What this is
- Hepatitis C virus (HCV) NS5A protein promotes the lysosomal degradation of diacylglycerol O-acyltransferase 1 (DGAT1).
- This degradation occurs via (), not chaperone-mediated autophagy (CMA).
- The study identifies the in DGAT1 as crucial for its interaction with the chaperone HSC70, which facilitates degradation.
Essence
- HCV NS5A protein induces the lysosomal degradation of DGAT1 through , driven by its interaction with HSC70 via the .
Key takeaways
- HCV NS5A protein interacts with DGAT1, promoting its lysosomal degradation via . This interaction is mediated by the found in DGAT1.
- Knockdown of VPS4B restores DGAT1 levels in HCV-infected cells, indicating its role in the pathway for DGAT1 degradation.
- Inhibition of macroautophagy does not affect DGAT1 levels, confirming that HCV-induced degradation occurs specifically through .
Caveats
- The study focuses on cellular mechanisms in vitro, which may not fully represent the complexities of HCV infection in vivo.
- Further research is needed to clarify the physiological significance of HCV-induced DGAT1 degradation and its implications for viral propagation.
Definitions
- endosomal microautophagy (eMI): A selective form of microautophagy where cytosolic components are directly ingested by lysosomes through invagination of the lysosomal membrane.
- KFERQ motif: A specific pentapeptide sequence crucial for the recognition of substrate proteins by the chaperone HSC70, facilitating their degradation.
Simplified
Introduction
Approximately 56 million people worldwide are estimated to be infected with hepatitis C virus (HCV), and ~20% of them develop liver cirrhosis or hepatocellular carcinoma [1]. HCV is an enveloped, positive-sense single-stranded RNA virus that belongs to the Flaviviridae family, Hepacivirus genus. The HCV genome consists of a 9.6-kb RNA encoding a polyprotein of 3,010 amino acids (aa). The polyprotein is cleaved into three structural proteins (core, envelope 1 [E1], and envelope 2 [E2] proteins) and seven nonstructural (NS) proteins (p7, NS2, NS3, NS4A, NS4B, NS5A, NS5B) by viral proteases and host signal peptidases [2]. The structural proteins are involved in the formation of viral particles, whereas the NS proteins participate in viral replication [3]. The development of HCV RNA replicon systems and HCV cell culture systems has enabled us to study HCV replication and the entire HCV-life cycle [4,5]. Recent advances in HCV research have resulted in the development of novel anti-HCV therapeutics, i.e., direct-acting antivirals (DAAs), which have dramatically improved the treatment of chronic hepatitis C. However, the emergence of resistance-associated substitutions raises new concerns [6].
We reported that HCV infection promotes the lysosomal degradation of hepatocyte nuclear factor-1α (HNF-1α) via chaperone-mediated autophagy (CMA) through an NS5A-mediated association of HNF-1α with cellular chaperone heat shock cognate 70 kDa (HSC70) protein; we demonstrated that lysosome-associated membrane protein type 2A (LAMP-2A) is required for the degradation of HNF-1α [7]. However, little is known about the roles of HCV-induced lysosomal degradation in the HCV life cycle and viral pathogenesis. In this study, we searched for a novel substrate of an HCV-induced lysosomal degradation pathway by examining the NS5A-interacting proteins that carry the KFERQ motif, which is important for the association with HSC70.
Autophagy is a cellular process that transports materials from the cytoplasm to the lysosome for degradation. This process contributes to cells’ survival by removing damaged organelles and protein aggregates and promoting cellular homeostasis [8]. There are at least three types of autophagy in mammalian cells: macroautophagy, microautophagy, and chaperone-mediated autophagy (CMA). In microautophagy, cytosolic components are directly ingested by lysosomes through an invagination of the lysosomal membrane. One of the selective forms of microautophagy is endosomal microautophagy (eMI). Both eMI and CMA utilize the cellular chaperone protein HSC70 to selectively recognize substrate proteins with a KFERQ motif [9-11].
