A novel ribozyme target site located in the HIV-1 nef open reading frame.
We have tested the sequence UUC CAG UCA GAC CU, at position 9016--9029 within the HIV-1(SF2) nef open reading frame, for accessibility to antisense and hammerhead ribozyme attack. The accessibility was first studied using a phosphorothioate-modified 14-nt DNA oligo (complementary to the nef9016--9029 site). A dose-dependent repression of HIV-1(SF2) growth was observed in human peripheral blood mononuclear cells after exogenous administration of the oligo to the cell culture medium. A hammerhead ribozyme with a 6+7-nt antisense specificity for the nef9016--9029 site (hhRz.nef9016--9029) was constructed and transfected into the human T-cell line HUT78. Again, a dose-dependent repression of virus growth was observed when different individual clones expressing hhRz.nef9016--9029 were infected with HIV-1(SF2). A complete abrogation of virus production was observed after infection with a low (0.5 TCID50) HIV-1 titer. Increasing doses (2.5 and 12.5 TCID50) of HIV-1 virus yielded a low production (10(3)-fold reduced) of virus particles in most cases; but a complete, or close to complete, abrogation was observed even in individual cultures infected with the highest dose. Presence of proviral pol and gag sequences in hhRz.nef9016--9029-expressing HUT78 clones was assayed, using PCR. Interestingly, since no pol and gag PCR products could be detected, the results strongly indicated that the hammerhead ribozyme was already acting on the infecting HIV RNA before its reverse transcription and integration as proviral DNA. In summary, the results obtained in this study support the nef9016--9029 site as a strong new candidate for ribozymal gene therapy against HIV-1 infection.
Larsson
S
S
Department of Medical Nutrition, Karolinska Institute, Huddinge, Sweden.
Hotchkiss
G
G
Su
J
J
Kebede
T
T
Andäng
M
M
Nyholm
T
T
Johansson
B
B
Sönnerborg
A
A
Vahlne
A
A
Britton
S
S
Ahrlund-Richter
L
L
D001483:Base Sequence; D002460:Cell Line; D002478:Cells, Cultured; D004279:DNA, Viral; D015693:Gene Products, nef / Q000235:genetics* / Q000378:metabolism; D016655:HIV Core Protein p24 / Q000378:metabolism; D015497:HIV-1 / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000166:cytology / Q000378:metabolism; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D011533:Proviruses / Q000235:genetics; D016337:RNA, Catalytic / Q000378:metabolism*; D012367:RNA, Viral / Q000378:metabolism*; D013379:Substrate Specificity; D013601:T-Lymphocytes / Q000166:cytology / Q000821:virology; D013873:Thionucleotides; D014162:Transfection; D054311:nef Gene Products, Human Immunodeficiency Virus
D004279:DNA, Viral; D015693:Gene Products, nef; D016655:HIV Core Protein p24; D016337:RNA, Catalytic; D012367:RNA, Viral; D013873:Thionucleotides; D054311:nef Gene Products, Human Immunodeficiency Virus