The nonstructural protein 5A (NS5A) encoded by the human hepatitis C virus RNA genome is shown here to induce the activation of NF-B and STAT-3 transcription factors from its cytoplasmic residence via oxidative stress. NS5A causes the disturbance of intracellular calcium. Ca 2؉ signaling triggers the elevation of reactive oxygen species in mitochondria, leading to the translocation of NF-B and STAT-3 into the nucleus. Evidence is presented for the constitutive activation of STAT-3 by NS5A. In the presence of antioxidants [pyrrolidine dithiocarbamate (PDTC), N-acetyl L-cysteine (NAC)] or Ca 2؉ chelators (EGTA-AM, TMB-8), NS5A-induced activation of NF-B and STAT-3 was eliminated. These results provide an insight into the mechanism by which NS5A can alter intracellular events relevant to liver pathogenesis associated with the viral infection.H epatitis C virus (HCV) causes acute͞chronic hepatitis with a significant risk of end-stage cirrhosis and hepatocellular carcinoma (1). The single-stranded RNA genome of the human HCV is a 9.6-kb-long positive-sense molecule, which encodes a polyprotein of about 3,000 aa (2, 3). The polyprotein is posttranslationally cleaved by both viral͞cellular proteases to produce about 10 polypeptides that include structural (core and E1 and E2) and nonstructural (NS2, NS3-NS5A͞B) proteins (2, 3). The single long ORF is preceded by 332 or 342 nt of the 5Ј untranslated region, which harbors an internal ribosome entry site capable of initiating translation at an internal site (4-6). The 3Ј end of the RNA genome contains a unique sequence of the untranslated region that is necessary for initiating RNA replication. Although the infectious cDNA clones that have been generated could infect chimpanzees, they failed to replicate in vitro in cultured cell lines (7). Lohmann et al. (8) described the generation of efficient replicating HCV RNA subgenomic replicons coexpressing a neomycin-resistance marker. A high level of replication of subgenomic replicons was characterized, resulting from adaptive mutations, which were scattered throughout the HCV genes of the replicon (9, 10).The HCV NS5A has generated a significant level of interest as several cellular targets have been identified. NS5A is a serine phosphoprotein, which exists as a polypeptide of p56 or p58 with varying degrees of phosphorylation (11,12). The identity of cellular kinase(s) responsible for NSS5A phosphorylation has not been firmly established. NS4A, an integral membrane protein, has been shown to modulate NS5A phosphorylation at least for some HCV subtypes (13). NS5A is localized to the cytoplasm in the perinuclear reticular network characteristic of the endoplasmic reticulum (ER) membrane (3,14). NS5A came into prominence because of its suggested role in IFN resistance. It was shown that NS5A directly interacted with double-stranded RNA-dependent kinase (PKR) and inactivated its function, thus modulating the IFN-stimulated antiviral response (15). Neither the sites of hyperphosphorylation nor the region designated ISDR (IFN-sen...

A number of highly regulated gene classes are regulated post-transcriptionally at the level of mRNA stability. A central feature in these mRNAs is the presence of A+U-rich elements (ARE) within their 39 UTRs. Two ARE binding proteins, HuR and AUF1, are associated with mRNA stabilization and destabilization, respectively. Previous studies have demonstrated homomultimerization of each protein and the capacity to bind simultaneous or competitively to a single ARE. To investigate this possibility further, cell biological and biophysical approaches were undertaken. Protein-protein interaction was monitored by fluorescence resonance energy transfer (FRET) and by immunocytochemistry in live and fixed cells using fluorescently labeled CFP/YFP fusion proteins of HuR and p37AUF1. Strong nuclear FRET between HuR/HuR and AUF1/AUF1 homodimers as well as HuR/AUF1 heterodimers was observed. Treatment with the MAP kinase activator, anisomycin, which commonly stabilizes ARE-containing mRNAs, caused rapid nuclear to cytoplasmic shuttling of HuR. AUF1 also underwent shuttling, but on a longer time scale. After shuttling, HuR/HuR, AUF1/AUF1, and HuR/AUF1, FRET was also observed in the cytoplasm. In further studies, arsenite rapidly induced the formation of stress granules containing HuR and TIA-1 but not AUF1. The current studies demonstrate that two mRNA binding proteins, HuR and AUF1, are colocalized and are capable of functional interaction in both the nucleus and cytoplasm. FRET-based detection of AUF1/HuR interaction may serve as a basis of opening up new dimensions in delineating the functional interaction of mRNA binding proteins with RNA turnover.