The selectivity of the target protein is determined by the presence of a specific pentapeptide motif, the KFERQ motif, in the amino acid sequence of the substrate protein [12-14]. The KFERQ motif contains one or two of the positively charged residues lysine (K) and arginine (R); one or two of the hydrophobic residues phenylalanine (F), isoleucine (I), leucine (L), and valine (V); one of the negatively charged residues aspartic acid (D) or glutamic acid (E); and one glutamine (Q) on either side of the pentapeptide [15-17]. Cuervo’s group developed free Web-based software, KFERQ finder V0.8 (https://rshine.einsteinmed.org/↗), to quickly identify this motif in any protein sequence [18].
The interaction between HSC70 and the target protein is necessary for two selective lysosomal autophagy pathways: CMA and eMI. In CMA, the protein complex formed by HSC70 interacts with LAMP-2A, causing the target protein to unfold and degrade in the lysosome. After the substrate transfer to LAMP-2A, HSC70 is released into the cytosol. In contrast to CMA, eMI does not require the unfolding or binding of cytosolic proteins to LAMP-2A. After binding to a substrate protein, HSC70 interacts with phosphatidylserine (PS) of the endosomal membrane. A substrate protein for eMI is sequestered by the formation of an invagination in the surface of the endosomal membrane, which is mediated by tumor susceptibility gene (TSG) 101 as an endosomal sorting complex required for transport (ESCRT)-I and three proteins: vacuolar protein sorting-associated protein (VPS) 4A, VPS4B, and Alix [19-21].
Diacylglycerol O-acyltransferase 1 (DGAT1) is an important enzyme in the final step of triglyceride synthesis. DGAT1 plays a crucial role in HCV infection by recruiting the HCV core protein onto the surface of cellular lipid droplets [22,23]. In this study, we observed that DGAT1 is degraded via the lysosomal degradation pathway. We aim to clarify the molecular mechanisms underlying the lysosomal degradation of DGAT1 protein induced by HCV NS5A protein. Here, we demonstrate that HCV NS5A protein promotes the lysosomal degradation of DGAT1 protein via eMI, but not CMA.
Results
HCV NS5A protein interacts with DGAT1
To examine the subcellular localization of NS5A and DGAT1 in Huh-7.5 cells, the cells were cotransfected with pCAG-FLAG-DGAT1 and pEF1A-NS5A-Myc-His6. Immunofluorescence staining revealed that FLAG-DGAT1 was colocalized with NS5A-Myc-His6 in the cytoplasm (Fig. 1C, bottom panel, merge). To further examine whether DGAT1 is colocalized with NS5A protein, we performed a proximity ligation assay (PLA). The PLA revealed a strong signal in the presence of both pCAG-FLAG-DGAT1 and pEF1-NS5A-Myc-His6 (Fig. 1D, bottom panel, merge). These results suggest that NS5A interacts with DGAT1 in Huh-7.5 cells.
