Hepatitis C Virus NS5A Protein Promotes the Lysosomal Degradation of Hepatocyte Nuclear Factor 1α via Chaperone-Mediated Autophagy

Abstract

Hepatitis C virus (HCV) infection is closely associated with type 2 diabetes. We reported that HCV infection induces the lysosomal degradation of hepatocyte nuclear factor 1 alpha (HNF-1α) via interaction with HCV nonstructural protein 5A (NS5A) protein, thereby suppressing GLUT2 gene expression. The molecular mechanisms of selective degradation of HNF-1α caused by NS5A are largely unknown. Chaperone-mediated autophagy (CMA) is a selective lysosomal degradation pathway. Here, we investigated whether CMA is involved in the selective degradation of HNF-1α in HCV-infected cells and observed that the pentapeptide spanning from amino acid (aa) 130 to aa 134 of HNF-1α matches the rule for the CMA-targeting motif, also known as KFERQ motif. A cytosolic chaperone protein, heat shock cognate protein of 70 kDa (HSC70), and a lysosomal membrane protein, lysosome-associated membrane protein type 2A (LAMP-2A), are key components of CMA. Immunoprecipitation analysis revealed that HNF-1α was coimmunoprecipitated with HSC70, whereas the Q130A mutation (mutation of Q to A at position 130) of HNF-1α disrupted the interaction with HSC70, indicating that the CMA-targeting motif of HNF-1α is important for the association with HSC70. Immunoprecipitation analysis revealed that increasing amounts of NS5A enhanced the association of HNF-1α with HSC70. To determine whether LAMP-2A plays a role in the degradation of HNF-1α protein, we knocked down LAMP-2A mRNA by RNA interference; this knockdown by small interfering RNA (siRNA) recovered the level of HNF-1α protein in HCV J6/JFH1-infected cells. This result suggests that LAMP-2A is required for the degradation of HNF-1α. Immunofluorescence study revealed colocalization of NS5A and HNF-1α in the lysosome. Based on our findings, we propose that HCV NS5A interacts with HSC70 and recruits HSC70 to HNF-1α, thereby promoting the lysosomal degradation of HNF-1α via CMA. Many viruses use a protein degradation system, such as the ubiquitin-proteasome pathway or the autophagy pathway, for facilitating viral propagation and viral pathogenesis. We investigated the mechanistic details of the selective lysosomal degradation of hepatocyte nuclear factor 1 alpha (HNF-1α) induced by hepatitis C virus (HCV) NS5A protein. Using site-directed mutagenesis, we demonstrated that HNF-1α contains a pentapeptide chaperone-mediated autophagy (CMA)-targeting motif within the POU-specific domain of HNF-1α. The CMA-targeting motif is important for the association with HSC70. LAMP-2A is required for degradation of HNF-1α caused by NS5A. We propose that HCV NS5A interacts with HSC70, a key component of the CMA machinery, and recruits HSC70 to HNF-1α to target HNF-1α for CMA-mediated lysosomal degradation, thereby facilitating HCV pathogenesis. We discovered a role of HCV NS5A in CMA-dependent degradation of HNF-1α. Our results may lead to a better understanding of the role of CMA in the pathogenesis of HCV.

The Multifaceted Roles of Autophagy in Flavivirus-Host Interactions

“…Gregoire et al showed that autophagy can be activated by ectopic expression of HCV NS3 alone (Table 1) [420]. In addition to NS4B and NS3, expression of the HCV core protein can induce complete autophagy (Table 1) [421,422] and another later study showed that HCV NS5A can trigger autophagy (Table 1) [423,424]. Although these results collectively suggest that HCV can activate autophagy via individual viral proteins, many conclusions were drawn based on the transient expression of viral proteins in cells that are not permissive to complete HCV life cycle, which may have led to large discrepant results.…”

“…On the other hand, HCV-induced CMA was shown to participate in the degradation of IFN-alpha receptor-1 (IFNAR1), which is stimulated by free fatty acid (FFA) (Table 1) [433]. Moreover, Matsui and colleagues recently reported that HCV NS5A can interact with Hsc70, a regulator of CMA, to target hepatocyte nuclear factor 1α (HNF1α) for lysosomal degradation (Table 1) [424]. A recent study implied that HCV-induced IFN-β-inducible SCOTIN recruits NS5A to autolysosomes for degradation, thus restricting HCV replication (Table 1) [434].…”

“…In addition to promoting HCV growth, viral-triggered autophagy seems to degrade a panel of intracellular components, including LDs, mitochondria and host and viral proteins [424,429,430,434,443]. However, these conclusions are largely debated to the previous study shown that HCV activates incomplete autophagy by inhibiting autophagic flux [402,418].…”

Mechanism and Complex Roles of HSC70 in Viral Infections

“…In HCV-infected Huh7.5 cells with FFA treatment, IFNAR1 is selectively degraded through interacting with HSC70 and LAMP2A on the lysosome membrane, suggesting that HSC70 inhibits host anti-HCV immunity through mediating autophagy (Ramazan et al, 2015). Another study suggests that HCV NS5A protein interacts with HSC70 and recruits HSC70 to hepatocyte nuclear factor 1 alpha (HNF-α), thereby promoting the CMA-dependent lysosomal degradation of HNF-α and facilitating virus pathogenesis (Matsui et al, 2018). Interestingly, HSC70 can directly participate in cellular host innate immunity by the DNA-dependent protein kinase (DNA-PK) DNA sensing pathway, which induces a robust and broad antiviral response via a non-classical DNA sensing pathway.…”

Diverse Functions of Autophagy in Liver Physiology and Liver Diseases

“…However, CMA was also reported to be activated by HCV and implicated in the degradation of IFN-alpha receptor-1 (IFNAR1), which is stimulated by FFAs (Table 5) [570]. Another study demonstrated that HCV NS5A interacts with HSC70 to promote the degradation of hepatocyte nuclear factor 1α (HNF1α) (Table 5) [571]. Recently, IFN-β-inducible SCOTIN was shown to recruit HCV NS5A into autolysosomes for degradation, restricting the HCV infection (Table 5) [572].…”