The hepatitis C virus (HCV) genome contains numerous RNA elements that are required for its replication. Most of the identified RNA structures are located within the 5= and 3= untranslated regions (UTRs). One prominent RNA structure, termed the cis-acting replication element (CRE), is located within the NS5B coding region. Mutation of part of the CRE, the 5BSL3.2 stemloop, impairs HCV RNA replication. This loop has been implicated in a kissing interaction with a complementary stem-loop structure in the 3= UTR. Although it is clear that this interaction is required for viral replication, the function of the interaction, and its regulation are unknown. In order to gain insight into the CRE function, we isolated cellular proteins that preferentially bind the CRE and identified them using mass spectrometry. This approach identified EWSR1 as a CRE-binding protein. Silencing EWSR1 expression impairs HCV replication and infectious virus production but not translation. While EWRS1 is a shuttling protein that is extensively nuclear in hepatocytes, substantial amounts of EWSR1 localize to the cytosol in HCV-infected cells and colocalize with sites of HCV replication. A subset of EWRS1 translocates into detergent-resistant membrane fractions, which contain the viral replicase proteins, in cells with replicating HCV. EWSR1 directly binds the CRE, and this is dependent on the intact CRE structure. Finally, EWSR1 preferentially interacts with the CRE in the absence of the kissing interaction. This study implicates EWSR1 as a novel modulator of CRE function in HCV replication. Hepatitis C virus (HCV) is a major cause of chronic hepatitis, cirrhosis, and hepatocellular carcinoma. HCV is an enveloped, positive-strand RNA virus classified within the family Flaviviridae. The viral genome encodes an open reading frame of ϳ3,011 codons that is translated as a single polyprotein, which is cleaved by viral and host proteases into at least 10 distinct products. The HCV RNA genome is flanked by the 5= and 3= untranslated regions (UTR). An internal ribosome entry site (IRES) within the 5=UTR is essential for translational initiation of the viral RNA (1, 2). Other RNA elements in the 5=UTR, stem-loop 1, and the 3=UTR, including a variable poly-U/UC tract and three stem-loops contained in the terminal 98 nucleotides that are referred to as the 3=X region, are indispensable for RNA replication (3-7). Multiple viral and host proteins have been suggested to interact with the HCV 3=UTR. Components of the viral replicase complex, NS3, NS5A, and NS5B, have each been shown biochemically to interact with the 3=UTR (8-10). Several cellular proteins have also been implicated in 3=UTR binding, including polypyrimidine tract binding protein (PTB), heterogeneous nuclear ribonucleoprotein C, glyceraldehyde dehydrogenase, HuR, and La (11-17). PTB, HuR, and La have also been shown to be required for efficient HCV RNA replication (18-21). These have been proposed to play multiple roles in HCV replication, including the regulation of translation versus the ...

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We recently identified three novel thioredoxin-like genes in the genome of the protozoan parasite Plasmodium that belong to the Phosducin-like family of proteins (PhLP). PhLPs are small cytosolic proteins hypothesized to function in G-protein signaling and protein folding. Although PhLPs are highly conserved in eukaryotes from yeast to mammals, only a few representatives have been experimentally characterized to date. In addition, while PhLPs contain a thioredoxin domain, they lack a CXXC motif, a strong indicator for redox activity, and it is unclear whether members of the PhLP family are enzymatically active. Here, we describe PbPhLP-3 as the first phosducin-like protein of a protozoan organism, Plasmodium berghei. Initial transcription analysis revealed continuous low-level expression of pbphlp-3 throughout the complex Plasmodium life cycle. Attempts to knockout pbphlp-3 in P. berghei did not yield live parasites, suggesting an essential role for the gene in Plasmodium. We cloned, expressed and purified PbPhLP-3 and determined that the recombinant protein is redox active in vitro in a thioredoxin-coupled redox assay. It also has the capacity to reduce the organic compound tert-Butyl hydroperoxide (TBHP) in vitro, albeit at low efficiency. Sequence analysis, structural modeling, and site-directed mutagenesis revealed a conserved cysteine in the thioredoxin domain to be the redox active residue. Lastly, we provide evidence that recombinant human PhLP-3 exhibits redox activity similar to that of PbPhLP-3 and suggest that redox activity may be conserved in PhLP-3 homologs of other species. Our data provide new insight into the function of PhLP-3, which is hypothesized to act as co-chaperones in the folding and regulation of cytoskeletal proteins. We discuss the potential implications of PhLP-3 as a thioredoxin-target protein and possible links between the cellular redox network and the eukaryotic protein folding machinery.