Nonstructural 3 Protein of Hepatitis C Virus Modulates the Tribbles Homolog 3/Akt Signaling Pathway for Persistent Viral Infection

Abstract

Hepatitis C virus (HCV) infection often causes chronic hepatitis, liver cirrhosis, and ultimately hepatocellular carcinoma. However, the mechanisms underlying HCV-induced liver pathogenesis are still not fully understood. By transcriptome sequencing (RNA-Seq) analysis, we recently identified host genes that were significantly differentially expressed in cell culture-grown HCV (HCVcc)-infected cells. Of these, tribbles homolog 3 (TRIB3) was selected for further characterization. TRIB3 was initially identified as a binding partner of protein kinase B (also known as Akt). TRIB3 blocks the phosphorylation of Akt and induces apoptosis under endoplasmic reticulum (ER) stress conditions. HCV has been shown to enhance Akt phosphorylation for its own propagation. In the present study, we demonstrated that both mRNA and protein levels of TRIB3 were increased in the context of HCV replication. We further showed that promoter activity of TRIB3 was increased by HCV-induced ER stress. Silencing of TRIB3 resulted in increased RNA and protein levels of HCV, whereas overexpression of TRIB3 decreased HCV replication. By employing an HCV pseudoparticle entry assay, we further showed that TRIB3 was a negative host factor involved in HCV entry. Both in vitro binding and immunoprecipitation assays demonstrated that HCV NS3 specifically interacted with TRIB3. Consequently, the association of TRIB3 and Akt was disrupted by HCV NS3, and thus, TRIB3-Akt signaling was impaired in HCVinfected cells. Moreover, HCV modulated TRIB3 to promote extracellular signal-regulated kinase (ERK) phosphorylation, activator protein 1 (AP-1) activity, and cell migration. Collectively, these data indicate that HCV exploits the TRIB3-Akt signaling pathway to promote persistent viral infection and may contribute to HCV-mediated pathogenesis. IMPORTANCETRIB3 is a pseudokinase protein that acts as an adaptor in signaling pathways for important cellular processes. So far, the functional involvement of TRIB3 in virus-infected cells has not yet been demonstrated. We showed that both mRNA and protein expression levels of TRIB3 were increased in the context of HCV RNA replication. Gene silencing of TRIB3 increased HCV RNA and protein levels, and thus, overexpression of TRIB3 decreased HCV replication. TRIB3 is known to promote apoptosis by negatively regulating the Akt signaling pathway under ER stress conditions. Most importantly, we demonstrated that the TRIB3-Akt signaling pathway was disrupted by NS3 in HCV-infected cells. These data provide evidence that HCV modulates the TRIB3-Akt signaling pathway to establish persistent viral infection.

Prediction of SARS-CoV interaction with host proteins during lung aging reveals a potential role for TRIB3 in COVID-19

“…The reanalysis of lung single-cell RNA sequencing data 19,20 demonstrated TRIB3 expressed mainly in alveolar type I (AT1) and type II (AT2) cells and in ciliated cells ( Figure 1d-f, Figure S3), which also express SARS-CoV-2 receptor ACE2 7,8,21 . The involvement of TRIB3 in viral infection is poorly understood; however, its inhibition was associated with an increase of hepatitis C virus (HCV) replication 22 . Additionally, TRIB3 negatively regulates the entry step of the HCV life cycle and propagation 22 and thus may constitute a common protective host factor for other positive-sense single-strand RNA viruses.…”

“…The involvement of TRIB3 in viral infection is poorly understood; however, its inhibition was associated with an increase of hepatitis C virus (HCV) replication 22 . Additionally, TRIB3 negatively regulates the entry step of the HCV life cycle and propagation 22 and thus may constitute a common protective host factor for other positive-sense single-strand RNA viruses. TRIB3 is also one of the unfolded protein response (UPR)-related genes with the strongest positive correlation with the intracellular abundance of the flavivirus dengue and Zika 23 .…”

Hepatitis C Virus-Induced Rab32 Aggregation and Its Implications for Virion Assembly

“…Approximately 1.5 ϫ 10 4 cells/well seeded on 24-well plates were transfected with either a negative, positive, or Rab32-specific siRNA. Cell viability was measured by using water-soluble tetrazolium salt (WST) (Dail Lab) as we reported previously (57).…”

“…After three washes with PBS, cells were analyzed using a Zeiss LSM 700 laser confocal microscopy system (Carl Zeiss, Inc., Thornwood, NY). Pearson's correlation coefficient and the Manders' overlap coefficient were used to quantify the level of colocalization as described previously (57).…”

Hepatitis C Virus Downregulates Ubiquitin-Conjugating Enzyme E2S Expression To Prevent Proteasomal Degradation of NS5A, Leading to Host Cells More Sensitive to DNA Damage

“…HCV infection triggers a wide range of cellular stress responses, including cell cycle arrest, apoptosis, ER stress, and autophagy (28,29). Moreover, HCV induces TRIB3, TGF-␤1, and XBP1 through the activation of ER stress (30)(31)(32). We therefore examined the role of ER stress on UBE2S expression level.…”