HIV-1 TAR element is processed by Dicer to yield a viral micro-RNA involved in chromatin remodeling of the viral LTR.
BACKGROUND
RNA interference (RNAi) is a regulatory mechanism conserved in higher eukaryotes. The RNAi pathway generates small interfering RNA (siRNA) or micro RNA (miRNA) from either long double stranded stretches of RNA or RNA hairpins, respectively. The siRNA or miRNA then guides an effector complex to a homologous sequence of mRNA and regulates suppression of gene expression through one of several mechanisms. The suppression of gene expression through these mechanisms serves to regulate endogenous gene expression and protect the cell from foreign nucleic acids. There is growing evidence that many viruses have developed in the context of RNAi and express either a suppressor of RNAi or their own viral miRNA.


RESULTS
In this study we investigated the possibility that the HIV-1 TAR element, a hairpin structure of ~50 nucleotides found at the 5' end of the HIV viral mRNA, is recognized by the RNAi machinery and processed to yield a viral miRNA. We show that the protein Dicer, the enzyme responsible for cleaving miRNA and siRNA from longer RNA sequences, is expressed in CD4+ T-cells. Interestingly, the level of expression of Dicer in monocytes is sub-optimal, suggesting a possible role for RNAi in maintaining latency in T-cells. Using a biotin labeled TAR element we demonstrate that Dicer binds to this structure. We show that recombinant Dicer is capable of cleaving the TAR element in vitro and that TAR derived miRNA is present in HIV-1 infected cell lines and primary T-cell blasts. Finally, we show that a TAR derived miRNA is capable of regulating viral gene expression and may be involved in repressing gene expression through transcriptional silencing.


CONCLUSIONS
HIV-1 TAR element is processed by the Dicer enzyme to create a viral miRNA. This viral miRNA is detectable in infected cells and appears to contribute to viral latency.
Immunology, Microbiology and Tropical Medicine program, The George Washington University School of Medicine, Washington, District of Columbia 20037, USA. bcmzak@gwumc.edu <bcmzak@gwumc.edu>
Zachary
Z
Klase
Prachee
P
Kale
Rafael
R
Winograd
Madhur V
MV
Gupta
Mohammad
M
Heydarian
Reem
R
Berro
Timothy
T
McCaffrey
Fatah
F
Kashanchi
D015153:Blotting, Western; D015704:CD4 Antigens / Q000378:metabolism; D002460:Cell Line; D002478:Cells, Cultured; D042002:Chromatin Assembly and Disassembly; D047369:Chromatin Immunoprecipitation; D053487:DEAD-box RNA Helicases / Q000235:genetics / Q000378:metabolism*; D004722:Endoribonucleases / Q000235:genetics / Q000378:metabolism*; D015967:Gene Expression Regulation, Viral; D016325:HIV Long Terminal Repeat / Q000235:genetics*; D015497:HIV-1 / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D006801:Humans; D019169:Jurkat Cells; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D035683:MicroRNAs / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D011485:Protein Binding; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D043244:Ribonuclease III; D013601:T-Lymphocytes / Q000378:metabolism / Q000821:virology; D020298:U937 Cells
D015704:CD4 Antigens; D035683:MicroRNAs; D034741:RNA, Small Interfering; D012367:RNA, Viral; D011993:Recombinant Fusion Proteins; D008156:Luciferases; D004722:Endoribonucleases; C408087:DICER1 protein, human; D043244:Ribonuclease III; D053487:DEAD-box RNA Helicases