<i>gag</i> , <i>vif</i> , and <i>nef</i> 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

<jats:title>ABSTRACT</jats:title> <jats:p> We previously demonstrated that expression of the nonproducer F12-human immunodeficiency virus type 1 (HIV-1) variant induces a block in the replication of superinfecting HIV that does not depend on the down-regulation of CD4 HIV receptors. In order to individuate the gene(s) involved in F12-HIV-induced interference, vectors expressing each of the nine F12-HIV proteins were transfected in HIV-susceptible HeLa CD4 cells. Pools of cell clones stably producing each viral protein were infected with HIV-1, and virus release was measured in terms of reverse transcriptase activity in supernatants. We hereby demonstrate that HeLa CD4 cells expressing the F12-HIV <jats:italic>gag</jats:italic> , <jats:italic>vif</jats:italic> , or <jats:italic>nef</jats:italic> gene were resistant, to different degrees, to infection with T-cell-line-adapted HIV-1 strains. Conversely, expression of either the <jats:italic>tat</jats:italic> , <jats:italic>rev</jats:italic> , or <jats:italic>vpu</jats:italic> F12-HIV gene increased the rate of HIV release, and no apparent effects on HIV replication were observed in cells expressing either the F12-HIV <jats:italic>vpr</jats:italic> , <jats:italic>pol</jats:italic> , or <jats:italic>env</jats:italic> gene. No variation of CD4 exposure was detected in any of the uninfected HeLa CD4 pools. These data indicate that F12-HIV homologous viral interference is the consequence of the synergistic anti-HIV effects of Gag, Vif, and Nef proteins. Retrovirus vectors expressing F12-HIV <jats:italic>vif</jats:italic> or <jats:italic>nef</jats:italic> allowed us to further establish that the expression of each mutated protein (i) inhibits the replication of clinical HIV-1 isolates as well, (ii) impairs the infectivity of the virus released by cells chronically infected with HIV-1, and (iii) limitedly to F12-HIV Vif protein, induces HIV resistance in both <jats:italic>vif</jats:italic> -permissive and <jats:italic>vif</jats:italic> -nonpermissive cells. The levels of action of F12-HIV <jats:italic>vif</jats:italic> and <jats:italic>nef</jats:italic> anti-HIV effects were also determined. We observed that HIV virions emerging from the first viral cycle on F12-HIV <jats:italic>vif</jats:italic> -expressing cells, although released in unaltered amounts, had a strongly reduced ability to initiate the retrotranscription process when they reinfected parental HeLa CD4 cells. Differently, we observed that expression of F12-HIV Nef protein affects the HIV life cycle at the level of viral assembling and/or release. For the first time, an inhibitory effect on the HIV life cycle in both acutely and chronically infected cells induced by mutated Vif and Nef HIV-1 proteins is described. These genes could thus be proposed as new useful reagents for anti-HIV gene therapy. </jats:p>