Characterization of the TRBP domain required for dicer interaction and function in RNA interference.

BACKGROUND: Dicer, Ago2 and TRBP are the minimum components of the human RNA-induced silencing complex (RISC). While Dicer and Ago2 are RNases, TRBP is the double-stranded RNA binding protein (dsRBP) that loads small interfering RNA into the RISC. TRBP binds directly to Dicer through its C-terminal domain. RESULTS: We show that the TRBP binding site in Dicer is a 165 amino acid (aa) region located between the ATPase and the helicase domains. The binding site in TRBP is a 69 aa domain, called C4, located at the C-terminal end of TRBP. The TRBP1 and TRBP2 isoforms, but not TRBPs lacking the C4 site (TRBPsDeltaC4), co-immunoprecipitated with Dicer. The C4 domain is therefore necessary to bind Dicer, irrespective of the presence of RNA. Immunofluorescence shows that while full-length TRBPs colocalize with Dicer, TRBPsDeltaC4 do not. tarbp2-/- cells, which do not express TRBP, do not support RNA interference (RNAi) mediated by short hairpin or micro RNAs against EGFP. Both TRBPs, but not TRBPsDeltaC4, were able to rescue RNAi function. In human cells with low RNAi activity, addition of TRBP1 or 2, but not TRBPsDeltaC4, rescued RNAi function. CONCLUSION: The mapping of the interaction sites between TRBP and Dicer show unique domains that are required for their binding. Since TRBPsDeltaC4 do not interact or colocalize with Dicer, we suggest that TRBP and Dicer, both dsRBPs, do not interact through bound dsRNA. TRBPs, but not TRBPsDeltaC4, rescue RNAi activity in RNAi-compromised cells, indicating that the binding of Dicer to TRBP is critical for RNAi function.

Small interfering RNAs against the TAR RNA binding protein, TRBP, a Dicer cofactor, inhibit human immunodeficiency virus type 1 long terminal repeat expression and viral production.

RNA interference (RNAi) is now widely used for gene silencing in mammalian cells. The mechanism uses the RNA-induced silencing complex, in which Dicer, Ago2, and the human immunodeficiency virus type 1 (HIV-1) TAR RNA binding protein (TRBP) are the main components. TRBP is a protein that increases HIV-1 expression and replication by inhibition of the interferon-induced protein kinase PKR and by increasing translation of viral mRNA. After HIV infection, TRBP could restrict the viral RNA through its activity in RNAi or could contribute more to the enhancement of viral replication. To determine which function will be predominant in the virological context, we analyzed whether the inhibition of its expression could enhance or decrease HIV replication. We have generated small interfering RNAs (siRNAs) against TRBP and found that they decrease HIV-1 long terminal repeat (LTR) basal expression 2-fold, and the LTR Tat transactivated level up to 10-fold. In the context of HIV replication, siRNAs against TRBP decrease the expression of viral genes and inhibit viral production up to fivefold. The moderate increase in PKR expression and activation indicates that it contributes partially to viral gene inhibition. The moderate decrease in micro-RNA (miRNA) biogenesis by TRBP siRNAs suggests that in the context of HIV replication, TRBP functions other than RNAi are predominant. In addition, siRNAs against Dicer decrease viral production twofold and impede miRNA biogenesis. These results suggest that, in the context of HIV replication, TRBP contributes mainly to the enhancement of virus production and that Dicer does not mediate HIV restriction by RNAi.

Low TRBP levels support an innate human immunodeficiency virus type 1 resistance in astrocytes by enhancing the PKR antiviral response.

Acute human immunodeficiency virus type 1 (HIV-1) replication in astrocytes produces minimal new virus particles due, in part, to inefficient translation of viral structural proteins despite high levels of cytoplasmic viral mRNA. We found that a highly reactive double-stranded (ds) RNA-binding protein kinase (PKR) response in astrocytes underlies this inefficient translation of HIV-1 mRNA. The dsRNA elements made during acute replication of HIV-1 in astrocytes triggers PKR activation and the specific inhibition of HIV-1 protein translation. The heightened PKR response results from relatively low levels of the cellular antagonist of PKR, the TAR RNA binding protein (TRBP). Efficient HIV-1 production was restored in astrocytes by inhibiting the innate PKR response to HIV-1 dsRNA with dominant negative PKR mutants, or PKR knockdown by siRNA gene silencing. Increasing the expression of TRBP in astrocytes restored acute virus production to levels comparable to those observed in permissive cells. Therefore, the robust innate PKR antiviral response in astrocytes results from relatively low levels of TRBP expression and contributes to their restricted infection. Our findings highlight TRBP as a novel cellular target for therapeutic interventions to block productive HIV-1 replication in cells that are fully permissive for HIV-1 infection.