No abstract

Antisense oligomers constitute an attractive class of specific tools for genetic analysis and for potential therapeutic applications. Targets with different cellular locations have been described, such as mRNA translation initiation sites, pre-mRNA splicing sites, or the genes themselves. However the mechanism(s) of action and the intracellular distribution of antisense oligomers remain poorly understood. Antisense oligomers conjugated with various fluorochromes or with BrdUrd were microinected into the cytoplasm of somatic cells, and their cellular distribution was monitored by fluorescence microscopy in fixed and nonfixed cells. A fast translocation in the nuclei and a concentration on nuclear structures were observed whatever probe was used. Nuclear transport occurs by diffusion since it is not affected by depletion of the intracellular ATP pool, temperature, or excess unlabeled oligomer. Accumulation of the oligomers in the nuclei essentially takes place on a set of proteins preferentially extracted between 0.2 M and 0.4 M NaCI as revealed by crosslinking of photosensitive oligomers. The relationship between nuclear location of antisense oligomers and their mechanism of action remains to be ascertained and could be of major interest in the design of more efficient antisense molecules.The ability of antisense oligomers to interfere specifically with mRNAs or their precursors provides a valuable tool for the control of cellular and viral gene expression (for review, see ref. 1). Although attractive, this approach is restricted by the poor stability of natural oligomers and the low efficiency of their cellular uptake. These difficulties can at least be partially circumvented by the use of nuclease-resistant analogs or drug delivery techniques (2). Despite these advances, the design of optimal strategies for oligomer synthesis and delivery has remained difficult since not much is known about the mechanism through which antisense oligomers interfere with gene expression within intact cells or of their intracellular fate.The antisense concept is based on the assumption that complementary DNA or RNA sequences might interact with mRNA to block their translation. To overcome the complexity of investigation in intact cells, several laboratories have developed cell-free models to evaluate the inhibitory potential of these molecules. Nevertheless, cell-free studies have usually been limited to observations at the translational level and are not always representative of the intact cell situation. Antisense oligomers were successfully used on cultured cells against various targets, such as translation initiation (3), introns (4), and splice sites (5). The subcellular locations of these targets lead to the assumption that oligomers might act in the cytoplasm and/or the nuclei.Charged oligomers are internalized through an endocytic pathway (6, 7), and a punctuated cytoplasmic labeling characteristic of accumulation in the endocytic vesicles is indeed observed when fluorescently tagged oligomers are used (6). However...

Interferons (IFNs) encode a family of secreted proteins that provide the front-line defense against viral infections. Their diverse biological actions are thought to be mediated by the products of specific but usually overlapping sets of cellular genes induced in the target cells. We have recently isolated a new human IFN-induced gene that we have termed ISG20, which codes for a 3 to 5 exonuclease with specificity for single-stranded RNA and, to a lesser extent, for DNA. In this report, we demonstrate that ISG20 is involved in the antiviral functions of IFN. In the absence of IFN treatment, ISG20-overexpressing HeLa cells showed resistance to infections by vesicular stomatitis virus (VSV), influenza virus, and encephalomyocarditis virus (three RNA genomic viruses) but not to the DNA genomic adenovirus. ISG20 specifically interfered with VSV mRNA synthesis and protein production while leaving the expression of cellular control genes unaffected. No antiviral effect was observed in cells overexpressing a mutated ISG20 protein defective in exonuclease activity, demonstrating that the antiviral effects were due to the exonuclease activity of ISG20. In addition, the inactive mutant ISG20 protein, which is able to inhibit ISG20 exonuclease activity in vitro, significantly reduced the ability of IFN to block VSV development. Taken together, these data suggested that the antiviral activity of IFN against VSV is partly mediated by ISG20. We thus show that, besides RNase L, ISG20 has an antiviral activity, supporting the idea that it might represent a novel antiviral pathway in the mechanism of IFN action. Interferons (IFNs)1 are a family of multifunctional secreted proteins characterized by their abilities to interfere with virus infection and replication (1, 2). IFNs can indirectly inhibit viral production by reducing the growth of target cells and by stimulating their susceptibility to apoptotic processes (3, 4) or by promoting the recognition and the cytotoxic killing of infected cells by the immune system (5, 6). IFNs also act directly at various steps of the viral multiplication cycle through the products of specific but usually overlapping sets of cellular genes induced in the target cells and involved in RNA and protein metabolism and signaling as well (7,8). Until now, three IFNregulated pathways have been considered to be involved in these processes: the double-stranded RNA-dependent protein kinase R (PKR) (9 -11), the 2-5A/RNase L system (12, 13), and the Mx proteins (14 -16). PKR is a serine/threonine kinase that, after binding to dsRNA, phosphorylates the protein synthesis initiation factor eIF2 and the inhibitor of nuclear factor B (IB), resulting in the inhibition of protein synthesis and specific transcription regulation (reviewed in . RNase L is a dormant cytosolic endoribonuclease that is activated by short oligoadenylates produced by the 2Ј-5Ј oligoadenylate synthetase after viral infection or IFN exposure (reviewed in Refs. 2 and 13). Degradation of viral RNAs and cleavage of cellular 18 S and 28 S rRNA...

The human ISG20/HEM45 gene was identified independently on the basis of its increased level of expression in response to either interferon or estrogen hormone. Notably, the encoded protein is homologous with members of the 3' to 5' exonuclease superfamily that includes RNases T and D, and the proofreading domain of Escherichia coli DNA polymerase I. We provide here direct biochemical evidence that Isg20 acts as a 3' to 5' exonuclease in vitro. This protein displays a pH optimum of approximately 7.0, prefers Mn2+ as a metal cofactor, and degrades RNA at a rate that is approximately 35-fold higher than its rate for single-stranded DNA. Along with RNase L, Isg20 is the second known RNase regulated by interferon. Previous data showed that Isg20 is located in promyelocytic leukemia (PML) nuclear bodies, known sites of hormone-dependent RNA polymerase II transcription and oncogenic DNA viral transcription and replication. The combined data suggest a potential role for Isg20 in degrading viral RNAs as part of the interferon-regulated antiviral response and/or cellular mRNAs as a regulatory component of interferon and estrogen signaling.