Human hepatitis C virus (HCV),1 a member of the family Flaviviridae, is the leading cause of chronic hepatitis (Ͼ80%). HCV infection has been strongly linked with the development of hepatocellular carcinoma (1-3). HCV contains a singlestranded RNA genome (9600 nucleotides) with plus polarity that encodes a single polyprotein of 3010 amino acids (1). The polyprotein is processed into three structural (C (p21), E1 (p31), and E2 (p72)) and several nonstructural (NS2 (p23), p7, NS3 (p70), NS4A (p6), NS4B (p27), NS5A (p58), and NS5B (p65)) polypeptides. Analysis of the conserved and divergent regions of the HCV genomes thus far cloned from various geographical locations indicates that the 5Ј-noncoding region (5Ј-NCR) that precedes the open reading frame and the 3Ј terminus of the genome are highly conserved among all subtypes (1, 4). These elements function as promoters of HCV gene expression.The translation initiation of the HCV RNA genome occurs by internal ribosome entry located in the 5Ј-NCR (5, 6). It has been suggested that HCV RNA translation is regulated by at least three distinct elements: 1) the global structure of the viral internal ribosome entry site (IRES), which includes almost the entire sequence (except nucleotides 1-40) within the 5Ј-NCR and a few nucleotides downstream of the AUG initiator codon (see Fig. 1A) (5-13); 2) the 3Ј-terminal region, which has been shown to enhance IRES activity (14) (but the exact role of the entire 3Ј-NCR during translation regulation is still unclear); and 3) trans-acting cellular factors that interact with the viral IRES element and assist in internal initiation of translation (15-18). Several subunits of eukaryotic initiation factor-3 have been shown to bind the apical half of domain III of the 5Ј-NCR (18, 34). Notable among the non-canonical translation initiation factors that regulate the viral IRES elements are the polypyrimidine tract-binding protein (PTB) and La antigen (for review, see Ref. 19). PTB (p57 or hnRNP-1) has been shown to exist as a homodimer in solution and presents an oligomeric array of eight RNA recognition motifs (four in each monomer) (20). Structural analysis suggests that PTB possesses unusual features of RNP-1 and RNP-2 motifs within its RNA recognition motifs (35). The protein binds polypyrimidine tract near the 3Ј-splice site of many introns and can act as a repressor of splicing (21). In the cytoplasm, PTB has been shown to physically interact with viral and cellular 5Ј-NCRs (15,22,25,32,36). Furthermore, it has been shown to facilitate translation directed by picornaviral IRES elements in the rabbit reticulocyte translation system (22, 32). The overexpression of PTB was recently shown to have a considerable stimulatory effect on translation directed by IRES elements derived from hepatitis A virus, poliovirus, and HCV in different types of cells (24). We have previously shown interaction of PTB with the HCV 5Ј-NCR at multiple sites using a UV cross-linking assay (15). Interestingly, Ito and Lai (33) have demonstrated an additional PTB-b...

Genes encoding numerous proto-oncogenes and cytokines, as well as a number of G-protein coupled receptors, are regulated post-transcriptionally at the level of mRNA stability. A common feature of all of these genes is the presence of A + U-rich elements (AREs) within their 3' untranslated regions. We, and others, have demonstrated previously that mRNAs encoding beta-adrenergic receptors (beta-ARs) are destabilized by agonist stimulation of the beta-AR/Galphas/adenylylcyclase pathway. However, in addition to PK-A, beta-ARs can also activate or inhibit mitogen activated kinase (MAPK) cascades, in a cell-type dependent basis. Recent evidence points to an important role for MAPKs in regulating the turnover of cytokine mRNAs, such as TNFalpha. We hypothesized that activation of MAPK's may also regulate beta-AR mRNA stability. The studies conducted herein demonstrate that generalized stimulation of MAPKs (JNK, p38) with anisomycin resulted in marked stabilization of beta-AR mRNA. Reciprocally, selective inhibition of JNK with SP600125 significantly decreased beta-AR mRNA half-life. Similarly, inhibition of the MEK/ERK pathway with either PD98059 or U0126 decreased beta-AR mRNA stability substantially. However, inhibition of p38 MAPK with SB203580 produced destabilization of beta-AR mRNA only at higher, non pharmacologically selective concentrations. In contrast to their effects on several other ARE containing mRNAs, inhibition of tyrosine kinases by genistein or PI3K by wortmannin, had no detectable effect on beta-AR mRNA stability. In summary, these results demonstrate for the first time that modulation of MAPK pathways can bi-directionally influence beta-AR mRNA stability.

Angiotensin II exerts its cardiovascular effects mainly through the activation of AT(1) receptors. These receptors can be regulated at a post-transcriptional level, that is through the modulation of the mRNA stability. This regulation usually involves proteins which are able to bind the 3'UTR of the mRNA molecule. The experiments of the present paper were performed in order to characterize the RNA-binding proteins interacting with the hAT(1) receptor mRNA in human vascular smooth muscle cells. Immunoblot analysis allowed us to identify three different RNA-binding proteins, AUF1, HuR, and hnRNP A1. UV cross-linking and immunoprecipitation experiments demonstrated that AUF1 binds to the hAT(1)-receptor mRNA radiolabeled probes specifically, but in different ways in relation to the clinically important A/C gene polymorphism. Gel shift experiments using purified recombinant proteins confirmed the specificity of interaction of these proteins with the hAT(1)-receptor mRNA. In basal conditions the proteins were mainly located in the nuclei, but angiotensin II administration clearly induced their translocation to the cytosol. This observation was confirmed by transfection experiments using both GFP/AUF1 and GFP/HuR fusion proteins. Our findings allow identification of specific RNA-binding proteins possibly involved in the control of the hAT1-receptor mRNA stability and in the regulation of their expressions.