Abstract
BackgroundRNA 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.ResultsIn 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.ConclusionHIV-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.
Highlights
RNA interference (RNAi) is a regulatory mechanism conserved in higher eukaryotes
HIV-1 target cells express Dicer As Dicer is the major catalytic engine that generates micro RNA (miRNA) through the cleavage of dsRNA, we sought to determine if cell lines relevant to HIV-1 infection expressed Dicer
Our work has demonstrated the existence of an HIV-1 miRNA derived from the TAR RNA sequence
Summary
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 guides an effector complex to a homologous sequence of mRNA and regulates suppression of gene expression through one of several mechanisms. Double stranded RNA is recognized by the RNAi machinery and is processed into small, 21 nucleotide small-interfering RNA (siRNA) which are capable of suppressing gene expression. The small RNA associates with the RNAinduced silencing complex (RISC) and guides the complex to a complementary sequence of mRNA where a member of the Argonaute family of proteins cleaves the target mRNA [1,2,3,10]. The miRNA may guide the RISC complex to a complementary, but not perfectly matching, region in the 3'UTR of the mRNA This association inhibits protein translation without degrading the target mRNA [2,11,12]. The miRNA guides this complex to a complementary region of chromosomal DNA and recruits factors that modify the chromatin structure and induce transcriptional silencing [13,14,15,16]
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