Unconjugated interferon‐stimulated gene 15 specifically interacts with the hepatitis C virus NS5A protein via domain I

Minami, Nanae; Abe, Takayuki; Deng, Lin; Matsui, Chieko; Fukuhara, Takasuke; Matsuura, Yoshiharu; Shoji, Ikuo

doi:10.1111/1348-0421.12493

Cited by 8 publications

(8 citation statements)

References 33 publications

(36 reference statements)

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“…We previously demonstrated that HCV NS5A protein interacts with the unmodified ISG15, leading to acceptance of ISGylation via covalent bond [26,28]. Thus, we considered that HBx protein may interact with ISG15 via protein-protein interactions, leading to acceptance of ISG15 conjugation via covalent bond.…”

Section: Discussionmentioning

confidence: 99%

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HERC5 E3 ligase mediates ISGylation of hepatitis B virus X protein to promote viral replication

Bawono

Abe

et al. 2021

Journal of General Virology

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Ubiquitin and ubiquitin-like protein modification play important roles in modulating the functions of viral proteins in many viruses. Here we demonstrate that hepatitis B virus (HBV) X protein (HBx) is modified by ISG15, which is a type I IFN-inducible, ubiquitin-like protein; this modification is called ISGylation. Immunoblot analyses revealed that HBx proteins derived from four different HBV genotypes accepted ISGylation in cultured cells. Site-directed mutagenesis revealed that three lysine residues (K91, K95 and K140) on the HBx protein, which are well conserved among all the HBV genotypes, are involved in acceptance of ISGylation. Using expression plasmids encoding three known E3 ligases involved in the ISGylation to different substrates, we found that HERC5 functions as an E3 ligase for HBx-ISGylation. Treatment with type I and type III IFNs resulted in the limited suppression of HBV replication in Hep38.7-Tet cells. When cells were treated with IFN-α, silencing of ISG15 resulted in a marked reduction of HBV replication in Hep38.7-Tet cells, suggesting a role of ISG15 in the resistance to IFN-α. In contrast, the silencing of USP18 (an ISG15 de-conjugating enzyme) increased the HBV replication in Hep38.7-Tet cells. Taken together, these results suggest that the HERC5-mediated ISGylation of HBx protein confers pro-viral functions on HBV replication and participates in the resistance to IFN-α-mediated antiviral activity.

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Section: Discussionmentioning

confidence: 99%

“…The expression plasmids for pCAG-FLAG-ISG15 and its conjugation defective mutant (pCAG-FLAG-ISG15-AA) were previously described [28]. The cDNA fragments encoding UBE1L, UbcH8, and HERC5 were cloned into the NotI/ BglII and SmaI/KpnI site of pCAG-MCS2 using the In-Fusion HD-Cloning kit.…”

Section: Plasmidsmentioning

confidence: 99%

HERC5 E3 ligase mediates ISGylation of hepatitis B virus X protein to promote viral replication

Bawono

Abe

et al. 2021

Journal of General Virology

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“…There are two crucial requirements of the substrates for HCV-induced CMA; NS5A binding and a CMA-targeting motif. We and other groups have been investigating NS5A-interacting proteins ( Matsui et al., 2012 ; Sianipar et al., 2015 ; Ross-Thriepland and Harris, 2015 ; Chen et al., 2016 ; Minami et al., 2017 ; Abe et al., 2020 ). Using software, we can easily do a search to determine whether the NS5A-interacting proteins contain potential CMA-targeting motifs.…”

Section: Conclusion and Future Perspectivesmentioning

confidence: 99%

The Role of Chaperone-Mediated Autophagy in Hepatitis C Virus-Induced Pathogenesis

Matsui

Yuliandari

Deng

et al. 2021

Front. Cell. Infect. Microbiol.

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Lysosome incorporate and degrade proteins in a process known as autophagy. There are three types of autophagy; macroautophagy, microautophagy, and chaperone-mediated autophagy (CMA). Although autophagy is considered a nonselective degradation process, CMA is known as a selective degradation pathway. All proteins internalized in the lysosome via CMA contain a pentapeptide KFERQ-motif, also known as a CMA-targeting motif, which is necessary for selectivity. CMA directly delivers a substrate protein into the lysosome lumen using the cytosolic chaperone HSC70 and the lysosomal receptor LAMP-2A for degradation. Hepatitis C virus (HCV) NS5A protein interacts with hepatocyte-nuclear factor 1α (HNF-1α) together with HSC70 and promotes the lysosomal degradation of HNF-1α via CMA, resulting in HCV-induced pathogenesis. HCV NS5A promotes recruitment of HSC70 to the substrate protein HNF-1α. HCV NS5A plays a crucial role in HCV-induced CMA. Further investigations of HCV NS5A-interacting proteins containing CMA-targeting motifs may help to elucidate HCV-induced pathogenesis.

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“…Our group and many other groups have been investigating NS5A-interacting proteins. More than 130 host proteins have been identified as NS5A-interacting proteins (11,(38)(39)(40)(41). We are currently examining the potential CMA-targeting motif in NS5Ainteracting proteins.…”

Section: Figmentioning

confidence: 99%

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

Matsui

Deng

Minami

et al. 2018

J Virol

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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.

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Unconjugated interferon‐stimulated gene 15 specifically interacts with the hepatitis C virus NS5A protein via domain I

Cited by 8 publications

References 33 publications

HERC5 E3 ligase mediates ISGylation of hepatitis B virus X protein to promote viral replication

HERC5 E3 ligase mediates ISGylation of hepatitis B virus X protein to promote viral replication

The Role of Chaperone-Mediated Autophagy in Hepatitis C Virus-Induced Pathogenesis

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

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