Potent inhibition of HIV-1 gene expression and TAT-mediated apoptosis in human T cells by novel mono- and multitarget anti-TAT/Rev/Env ribozymes and a general purpose RNA-cleaving DNA-enzyme.
One of the hallmarks of progression of HIV-1/AIDS is the rapid depletion of CD4+T cells that is known to occur at the late stages of the disease when usually X4 tropic HIV-1 predominates. Besides direct killing of T lymphocytes, HIV-1 infection leads to extensive apoptosis of naïve/uninfected bystander T cells, which is predominantly mediated by HIV-1 TAT protein. Therefore, reduction of HIV-1 TAT protein is likely to reduce substantially the pathogenesis associated with HIV-1 infection. We designed two non-GUX hammerhead ribozymes (Rzs) and a Di-Rz by placing them in direct tandem. These were targeted against the most conserved second exon of HIV-1 TAT/Rev/Env region. Although very impressive in vitro cleavage of the target RNA by the two hammerhead Rzs was obtained under standard conditions of cleavage, only one of them was active under simulated physiological conditions. Sequence-specific cleavage by the Di-Rz was most efficient under standard conditions. Cleavage reactions carried out under simulated physiological conditions by the Di-Rz, however, indicated that both mono-Rzs were functional. We also assembled a 10-23 catalytic motif containing general purpose RNA-cleaving DNA-enzyme (Dz) against the same region, which cleaved the target RNA very efficiently. Both Rzs and Dz showed not only potent inhibition of HIV-1 gene expression but also showed remarkable protection against HIV-1 TAT protein-mediated apoptosis in Jurkat T cells. Possible implications of these findings are discussed.
Unwalla
Hoshang
H
National Institute of Immunology, JNU Campus, New Delhi 110067, India.
Chakraborti
Samitabh
S
Sood
Vikas
V
Gupta
Nidhi
N
Banerjea
Akhil C
AC
D019380:Anti-HIV Agents / Q000378:metabolism* / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects*; D002460:Cell Line; D002478:Cells, Cultured; D021881:DNA, Catalytic / Q000378:metabolism* / Q000494:pharmacology; D015870:Gene Expression / Q000187:drug effects*; D015696:Gene Products, tat / Q000037:antagonists & inhibitors*; D015752:Genes, env / Q000235:genetics; D015757:Genes, rev / Q000235:genetics; D015497:HIV-1 / Q000235:genetics*; D006367:HeLa Cells; D006801:Humans; D019169:Jurkat Cells; D008264:Macrophages / Q000821:virology*; D016337:RNA, Catalytic / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D012367:RNA, Viral / Q000378:metabolism; D054322:tat Gene Products, Human Immunodeficiency Virus
D019380:Anti-HIV Agents; D021881:DNA, Catalytic; D015696:Gene Products, tat; D016337:RNA, Catalytic; D012367:RNA, Viral; C427204:hammerhead ribozyme; D054322:tat Gene Products, Human Immunodeficiency Virus