NS5A protein interacts with DGAT1 protein in Huh-7.5 cells. () Huh-7.5 cells were transfected with either pCAG-FLAG-DGAT1 or empty plasmid together with pEF1A-NS5A-Myc-Hisas indicated, and cultured. At 48 h after transfection, cells were harvested. Cell lysates were assayed for immunoprecipitation with mouse anti-FLAG mAb, followed by immunoblotting with mouse anti-FLAG mAb (3rd panel) or mouse anti-c-myc mAb (4th panel). The western blots are representative of three independent experiments. *: IgG heavy chain. () Huh-7.5 cells were plated and cultured for 12 h. Cells were infected with HCV J6/JFH1 at a multiplicity of infection (MOI) of 2. At 5 days post-infection, the cells were stained with anti-DGAT1 mAb followed by Alexa Fluor 488-conjugated goat anti-mouse IgG () and anti-NS5A pAb followed by Alexa Fluor 594-conjugated goat anti-rabbit IgG (). The cells were stained with Hoechst 33342 for the nuclei (). The stained cells were examined by scanning laser confocal microscopy and image software. The images are representative of three independent experiments. () Huh-7.5 cells were transfected with pCAG-FLAG-DGAT1 together with pEF1A-NS5A-Myc-Hisas indicated, and cultured. At 48 h after transfection, the cells were stained with anti-DGAT1 mAb followed by Alexa Fluor 488-conjugated goat anti-mouse IgG () and anti-NS5A pAb followed by Alexa Fluor 594-conjugated goat anti-rabbit IgG (). The images are representative of three independent experiments. () Huh-7.5 cells were transfected with pEF1A-NS5A-Myc-Histogether with pCAG-FLAG-DGAT1, and cultured. At 72 h after transfection, the cells were stained with anti-DDDDK rabbit pAb and anti-c-myc mouse mAb followed by a proximity ligation assay (Duolink). The images are representative of three independent experiments. A B C D 6 6 6 green red blue green red
NS5A domain I binds to DGAT1 protein
We further mapped the NS5A-binding region on DGAT1 protein using a series of FLAG-tagged DGAT1 deletion mutants (Fig. 2C). All of the FLAG-DGAT1 deletion mutants except FLAG-DGAT1 (aa 1–156) and FLAG-DGAT1 (aa 400–488) (Fig. 2D, upper panel, lane 8; Fig. 2F, upper panel, lane 6) were coimmunoprecipitated with NS5A-Myc-His6 with the use of anti-HA pAb. These results suggest that the region from aa 157 to 399 on DGAT1 is important for the interaction with NS5A protein.
Mapping of the DGAT1-binding domain on NS5A protein and the NS5A-binding domain on DGAT1 protein. () Schematic representation of the NS5A protein. AH: amphipathic helix, LCS: low-complexity sequences. Each NS5A deletion mutants (a–h) contains an HA-tag in the N-terminal region. () Huh-7.5 cells were plated and cultured for 12 h. Cells were transfected with pCAG-FLAG-DGAT1 together with each HA-NS5A mutant plasmid as indicated (Fig. 2A, a–h). At 48 h post-transfection, cells were harvested and cell lysates were immunoprecipitated with anti-FLAG beads. Input samples and immunoprecipitated samples were analyzed by immunoblotting with anti-HA rabbit pAb (), or anti-DDDDK mouse mAb (). The western blots are representative of three independent experiments. IP: immunoprecipitation, IB: immunoblotting. *: IgG light chain. () Schematic representation of the DGAT1 protein. Each DGAT1 deletion mutant (a–i) contains a FLAG-tag in the N-terminal region. AH: amphipathic helix, EL: extracellular lumen, IL: intracellular lumen, TM: transmembrane. () Huh-7.5 cells were plated and cultured for 12 h. Cells were transfected with pCAG-HA-NS5A together with each DGAT1 mutant plasmid as indicated (Fig. 2C, a–i). At 48 h post-transfection, cells were harvested and cell lysates were immunoprecipitated with protein A Sepharose beads. Input samples and immunoprecipitated samples were analyzed by immunoblotting with anti-DDDDK mouse mAb () or anti-HA rabbit pAb (). The western blots are representative of three independent experiments. A B C D, E, F upper panel lower panel upper panel lower panel
HCV NS5A is important for the lysosomal degradation of DGAT1 protein
Next, to determine whether protein degradation is involved in the HCV-induced reduction of DGAT1 protein, we assessed the potential role of proteasomal or lysosomal protease on DGAT1 protein. We treated the cells with a proteasome inhibitor, MG132, or a lysosomal protease inhibitor, ammonium chloride (NH4Cl). MG132 did not increase the levels of DGAT1 protein (Fig. 3C, upper panel, lanes 6, 8 and 10), whereas NH4Cl restored the levels of DGAT1 protein (Fig. 3D, upper panel, lane 6). These results suggest that HCV infection induces the lysosomal degradation of DGAT1 protein.
