HIV-1 RRE RNA acts as an RNA silencing suppressor by competing with TRBP-bound siRNAs.

Several proteins and RNAs expressed by mammalian viruses have been reported to interfere with RNA interference (RNAi) activity. We investigated the ability of the HIV-1-encoded RNA elements Trans-Activation Response (TAR) and Rev-Response Element (RRE) to alter RNAi. MicroRNA let7-based assays showed that RRE is a potent suppressor of RNAi activity, while TAR displayed moderate RNAi suppression. We demonstrate that RRE binds to TAR-RNA Binding Protein (TRBP), an essential component of the RNA Induced Silencing Complex (RISC). The binding of TAR and RRE to TRBP displaces small interfering (si)RNAs from binding to TRBP. Several stem-deleted RRE mutants lost their ability to suppress RNAi activity, which correlated with a reduced ability to compete with siRNA-TRBP binding. A lentiviral vector expressing TAR and RRE restricted RNAi, but RNAi was restored when Rev or GagPol were coexpressed. Adenoviruses are restricted by RNAi and encode their own suppressors of RNAi, the Virus-Associated (VA) RNA elements. RRE enhanced the replication of wild-type and VA-deficient adenovirus. Our work describes RRE as a novel suppressor of RNAi that acts by competing with siRNAs rather than by disrupting the RISC. This function is masked in lentiviral vectors co-expressed with viral proteins and thus will not affect their use in gene therapy. The potent RNAi suppressive effects of RRE identified in this study could be used to enhance the expression of RNAi restricted viruses used in oncolysis such as adenoviruses.

Inhibitory Killer Immunoglobulin-like Receptors to self HLA-B and HLA-C ligands contribute differentially to Natural Killer cell functional potential in HIV infected slow progressors.

Inhibitory Killer Immunoglobulin-like Receptors (iKIR) interact with their ligands, HLA molecules, to license Natural Killer (NK) cells for functional competence. Previous studies stimulating peripheral blood mononuclear cells (PBMCs) with the HLA-devoid K562 cell line revealed that NK cells from individuals with an iKIR encoded by the KIR3DL1 locus with self HLA-Bw4 as their ligands, had higher frequencies of tri-functional NK cells that expressed the degranulation marker CD107a and secreted Interferon-γ and Tumor Necrosis Factor-α than those from individuals who were homozygous for HLA-Bw6 alleles, which are not ligands for these iKIR. To assess the effect of other iKIR to self-HLA (S-iKIR) on the NK cell response, we compared HIV-infected slow progressors (SP) carrying S-iKIR to HLA-C alleles with or without S-iKIR to HLA-Bw4. We show that S-iKIR to HLA-B and C alleles differ in their contribution to NK cell functional potential in HIV-infected SP upon stimulation with K562 targets.

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.

Interactions between the double-stranded RNA-binding proteins TRBP and PACT define the Medipal domain that mediates protein-protein interactions.

The double-stranded (ds) RNA binding proteins, TRBP and PACT bind the interferon-induced protein kinase PKR and dsRNA. TRBP inhibits, whereas PACT activates PKR. They have two dsRNA binding domains (dsRBDs) and a C-terminal domain that does not bind RNA. All three domains show a strong homology between the two proteins. Interaction assays by in vitro binding, yeast two-hybrid, and immunoprecipitations show that TRBP and PACT form heterodimers in the absence of dsRNA. In cells, TRBP and PACT colocalize in specific dots of the perinuclear space. Analysis of the individual domains shows that the two dsRBDs of each protein interact with each other. In contrast, the C-terminal domain of PACT homodimerizes and interacts with its homologous region in TRBP, but the same domain in TRBP does not homodimerize. Because the C-terminal domain in TRBP binds to the tumor suppressor Merlin, the RNase III Dicer and PACT, we name it the Merlin Dicer PACT liaison (Medipal) domain. Based on known interactions Medipal is defined as aminoacids 228-366 in TRBP and 195-313 in PACT. TRBP-PACT interaction correlates with an absence of eIF2alpha activation by PACT, suggesting that the heterodimer does not activate PKR. We propose that the Medipal domain mediates specialized functions through protein-protein interactions and contributes to the RNA interference pathway and to PKR activation.

Proteomic fingerprinting in HIV/HCV co-infection reveals serum biomarkers for the diagnosis of fibrosis staging.

BACKGROUND: Hepatic complications of hepatitis C virus (HCV), including fibrosis and cirrhosis are accelerated in human immunodeficiency virus (HIV)-infected individuals. Although, liver biopsy remains the gold standard for staging HCV-associated liver disease, this test can result in serious complications and is subject to sampling errors. These challenges have prompted a search for non-invasive methods for liver fibrosis staging. To this end, we compared serum proteome profiles at different stages of fibrosis in HIV/HCV co- and HCV mono-infected patients using surface-enhanced laser desorption ionization time-of-flight mass spectrometry (SELDI-TOF MS). METHODS: Sera from 83 HIV/HCV co- and 68 HCV mono-infected subjects in 4 stages of fibrosis were tested. Sera were fractionated, randomly applied to protein chip arrays (IMAC, CM10 and H50) and spectra were generated at low and high laser intensities. RESULTS: Sixteen biomarkers achieved a p value < 0.01 (ROC values > 0.75 or < 0.25) predictive of fibrosis status in co-infected individuals and 14 in mono infected subjects. Five of these candidate biomarkers contributed to both mono- and co-infected subjects. Candidate diagnostic algorithms were created to distinguish between non-fibrotic and fibrotic individuals using a panel of 4 biomarker peaks. CONCLUSION: These data suggest that SELDI MS profiling can identify diagnostic serum biomarkers for fibrosis that are both common and distinct in HIV/HCV co-infected and HCV mono-infected individuals.

TRBP control of PACT-induced phosphorylation of protein kinase R is reversed by stress.

The TAR RNA binding Protein, TRBP, inhibits the activity of the interferon-induced protein kinase R (PKR), whereas the PKR activator, PACT, activates its function. TRBP and PACT also bind to each other through their double-stranded RNA binding domains (dsRBDs) and their Medipal domains, which may influence their activity on PKR. In a human immunodeficiency virus (HIV) long terminal repeat-luciferase assay, PACT unexpectedly reversed PKR-mediated inhibition of gene expression. In a translation inhibition assay in HeLa cells, PACT lacking the 13 C-terminal amino acids (PACTDelta13), but not full-length PACT, activated PKR and enhanced interferon-mediated repression. In contrast, in the astrocytic U251MG cells that express low TRBP levels, both proteins activate PKR, but PACTDelta13 is stronger. Immunoprecipitation assays and yeast two-hybrid assays show that TRBP and PACTDelta13 interact very weakly due to a loss of binding in the Medipal domain. PACT-induced PKR phosphorylation was restored in Tarbp2(-/-) murine tail fibroblasts and in HEK293T or HeLa cells when TRBP expression was reduced by RNA interference. In HEK293T and HeLa cells, arsenite, peroxide, and serum starvation-mediated stresses dissociated the TRBP-PACT interaction and increased PACT-induced PKR activation, demonstrating the relevance of this control in a physiological context. Our results demonstrate that in cells, TRBP controls PACT activation of PKR, an activity that is reversed by stress.