Nonstructural 5A Protein Of Hepatitis Research Articles

Nonstructural 5A Protein of Hepatitis C Virus Interacts with Pyruvate Carboxylase and Modulates Viral Propagation

Hepatitis C virus (HCV) is highly dependent on cellular factors for its own propagation. By employing tandem affinity purification method, we identified pyruvate carboxylase (PC) as a cellular partner for NS5A protein. NS5A interacted with PC through the N-terminal region of NS5A and the biotin carboxylase domain of PC. PC expression was decreased in cells expressing NS5A and HCV-infected cells. Promoter activity of PC was also decreased by NS5A protein. However, FAS expression was increased in cells expressing NS5A and cell culture grown HCV (HCVcc)-infected cells. Silencing of PC promoted fatty acid synthase (FAS) expression level. These data suggest HCV may modulate PC via NS5A protein for its own propagation.

Nonstructural 5A protein of hepatitis C virus regulates heat shock protein 72 for its own propagation

We identified heat shock protein 72 (Hsp72) as a host factor that was differentially expressed in cells expressing nonstructural 5A (NS5A) protein. To investigate how NS5A modulates Hsp72 in hepatitis C virus (HCV) life cycle, we examined the role of Hsp72 in HCV replication and virus production. NS5A specifically interacted with Hsp72. Both Hsp72 and nuclear factor of activated T cells 5 (NFAT5) levels were increased in cells expressing NS5A protein. Treatments of N-acetylcysteine and glutathione markedly reduced protein levels of both NFAT5 and Hsp72. Knockdown of NFAT5 resulted in decrease in Hsp72 level in cells expressing NS5A. Importantly, silencing of Hsp72 expression resulted in decrease in both RNA replication and virus production in HCV-infected cells. These data indicate that NS5A modulates Hsp72 via NFAT5 and reactive oxygen species activation for HCV propagation.

Characterization of monoclonal antibodies against the nonstructural 5A protein of hepatitis C virus

The nonstructural 5A (NS5A) protein of hepatitis C virus (HCV) is a multifunctional protein that leads to pleiotropic responses, in part by regulating cell growth and cellular signaling pathways. Here, we produced monoclonal antibodies (mAbs) directed against the HCV NS5A protein. The N-terminal epitope was mapped to amino acids 60-80 of the NS5A protein, and the epitope in the middle region was mapped to amino acids 221-236. Because these epitopes overlap with binding regions of human vesicle-associated membrane-protein-associated protein (hVAP)-B and TNF-receptor-associated factor 2 (TRAF2), respectively, we investigated these mAbs for their potential capacity to inhibit viral and cellular interactions. We found that NS5A and hVAP-B interaction was abolished by mAb E5D3, and NS5A and TRAF2 interaction was inhibited by mAb C6D4. Since hVAP-B is necessary for HCV replication and TRAF2 is the major transducer in TNF signaling cascades, these data may provide further insights into the mechanisms underlying HCV replication and viral modulation of host signal transduction.

The non-structural 5A protein of hepatitis C virus exhibits genotypic differences in interferon antagonism

Patients infected with hepatitis C virus (HCV) genotype 2 or 3 usually respond better to interferon (IFN) treatment than those infected with genotype 1. In this study, we investigated whether the non-structural 5A protein (NS5A) of HCV genotypes 1 and 2 (1b-NS5A and 2a-NS5A, respectively) exerted differential counteractivities against IFN treatment. We compared the inhibitory effects of 1b-NS5As and 2a-NS5As on IFN activity. We also investigated the replication inhibition of HCV subgenomic replicons containing 1b-NS5A or 2a-NS5A in response to IFN treatment. 1b-NS5As exerted more profound inhibitory effects on IFN activity than 2a-NS5As. The replication of the 2a-NS5A-containing replicons was more sensitive to IFN treatment than that of the 1b-NS5A-containing replicons. Deletion of the interferon sensitivity-determining region/protein kinase R-binding domain (PKR-BD), the V3 domain, or the C-terminus region of NS5A significantly abrogated its anti-IFN activity. Domain swapping between 1b-NS5A and 2a-NS5A in the V3 domain and/or the C-terminus region resulted in a transfer of their anti-IFN activity. 1b-NS5As exert higher magnitudes of IFN antagonism than do 2a-NS5As. The V3 and the C-terminus regions are responsible for the differential anti-IFN effects. This phenomenon may partly explain the genotype-linked differences in the response of HCV to IFN treatment.

446 Insertion of a new V3-like domain in nonstructural 5A protein of hepatitis C virus genotype 1B

446 Insertion of a new V3-like domain in nonstructural 5A protein of hepatitis C virus genotype 1B

The non-structural 5A protein of hepatitis C virus.

The non-structural (NS)5A protein of hepatitis C virus (HCV) is cleaved, after translation, by the NS3-encoded zinc-dependent serine proteinase, from the NS4B protein upstream and the NS5B protein downstream. The released, mature NS5A protein is a 56 000 MW phosphoprotein (p56), which also exists within infected cells in a hyperphosphorylated form (p58). The NS5A gene has a quasispecies distribution, meaning that various NS5A sequences co-exist, in various proportions, in infected individuals. HCV NS5A appears to be located in cytoplasmic membranes surrounding the nucleus. Its precise functions are not known. HCV non-structural proteins, including NS5A, form a large multiprotein replication complex, which probably directs the replication of the HCV genome. HCV NS5A lacking the 146 N-terminal amino acids is a potent transcriptional activator in vitro. NS5A can also bind to single-strand RNA-dependent protein kinase (PKR) and inhibit its antiviral function. An 'interferon (IFN) sensitivity-determining region' has recently been postulated in the NS5A protein central region in hepatitis C virus (HCV) genotype 1b, but strongly conflicting evidence has been published. In fact, there would seem to be no such region in the NS5A protein, even though NS5A plays an important and complex role in HCV resistance to IFN. Structure-function studies are required to identify precisely how NS5A and IFN interact.