To determine whether macroautophagy is involved in HCV-induced reduction of DGAT1 protein, we treated the cells with a macroautophagy inhibitor, 3-methyladenine (3-MA). Inhibition of macroautophagy, as evidenced by the accumulation of SQSTM1/p62 and a reduction in LC3-II form (Fig. 3E, 2nd and 3rd panels, lane 4), did not increase DGAT1 protein levels (Fig. 3E, upper panel, lane 4). This result suggests that macroautophagy is not involved in the HCV-induced reduction of DGAT1 protein.
For the investigation of a possible role of NS5A in the degradation of the endogenous DGAT1 protein, Huh-7.5 cells were transfected with increasing amounts of either pEF1A-NS5A-Myc-His6 or pEF1A-NS5B-Myc-His6. Overexpression of NS5A-Myc-His6, but not NS5B-Myc-His6, significantly reduced DGAT1 protein (Fig. 3F, upper panels). These results indicate that NS5A protein specifically reduces endogenous DGAT1 protein.
HCV infection induces the lysosomal degradation of DGAT1 protein. () Huh-7.5 cells were infected with HCV J6/JFH1 at an MOI of 2. Cells were cultured and harvested at 2, 4, and 6 days post-infection. Cells were analyzed by immunoblotting with anti-DGAT1, anti-NS5A, and anti-β-actin mAbs. The level of β-actin served as a loading control. The relative levels of the proteins were quantified by densitometry and are indicated below the respective lanes. Mock cells served as negative controls. () Huh-7.5 cells were infected with HCV J6/JFH1 at an MOI of 2. Cells were cultured and harvested at the indicated times. Total cellular RNA was extracted, and the levels of DGAT1 mRNA were quantified by the real-time quantitative RT-PCR. The amounts of DGAT1 mRNA were normalized to the amounts of GAPDH mRNA. Mock cells served as negative controls. Data represent the mean ± SEM obtained from three independent experiments performed in triplicate. () Huh-7.5 cells were infected with HCV J6/JFH1 at an MOI of 2. At 3 days post-infection, 25 μM MG132 was administered to the cells. Cells were cultured for 3, 6, and 9 h, harvested, and analyzed by immunoblotting as indicated. The level of β-actin served as a loading control. The relative levels of the proteins were quantified by densitometry and are indicated below the respective lanes. DMSO: dimethyl sulfoxide. () Huh-7.5 cells were infected with HCV J6/JFH1 at an MOI of 2. At 3 days post-infection, 10 mM NHCl was administered to the cells. Cells were cultured for 12 h, harvested, and analyzed by immunoblotting as indicated. The relative levels of the proteins were quantified by densitometry and are indicated below the respective lanes. () Huh-7.5 cells were infected with HCV J6/JFH1 at an MOI of 2. At 4 days post-infection, 2 mM 3-MA was administered to the cells. Cells were harvested, and analyzed by immunoblotting as indicated. The relative levels of the proteins were quantified by densitometry and are indicated below the respective lanes. 3-MA: 3-methyladenine. () Huh-7.5 cells were plated and cultured for 12 h. Cells were transfected with increasing amounts of either NS5A plasmid or NS5B plasmid as indicated. At 48 h post-transfection, cells were harvested. Cell lysates were analyzed by immunoblotting with anti-DGAT1 and anti-c-myc mAbs. The relative levels of protein expression were quantitated by densitometry and are indicated below the respective lanes. (A, C-F) The western blots are representative of three independent experiments. The densitometry data corresponds to the representative blot. A B C D E F 4
The KFERQ motif of DGAT1 is required for the interaction with HSC70
