No abstract

Synthetic small interfering RNAs (siRNAs) have been shown to induce the degradation of specific mRNA targets in human cells by inducing RNA interference (RNAi). Here, we demonstrate that siRNA duplexes targeted against the essential Tat and Rev regulatory proteins encoded by human immunodeficiency virus type 1 (HIV-1) can specifically block Tat and Rev expression and function. More importantly, we show that these same siRNAs can effectively inhibit HIV-1 gene expression and replication in cell cultures, including those of human T-cell lines and primary lymphocytes. These observations demonstrate that RNAi can effectively block virus replication in human cells and raise the possibility that RNAi could provide an important innate protective response, particularly against viruses that express double-stranded RNAs as part of their replication cycle.The phenomenon now generally termed RNA interference (RNAi) is an evolutionarily conserved process whereby the expression or introduction of double-stranded RNA (dsRNA) sequences results in the specific posttranscriptional inactivation of genes which are complementary to the dsRNA sequence used (reviewed in reference 37). In several organisms, including plants, nematodes, and fruit flies, RNAi can be used to inactivate a specific host gene subsequent to microinjection or transfection of the appropriate dsRNA (16,21,39). However, in mammalian cells, dsRNAs consisting of more than 30 nucleotides (nt) induce not only RNAi but also a set of responses, including activation of the interferon response, that collectively result in a global, nonspecific inhibition of host cell mRNA expression (32,36).Efforts to resolve the mechanisms underlying RNAi in the plant and drosophila systems revealed that longer dsRNAs were processed by a host RNase, termed dicer, into ϳ21-nt dsRNAs, called small interfering RNAs (siRNAs), that contain 2-nt 3Ј overhangs (14, 19-21, 25, 26, 28, 43). These siRNAs are key mediators of RNAi and are thought to serve as guide RNAs for a large protein complex, termed the RNA-induced silencing complex, that is essential for target mRNA degradation (22). Importantly, it soon became apparent that direct introduction of synthetic siRNAs could also induce RNAi not only in drosophila cells but also in human cells (8,14,15). Subsequently, transfection of synthetic siRNAs has been used to selectively block the expression of a small but rapidly growing list of human genes and to determine the phenotypic consequences of this inhibition (1,11,18,23,25,33).RNAi is believed to have evolved as a host defense mechanism directed at transposable elements and infecting viruses (12,27,34,38). We therefore wondered if RNAi might provide an effective defense against a pathogenic human virus. Here, we describe siRNAs targeted against the essential human immunodeficiency virus type 1 (HIV-1) regulatory proteins Tat and Rev (13) and show that these siRNAs can effectively and specifically block Tat or Rev expression and function in transfected 293T cells. More importantly, we show that...

The RNA degradosome is a multiprotein complex required for the degradation of highly structured RNAs. We have developed a method for reconstituting a minimal degradosome from purified proteins. Our results demonstrate that a degradosome-like complex containing RNase E, PNPase, and RhlB can form spontaneously in vitro in the absence of all other cellular components. Moreover, ATP-dependent degradation of the malEF REP RNA by the reconstituted, minimal degradosome is indistinguishable from that of degradosomes isolated from whole cells. The Rne protein serves as an essential scaffold in the reconstitution process; however, RNase E activity is not required. Rather, Rne coordinates the activation of RhlB dependent on a 3 single-stranded extension on RNA substrates. A model for degradosome-mediated degradation of structured RNA is presented with its implications for mRNA decay in Escherichia coli.