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Human immunodeficiency virus type 1 (HIV-1) gene expression is activated by Tat, a virally encoded protein. Tat trans-activation requires viral (trans-activation—responsive; TAR) RNA sequences located in the R region of the long terminal repeat (LTR). Existing evidence suggests that Tat probably cooperates with cellular factors that bind to TAR RNA in the overall trans-activation process. A HeLa complementary DNA was isolated and characterized that encodes a TAR RNA-binding protein (TRBP). TRBP activated the HIV-1 LTR and was synergistic with Tat function.

Despite the great potential of RNAi, ectopic expression of shRNA or siRNAs holds the inherent risk of competition for critical RNAi components, thus altering the regulatory functions of some cellular microRNAs. In addition, specific siRNA sequences can potentially hinder incorporation of other siRNAs when used in a combinatorial approach. We show that both synthetic siRNAs and expressed shRNAs compete against each other and with the endogenous microRNAs for transport and for incorporation into the RNA induced silencing complex (RISC). The same siRNA sequences do not display competition when expressed from a microRNA backbone. We also show that TAR RNA binding protein (TRBP) is one of the sensors for selection and incorporation of the guide sequence of interfering RNAs. These findings reveal that combinatorial siRNA approaches can be problematic and have important implications for the methodology of expression and use of therapeutic interfering RNAs.

TAR RNA binding protein (TRBP) belongs to an RNA binding protein family that includes the double‐stranded RNA‐activated protein kinase (PKR), Drosophila Staufen and Xenopus xlrbpa . One member of this family, PKR, is a serine/threonine kinase which has anti‐viral and anti‐proliferative effects. In this study we show that TRBP is a cellular down‐regulator of PKR function. Assaying expression from an infectious HIV‐1 molecular clone, we found that PKR inhibited viral protein synthesis and that over‐expression of TRBP effectively countered this inhibition. In intracellular and in cell‐free assays we show that TRBP directly inhibits PKR autophosphorylation through an RNA binding‐independent pathway. Biologically, TRBP serves a growth‐promoting role; cells that over‐express TRBP exhibit transformed phenotypes. Our results demonstrate the oncogenic potential of TRBP and are consistent with the notion that intracellular PKR function contributes physiologically towards regulating cellular proliferation.

HIV-1 TAR RNA is the binding site of the viral protein Tat, the trans-activator of the HIV-1 LTR. It is present at the 5' end of all HIV-1 spliced and unspliced mRNAs in the nucleus as well as in the cytoplasm. It has a highly folded stem-bulge-loop structure, which also binds cellular proteins to form ribonucleoprotein complexes. The Tat-Cyclin T1-CDK9 complex is the main component in the trans-activation of HIV-1 and its affinity for TAR is regulated through Tat acetylation by histone acetyl transferases. Recent studies show that this complex is able to recruit other cellular partners to mediate efficient transcriptional elongation. TRBP, PKR and La bind directly to the TAR RNA structure and influence translation of HIV-1 in either positive or negative manners. Some mutations in TAR RNA severely impair HIV-1 trans-activation, translation and viral production, showing its functional importance. The overexpression or suppression of several TAR RNAbinding proteins has a strong impact on viral replication pointing out their major role in the viral life cycle. TAR RNA has been the target of drug development to inhibit viral replication. Recent data using small molecules or RNA-based technologies show that acting on the TAR RNA or on its viral and cellular binding factors effectively decreases virion production.