Arginine rich short linear motif of HIV-1 regulatory proteins inhibits Dicer dependent RNA interference (original) (raw)
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BMC Molecular Biology, 2007
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.
HIV-1 RRE RNA acts as an RNA silencing suppressor by competing with TRBP-bound siRNAs
RNA biology, 2015
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-Associate...
Journal of Virology, 2007
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
Evidence that HIV-1 Encodes an siRNA and a Suppressor of RNA Silencing
Immunity, 2005
and invertebrate animals lack protein-based Laboratory of Molecular Microbiology adaptive immunity and do not conserve a typical in-National Institute of Allergy and Infectious Diseases terferon response. For these less-developed systems, National Institutes of Health a biological rationale exists for RNA-based defense Bethesda, Maryland 20892 against invading pathogens. RNA silencing is pos-2 Laboratory of Experimental and Computational tulated to be an ancient immune mechanism used by Biology cells to restrict viruses, transgenes, and transposons NCI Center for Cancer Research (Carmichael, 2002; Ding et al., 2004; Saksela, 2003). National Cancer Institute Consistent with this notion, RNA silencing was first Frederick, Maryland 21702 documented as a natural antiviral response in plants 3 Laboratoire de Virologie Moleculaire (Zamore, 2004). More recently, evidence for this type of Institut de Genetique Humaine functional immunity has been extended to insect cells CNRS UPR1142, Montpellier (Li et al., 2002; Li et al., 2004). France The emergence of protein-based adaptive immunity in higher-jawed vertebrates questions whether nucleic acid-based immunity has become extinguished. That Summary this defense might still persist comes from findings that show that in various artificial contexts, RNAi can be In plants and invertebrate animals, RNA silencing is used as a tool for the efficient suppression of gene exa form of nucleic acid-based adaptive immunity. By pression in human cells (Joost Haasnoot et al., 2003; contrast, jawed vertebrates have evolved complex McManus and Sharp, 2002; Novina et al., 2002; Silva et protein-based adaptive immunity. Although short inal., 2002; Xia et al., 2002). However, to date, there has terfering RNAs (siRNAs) have been used as artificial been no evidence that physiological infection of vertetools to silence viral infection in human cells, it rebral/mammalian cells by viruses produces viral-specific mains unknown whether mammalian viruses naturally siRNA, which triggers a RISC-based restriction on forelicit such immunity in vertebral cells. Here, we report eign transcripts. Should a RISC-based restriction be the evidence that HIV-1 encodes viral siRNA precuroperative, then the evolution of mammalian viral gesors in its genome and that natural HIV-1 infection nomes would be driven by nucleic acid-based selection provokes nucleic acid-based immunity in human cells. (against dsRNA helices) in addition to classical protein-To combat this cellular defense, HIV-1 has evolved in and cellular-based adaptive immunities. its Tat protein a suppressor of RNA silencing (SRS) The genome of HIV-1 contains a plethora of dsRNA function. Tat abrogates the cell's RNA-silencing deregions (Mujeeb et al., 1999; Phuphuakrat and Auewarfense by subverting the ability of Dicer to process akul, 2003; Rana and Jeang, 1999); each, at first glance, precursor double-stranded RNAs into siRNAs. presumptively capable of being processed by Dicer into siRNA. Interestingly, because HIV-1 is potently lytic
Suppression of HIV-1 replication by microRNA effectors
Retrovirology, 2009
The rate of HIV-1 gene expression is a key step that determines the kinetics of virus spread and AIDS progression. Viral entry and gene expression were described to be the key determinants for cell permissiveness to HIV. Recent reports highlighted the involvement of miRNA in regulating HIV-1 replication post-transcriptionally. In this study we explored the role of cellular factors required for miRNA-mediated mRNA translational inhibition in regulating HIV-1 gene expression. Here we show that HIV-1 mRNAs associate and co-localize with components of the RNA Induced Silencing Complex (RISC), and we characterize some of the proteins required for miRNA-mediated silencing (miRNA effectors). RCK/p54, GW182, LSm-1 and XRN1 negatively regulate HIV-1 gene expression by preventing viral mRNA association with polysomes. Interestingly, knockdown of RCK/p54 or DGCR8 resulted in virus reactivation in PBMCs isolated from HIV infected patients treated with suppressive HAART.
