Summary Piwi-interacting RNAs (piRNAs) silence transposons in the germ line of animals. They are thought to derive from long primary transcripts spanning transposon-rich genomic loci, “piRNA clusters.” piRNAs are proposed to direct an auto-amplification loop in which an antisense piRNA, bound to Aubergine or Piwi protein, directs the cleavage of sense RNA, triggering production of a sense piRNA bound to the PIWI protein Argonaute3 (Ago3). In turn, the new piRNA is envisioned to direct cleavage of a cluster transcript, initiating production of a second antisense piRNA. Here, we describe strong loss-of-function mutations in ago3, allowing a direct genetic test of this model. We find that Ago3 acts to amplify piRNA pools and to enforce on them an antisense bias, increasing the number of piRNAs that can act to silence transposons. We also detect a second piRNA pathway centered on Piwi and functioning without benefit of Ago3-catalyzed amplification. Transposons targeted by this second pathway often reside in the flamenco locus, which is expressed in somatic ovarian follicle cells, suggesting a role for piRNAs beyond the germ line.

Small interfering RNAs (siRNAs) direct RNA interference (RNAi) in eukaryotes. In flies, somatic cells produce siRNAs from exogenous double-stranded RNA (dsRNA) as a defense against viral infection. We identified endogenous siRNAs (endo-siRNAs), 21 nucleotides in length, that correspond to transposons and heterochromatic sequences in the somatic cells of Drosophila melanogaster. We also detected endo-siRNAs complementary to messenger RNAs (mRNAs); these siRNAs disproportionately mapped to the complementary regions of overlapping mRNAs predicted to form double-stranded RNA in vivo. Normal accumulation of somatic endo-siRNAs requires the siRNA-generating ribonuclease Dicer-2 and the RNAi effector protein Argonaute2 (Ago2). We propose that endo-siRNAs generated by the fly RNAi pathway silence selfish genetic elements in the soma, much as Piwi-interacting RNAs do in the germ line.

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The human immunodeficiency virus type 1 (HIV-1) Rev protein acts post-transcriptionally to increase the amounts of the viral gag-pol and env messenger RNAs in the cytoplasm of infected cells. The mechanism of Rev action is uncertain. Possibilities include an accelerating effect on the rate of export of its mRNA targets from the nucleus and/or modulation of the splicing of pre-mRNAs. Both the gag-pol and env mRNAs contain a sequence that is required for responsiveness to Rev--the Rev responsive element, RRE. Here we show that Rev is a sequence-specific binding protein, whose binding site is the RRE. This information should help to clarify the mechanism by which Rev acts.

The Rev protein of human immunodeficiency virus type 1 is a sequence-specific RNA binding protein that is essential for viral replication. Here we present evidence that Rev is a stable oligomer both in vitro and in vivo. Analysis ofRev mutants indicates that oligomerization is essential for RNA binding and hence Rev function. The oligomerization and RNA binding domains overlap over 47 amino acids. Within this region is a short arginine-rich motif found in a large class of RNA binding proteins. Substitution of multiple residues within the arginine-rich motif abolishes oligomerization, whereas several single-amino-acid substitution mutants oligomerize but do not bind RNA. Thus, Rev's arginine-rich motif participates in two distinct functions: oligomerization and RNA binding.The human immunodeficiency virus type 1 Rev protein acts posttranscriptionally to increase selectively the cytoplasmic levels of the gag-pol and env mRNAs (1-5). A specific RNA region that is required for Rev to function (Rev Response Element; RRE) has been mapped within the gag-pol and env mRNAs (3,6,7). Rev is a sequence-specific RNA binding protein that interacts directly with the RRE (8)(9)(10)(11)(12)(13)(14). This sequence-specific RNA binding activity explains the basis for Rev's selective action. Rev is a member of a class of RNA binding proteins characterized by the presence of an argininerich motif (15,16).In NaCI/20%o (vol/vol) glycerol], cell debris was removed by centrifugation, and the supernatant was loaded directly onto a 400-ml DEAE-Sepharose CL-6B column. The 200 mM salt eluate from this column was fractionated on a 2-ml heparinSepharose affinity column. Rev protein was removed by 2 M salt, dialyzed against buffer A, and stored at -80oC. As judged by staining with Coomassie blue, Rev was >98% pure and retained sequence-specific RNA binding activity (data not shown). RESULTSRev Binds to RNA as an Oligomer. Previous studies have shown that Rev binds specifically and tightly to the RRE (8)(9)(10)(11)(12)(13)(14). To address whether Rev interacts with the RRE as a monomer, or as an oligomer, we determined the sedimentation coefficient of the Rev-RRE complex. E. coli-derived Rev was incubated with a 32P-labeled RRE-containing RNA transcript, the reaction mixture was treated with RNase A and T1, and the products were fractionated on a glycerol gradient. Each gradient fraction was analyzed on a native gel to resolve the 32P-labeled Rev-RRE complex from the RNase digestion products. The results in Fig. 1 tTo whom reprint requests should be addressed. 7734The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Antiviral immunity requires recognition of viral pathogens and activation of cytotoxic and Th cells by innate immune cells. In this study, we demonstrate that hepatitis C virus (HCV) core and nonstructural protein 3 (NS3), but not envelope 2 proteins (E2), activate monocytes and myeloid dendritic cells (DCs) and partially reproduce abnormalities found in chronic HCV infection. HCV core or NS3 (not E2) triggered inflammatory cytokine mRNA and TNF-α production in monocytes. Degradation of I-κBα suggested involvement of NF-κB activation. HCV core and NS3 induced production of the anti-inflammatory cytokine, IL-10. Both monocyte TNF-α and IL-10 levels were higher upon HCV core and NS3 protein stimulation in HCV-infected patients than in normals. HCV core and NS3 (not E2) inhibited differentiation and allostimulatory capacity of immature DCs similar to defects in HCV infection. This was associated with elevated IL-10 and decreased IL-2 levels during T cell proliferation. Increased IL-10 was produced by HCV patients’ DCs and by core- or NS3-treated normal DCs, while IL-12 was decreased only in HCV DCs. Addition of anti-IL-10 Ab, not IL-12, ameliorated T cell proliferation with HCV core- or NS3-treated DCs. Reduced allostimulatory capacity in HCV core- and NS3-treated immature DCs, but not in DCs of HCV patients, was reversed by LPS maturation, suggesting more complex DC defects in vivo than those mediated by core or NS3 proteins. Our results reveal that HCV core and NS3 proteins activate monocytes and inhibit DC differentiation in the absence of the intact virus and mediate some of the immunoinhibitory effects of HCV via IL-10 induction.

The Rev protein of human immunodeficiency virus type 1 (HIV-1) facilitates the nuclear export of unspliced and partly spliced viral RNAs. Rev contains an RNA binding domain, required for interaction with HIV-1 RNA, and an effector domain, required for RNA-bound Rev to function. The Rev effector domain is believed to interact with a cellular cofactor required for the Rev response and thus HIV-1 replication. Here we report the use of a yeast two-hybrid screen to clone human Rev interacting protein (hRIP), which specifically interacts with the Rev effector domain. This hRIP protein has homology with nucleoporins, a class of proteins that mediate nucleocytoplasmic transport. These and other properties of hRIP are those expected of a Rev cellular cofactor.