Nonstructural Protein 5A of Hepatitis C Virus Inhibits the Function of Karyopherin β3

Abstract

It has been suggested that nonstructural protein 5A (NS5A) of hepatitis C virus (HCV) plays a role in the incapacitation of interferon by inactivation of RNA-dependent protein kinase PKR. In order to further investigate the role of NS5A, we tried to identify cellular proteins interacting with NS5A by using the yeast two-hybrid system. The karyopherin ␤3 gene was isolated from a human liver cell library as a protein interacting with NS5A. The protein-protein interaction between NS5A and karyopherin ␤3 was confirmed by in vitro binding assay and an in vivo coimmunoprecipitation method. The effect of NS5A on the karyopherin ␤3 activity was investigated using a yeast cell line containing mutations in both PSE1 and KAP123, genes that are homologous to the human karyopherin ␤3 gene. Human karyopherin ␤3 complemented the loss of the PSE1 and KAP123 functions, supporting growth of the double mutant cells. However, expression of NS5A hampered the growth of the double mutant cells supplemented with human karyopherin ␤3. On the other hand, expression of NS5A by itself had no effect on the growth of the double mutant expressing wild-type yeast PSE1. This indicates that NS5A may inhibit karyopherin ␤3 function via protein-protein interaction. The role of NS5A in HCV replication is discussed.Hepatitis C virus (HCV) is the major etiologic agent of non-A, non-B hepatitis (1,8,38). Chronic infection with HCV results in liver cirrhosis and hepatocellular carcinoma (7,45). HCV belongs to the family Flaviviridae, having a positive-sense RNA genome (32,42,47). The RNA encodes a polyprotein (ϳ3,010 amino acids) with the following gene order: 5Ј-C-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B-3Ј. During and/or after translation, the polyprotein is processed into functional proteins by host-and virus-encoded proteases. Core (C) and envelope (E1 and E2) proteins are believed to compose the structural elements of the virion particle. Nonstructural protein 2 (NS2), NS3, and NS4A are involved in the proteolytic processing of the HCV polyprotein (4,5,15,18,25,26,27,28,30,40,50,52). RNA-dependent RNA polymerase and RNA helicase activities are assigned to NS5B and the C-terminal two-thirds of NS3, respectively (6,36). No function has yet been assigned to NS4B.NS5A exists in two different forms (p56 and p58) in cells. The proteins differ in their phosphorylation status (2, 34, 51). NS4A or NS3-4A-4B augments hyperphosphorylation of NS5A (43, 51). The sequence around the middle part of NS5A (amino acids 2209 to 2248) is termed the interferon sensitivity-determining region, since it correlates with interferon sensitivity of the HCV genotype 1b (16,17,39). The sequence in the interferon sensitivity-determining region was shown to play a key role in the inhibition of the protein kinase PKR, a mediator of interferon-induced resistance, through protein-protein interaction (20, 21). NS5A was also shown to interact with a SNARE-like protein (53). The C-terminal region of NS5A contains a potential transcriptional activation domain, but the role of its activity ...

Expression of hepatitis C virus NS5A natural mutants in a hepatocytic cell line inhibits the antiviral effect of interferon in a PKR-independent manner

“…30 Finally, NS5A might interfere with the nuclear transport of RNA and proteins by interacting with Karyopherin ␤ 3. 31 Thus, NS5A expression may play a crucial role in control of the antiviral and proliferative status of the HCV-related liver cell by interacting with several cellular signaling pathways.Abbreviations: HCV, hepatitis C virus; IFN-␣, interferon alfa; ISDR, interferon sensitivity-determining region; EMCV, encephalomyocarditis virus; VSV, vesicular stomatitis virus; PKR, double-stranded RNA-dependent kinase; PCR, polymerase chain reaction; FCS, fetal calf serum; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; eIF2, eukaryotic initiation factor 2; PFU, plaque-forming unit; PBS, phosphatebuffered saline; TNF, tumor necrosis factor. From…”

“…30 Finally, NS5A might interfere with the nuclear transport of RNA and proteins by interacting with Karyopherin ␤ 3. 31 Thus, NS5A expression may play a crucial role in control of the antiviral and proliferative status of the HCV-related liver cell by interacting with several cellular signaling pathways.…”

Insertion and deletion analyses identify regions of non-structural protein 5A of Hepatitis C virus that are dispensable for viral genome replication

“…In addition, the phosphorylation pattern of NS5A plays an important role in HCV genome replication (Evans et al, 2004b;Appel et al, 2005). It also interacts with a multitude of cellular proteins and alters the host cells to support viral RNA replication (Gale et al, 1998;Chung et al, 2000;Park et al, 2002;Evans et al, 2004a;Gao et al, 2004).…”

“…The aminoterminal domain of NS5A appears to interact with several cellular proteins (Gale et al, 1998;Chung et al, 2000;Park et al, 2002;Waris et al, 2003;Evans et al, 2004a;Gao et al, 2004) that may be necessary for replication. In addition, an amphipathic a-helix and a zinc-binding site located at the amino terminus are required for replication (Brass et al, 2002; Elazar et al, 2003;Tellinghuisen et al, 2004Tellinghuisen et al, , 2005 et al, 2000) and the carboxyterminal sequences (residues 399-441) have been demonstrated (Zhu et al, 2003).…”

“…In addition, the phosphorylation pattern of NS5A plays an important role in HCV genome replication (Evans et al, 2004b;Appel et al, 2005). It also interacts with a multitude of cellular proteins and alters the host cells to support viral RNA replication (Gale et al, 1998;Chung et al, 2000;Park et al, 2002;Evans et al, 2004a;Gao et al, 2004).Transposon-mediated insertion mutagenesis (Goryshin & Reznikoff, 1998) is a powerful tool that allows insertion of short peptides into proteins encoded in cloned DNA to evaluate permissive sites in the protein. It can also be used to assess regions of the protein that are dispensable for its functions.…”