing the F12-HIV gag, vif, or nef gene were resistant, to different degrees, to infection,vith T-cell-line-adapted HIV-1 stra ins. Conversely, expression of either the tat, rev, or vpu F12-HIV gen e increased the rate of HIV release, and no apparent effects on HIV re plication were observed in cells expressing either the F12-HIV vpr, po l, or env gene. No variation of CD4 exposure was detected in any of th e uninfected HeLa CD4 pools. These data indicate that F12-HIV homologo us viral interference is the consequence of the synergistic anti-HIV e ffects of Gag, Vif, and Nef proteins. Retrovirus vectors expressing F1 2-HIV vif or nef allowed us to further establish that the expression o f each mutated protein (i) inhibits the replication of clinical HIV-1 isolates as well, (ii) impairs the infectivity of the virus released b y cells chronically infected with HIV-1, and (iii) limitedly to F12-HI V Vif protein, induces HIV resistance in both vif-permissive and vif-n onpermissive cells. The levels of action of F12-HIV vif and nef anti-H IV effects were also determined. We observed that HIV virions emerging from the first viral cycle on F12-HIV vif-expressing cells, although released in unaltered amounts, had a strongly reduced ability to initi ate the retrotranscription process when they reinfected parental HeLa CD4 cells. Differently, we observed that expression of F12-HIV Nef pro tein affects the HIV life cycle at the level of viral assembling and/o r release. For the first time, an inhibitory effect on the HIV life cy cle in both acutely and chronically infected cells induced by mutated Vif and Nef HIV-1 proteins is described. These genes could thus be pro posed as new useful reagents for anti-HIV gene therapy.