Inducible expression of IkappaBalpha repressor mutants interferes with NF-kappaB activity and HIV-1 replication in Jurkat T cells.
Human immunodeficiency virus (HIV-1) utilizes the NF-kappaB/Rel proteins to regulate transcription through NF-kappaB binding sites in the HIV-1 long terminal repeat (LTR). Normally, NF-kappaB is retained in the cytoplasm by inhibitory IkappaB proteins; after stimulation by multiple activators including viruses, IkappaBalpha is phosphorylated and degraded, resulting in NF-kappaB release. In the present study, we examined the effect of tetracycline-inducible expression of transdominant repressors of IkappaBalpha (TD-IkappaBalpha) on HIV-1 multiplication using stably selected Jurkat T cells. TD-IkappaBalpha was inducibly expressed as early as 3 h after doxycycline addition and dramatically reduced both NF-kappaB DNA binding activity and LTR-directed gene activity. Interestingly, induced TD-IkappaBalpha expression also decreased endogenous IkappaBalpha expression to undetectable levels by 24 h after induction, demonstrating that TD-IkappaBalpha repressed endogenous NF-kappaB-dependent gene transcription. TD-IkappaBalpha expression also sensitized Jurkat cells to tumor necrosis factor-induced apoptosis. De novo HIV-1 infection of Jurkat cells was dramatically altered by TD-IkappaBalpha induction, resulting in inhibition of HIV-1 multiplication, as measured by p24 antigen, reverse transcriptase, and viral RNA. Given the multiple functions of the NF-kappaB/IkappaB pathway, TD-IkappaBalpha expression may interfere with HIV-1 multiplication at several levels: LTR-mediated transcription, Rev-mediated export of viral RNA, inhibition of HIV-1-induced pro-inflammatory cytokines, and increased sensitivity of HIV-1-infected cells to apoptosis.
Kwon
H
H
Lady Davis Institute for Medical Research, McGill University, Montreal, Quebec H3T 1E2, Canada.
Pelletier
N
N
DeLuca
C
C
Genin
P
P
Cisternas
S
S
Lin
R
R
Wainberg
M A
MA
Hiscott
J
J
D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D017209:Apoptosis; D004279:DNA, Viral / Q000187:drug effects / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors* / Q000096:biosynthesis / Q000235:genetics*; D015536:Down-Regulation / Q000187:drug effects; D004318:Doxycycline / Q000494:pharmacology; D015967:Gene Expression Regulation, Viral / Q000187:drug effects; D016655:HIV Core Protein p24 / Q000378:metabolism; D054303:HIV Reverse Transcriptase / Q000378:metabolism; D015497:HIV-1 / Q000235:genetics / Q000502:physiology*; D006801:Humans; D020672:I-kappa B Proteins; D019169:Jurkat Cells; D008969:Molecular Sequence Data; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000037:antagonists & inhibitors*; D012367:RNA, Viral / Q000187:drug effects / Q000378:metabolism; D012097:Repressor Proteins / Q000494:pharmacology*; D013601:T-Lymphocytes / Q000821:virology; D014779:Virus Replication / Q000187:drug effects*
D000900:Anti-Bacterial Agents; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D016655:HIV Core Protein p24; D020672:I-kappa B Proteins; D016328:NF-kappa B; C000606204:NFKBIA protein, human; D012367:RNA, Viral; D012097:Repressor Proteins; D000072000:NF-KappaB Inhibitor alpha; D054303:HIV Reverse Transcriptase; D004318:Doxycycline