RNAi-mediated inhibition of HIV-1 by targeting partially complementary viral sequences
Nucleic Acids Research, 2009
Potent antiviral RNAi can be induced by intracellular expression of short hairpin RNAs (shRNAs) and artificial microRNAs (miRNAs). Expression of shRNA and miRNA results in target mRNA degradation (perfect base pairing) or translational repression (partial base pairing). Although efficient inhibition can be obtained, error-prone viruses such as human immunodeficiency virus type 1 (HIV-1) can escape from RNAi-mediated inhibition by mutating the target sequence. Recently, artificial miRNAs have been shown to be potent RNAi inducers due to their efficient processing by the RNAi machinery. Furthermore, miRNAs may be more proficient in suppressing imperfect targets than shRNAs. In this study, we tested the knockdown efficiency of miRNAs and shRNAs against wild-type and RNAi-escape HIV-1 variants with one or two mutations in the target sequence. ShRNAs and miRNAs can significantly inhibit the production of HIV-1 variants with mutated target sequences in the open reading frame. More pronounced mutation-tolerance was measured for targets in the 3' untranslated region (3' UTR). Partially complementary sequences within the 3' UTR of the HIV-1 RNA genome efficiently act as target sites for miRNAs and shRNAs. These data suggest that targeting imperfect target sites by antiviral miRNAs or shRNAs provides an alternative RNAi approach for inhibition of pathogenic viruses.
HIV-1 can escape from RNA interference by evolving an alternative structure in its RNA genome
Nucleic Acids Research, 2005
HIV-1 replication can be efficiently inhibited by intracellular expression of an siRNA targeting the viral RNA. However, HIV-1 escape variants emerged after prolonged culturing. These RNAi-resistant viruses contain nucleotide substitutions or deletions in or near the targeted sequence. We observed an inverse correlation between the level of resistance and the stability of the siRNA/target-RNA duplex. However, two escape variants showed a higher level of resistance than expected based on the duplex stability. We demonstrate that these mutations induce alternative folding of the RNA such that the target sequence is occluded from binding to the siRNA, resulting in reduced RNAi efficiency. HIV-1 can thus escape from RNAi-mediated inhibition not only through nucleotide substitutions or deletions in the siRNA target sequence, but also through mutations that alter the local RNA secondary structure. The results highlight the enormous genetic flexibility of HIV-1 and provide detailed molecular insight into the sequence specificity of RNAi and the impact of target RNA secondary structure.
Tat-dependent production of an HIV-1 TAR-encoded miRNA-like small RNA
Nucleic acids research, 2016
Evidence is accumulating that retroviruses can produce microRNAs (miRNAs). To prevent cleavage of their RNA genome, retroviruses have to use an alternative RNA source as miRNA precursor. The transacting responsive (TAR) hairpin structure in HIV-1 RNA has been suggested as source for miRNAs, but how these small RNAs are produced without impeding virus replication remained unclear. We used deep sequencing analysis of AGO2-bound HIV-1 RNAs to demonstrate that the 3' side of the TAR hairpin is processed into a miRNA-like small RNA. This ∼21 nt RNA product is able to repress the expression of mRNAs bearing a complementary target sequence. Analysis of the small RNAs produced by wild-type and mutant HIV-1 variants revealed that non-processive transcription from the HIV-1 LTR promoter results in the production of short TAR RNAs that serve as precursor. These TAR RNAs are cleaved by Dicer and processing is stimulated by the viral Tat protein. This biogenesis pathway differs from the cano...
Journal of Virology, 2002
To define the human immunodeficiency virus type 1 (HIV-1) RNA maturation pathways, we analyzed the intracellular distribution of HIV-1 RNA and the viral regulatory proteins Rev and Tat in transfected COS cells and HIV-1-infected lymphoid C8166 cells by means of ultrastructural in situ hybridization using antisense RNA probes and immunoelectron microscopy. The intranuclear viral RNA occurs in ribonucleoprotein fibrils in the perichromatin and interchromatin regions. The simultaneous demonstration of Rev, Tat, Br-labeled RNA, and cellular proteins SC35 and CRM1 in such fibrils reveals the potential of Rev to associate with nascent HIV pre-mRNA and its splicing complex and transport machinery. In a rev-minus system, the env intron-containing, incompletely spliced viral RNAs are revealed only in the nucleus, indicating that Rev is required to initiate the transport to the cytoplasm. Moreover, env intron sequences frequently occur in the periphery of interchromatin granule clusters, while the probe containing the rev exon sequence does not associate with this nucleoplasmic domain. When cells were treated with the CRM1 inhibitor leptomycin B in the presence of Rev protein, the env intron containing HIV RNAs formed clusters throughout the nucleoplasm and accumulated at the nuclear pores. This suggests that Rev is necessary and probably also sufficient for the accumulation of incompletely spliced HIV RNAs at the nuclear pores while CRM1 is needed for translocation across the nuclear pore complex.