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-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 gag, vif, or nef gene were resistant, to different degrees, to infection with T-cell-line-adapted HIV-1 strains. Conversely, expression of either the tat, rev, or vpu 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 vpr, pol, or env 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 vif or nef 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 vif-permissive and vif-nonpermissive cells. The levels of action of F12-HIV vif and nef anti-HIV 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 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.
D'Aloja
P
P
Laboratory of Virology, Istituto Superiore di Sanità, Rome, Italy.
Olivetta
E
E
Bona
R
R
Nappi
F
F
Pedacchia
D
D
Pugliese
K
K
Ferrari
G
G
Verani
P
P
Federico
M
M
D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002999:Clone Cells; D015870:Gene Expression; D015683:Gene Products, gag / Q000235:genetics / Q000378:metabolism*; D015693:Gene Products, nef / Q000235:genetics / Q000378:metabolism*; D016346:Gene Products, vif / Q000235:genetics / Q000378:metabolism*; D015757:Genes, rev; D014644:Genetic Variation; D015497:HIV-1 / Q000235:genetics / Q000302:isolation & purification / Q000502:physiology*; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D013601:T-Lymphocytes / Q000821:virology; D014762:Viral Interference; D014779:Virus Replication; D054311:nef Gene Products, Human Immunodeficiency Virus; D054320:vif Gene Products, Human Immunodeficiency Virus
D015683:Gene Products, gag; D015693:Gene Products, nef; D016346:Gene Products, vif; D054311:nef Gene Products, Human Immunodeficiency Virus; D054320:vif Gene Products, Human Immunodeficiency Virus