EWSR1 Binds the Hepatitis C Virus cis -Acting Replication Element and Is Required for Efficient Viral Replication

Abstract

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 ...

Detergent-Resistant Membrane Association of NS2 and E2 during Hepatitis C Virus Replication

“…and 4). Considering that NS5A interacts with ApoE and was shown to associate with the DRM whether it was expressed singly in the context of the subgenomic replicon or infectious virus (28,67), NS5A is likely responsible to recruit ApoE to the DRM. This will then allow the interaction of ApoE and E1/E2 complexes at the DRM, promoting infectious virus assembly.…”

The Coding Region of the HCV Genome Contains a Network of Regulatory RNA Structures

“…By using bioinformatic tools and ribonuclease mapping, several groups have contributed to the identification of RNA structures in untranslated regions (UTRs) as well as the core and NS5B-encoding regions (Fricke et al, 2015; Tuplin et al, 2004). Many of these structures have since been validated by mutational analysis in cell culture models of HCV replication and infectivity, and they have helped assign functional roles for specific RNA structural elements (Diviney et al, 2008; Friebe and Bartenschlager, 2002; Friebe et al, 2005; Friebe et al, 2001; Kolykhalov et al, 2000; Lee et al, 2004; McMullan et al, 2007; Oakland et al, 2013; Vassilaki et al, 2008; You and Rice, 2008; You et al, 2004). However, these methodologies have been less successful in identifying and characterizing RNA structures that occur elsewhere in the HCV genome (Chu et al, 2013).…”

Hepatitis C Virus Is Released via a Noncanonical Secretory Route

“…Recently, it has been proposed that the cellular Ewing sarcoma breakpoint region 1 (EWSR1) protein is important for regulation of the switch from translation to replication by binding to the cis-acting replication element of HCV (14). Furthermore, transport of HCV Core toward LDs by the enzyme diacylglycerol acyltransferase-1 (DGAT1) is crucial for production of newly formed virions (15,16), and the NS2 protein, together with p7, might be a major player in coordinating assembly (11,17,18).…”