DGAT1 protein interacts with HSC70 via the KFERQ motif. () Schematic representation of DGAT1 protein and its amino acid sequences. The KFERQ motif resides in the region ranging from aa 149 to aa 153 of DGAT1 protein. An expression plasmid for FLAG-DGAT1 Q149A was constructed. () Huh-7.5 cells were plated and cultured for 12 h. Cells were transfected with pCAG-FLAG-DGAT1 or pCAG-FLAG-DGAT1 Q149A. At 48 h post-transfection, cells were harvested and cell lysates were immunoprecipitated with anti-HSC70 mouse mAb. Input samples and immunoprecipitated samples were analyzed by immunoblotting with anti-HSC70 mouse mAb and anti-FLAG mouse mAb. The western blots are representative of three independent experiments. A B
HCV NS5A is colocalized with DGAT1 in the late endosome and the lysosome
HCV NS5A is colocalized with DGAT1 in the late endosome and the lysosome. () Huh-7.5 cells were infected with HCV J6/JFH1 at an MOI of 2. At 4 days post infection, 10 mM NHCl was administered to the cells for 12 hours. The cells were stained with anti-DGAT1 mAb followed by Alexa Fluor 405-conjugated goat anti-mouse IgG (), anti-NS5A pAb followed by Alexa Fluor 488-conjugated goat anti-rabbit IgG (), and LysoTracker (). The stained cells were examined using a Zeiss LSM 700 scanning laser confocal microscope and image software. Scale bars, 10 μm. () Huh-7.5 cells were infected with HCV J6/JFH1 at an MOI of 2. At 3 days post infection, cells were transfected with pDsRed-Monomer-Rab7-C3 plasmid (). At 12 h post-transfection, 10 mM NHCl was administered to the cells for 12 hours. The cells were stained with anti-DGAT1 mAb followed by Alexa Fluor 405-conjugated goat anti-mouse IgG (), anti-NS5A pAb followed by Alexa Fluor 488-conjugated goat anti-rabbit IgG (). The stained cells were examined using a Zeiss LSM 700 scanning laser confocal microscope and image software. Scale bars, 10 μm. () Huh-7.5 cells were infected with HCV J6/JFH1 at an MOI of 2. At 3 days post infection, cells were transfected with pLEF-GFP-LC3 (). At 12 h post-transfection, 10 mM NHCl was administered to the cells for 12 hours. The cells were stained with anti-DGAT1 mAb followed by Alexa Fluor 405-conjugated goat anti-mouse IgG (), anti-NS5A pAb followed by Alexa Fluor 594-conjugated goat anti-rabbit IgG (). The stained cells were examined using a Zeiss LSM 700 scanning laser confocal microscope and image software. Scale bars, 10 μm. The images are representative of three independent experiments. A B C 4 4 4 blue green red red blue green green blue red
HCV-induced degradation of DGAT1 protein was restored by knockdown of VPS4B but not LAMP-2A
To investigate whether HCV induces DGAT1 protein degradation via eMI, we made stable VPS4A-knockdown cells and VPS4B-knockdown cells using shRNA. DGAT1 protein was recovered in shVPS4B-Huh-7.5 cells (Fig. 6B, upper panel, lane 6). On the other hand, DGAT1 protein was not recovered in VPS4A- Huh-7.5 cells (Fig. 6B, upper panel, lane 4). These results suggest that HCV induces the lysosomal degradation of DGAT1 protein via eMI and that VPS4B plays an important role in eMI.
The HCV infection-induced reduction of DGAT1 is restored in VPS4B knockdown cells. () shControl and shLAMP2A Huh-7.5 cells were infected with HCV J6/JFH1 at an MOI of 2. At 4 days post-infection, the cells were harvested and the cell lysates were analyzed by immunoblotting with anti-DGAT1 mAb, anti-LAMP-2A pAb, anti-NS5A pAb, and anti-β-actin mAb. The level of β-actin served as a loading control. The relative levels of the proteins were quantified by densitometry and are indicated below the respective lanes. Mock cells served as negative control. () shControl, shVPS4A, and shVPS4B Huh-7.5 cells were infected with HCV J6/JFH1 at an MOI of 2. At 4 days post-infection, the cells were harvested, and the cells lysates were analyzed by immunoblotting with anti-DGAT1 mAb, anti-VPS4A pAb, anti-VPS4B mAb, anti-NS5A pAb, and anti-β-actin mAb. The level of β-actin served as a loading control. The relative levels of the proteins were quantified by densitometry and are indicated below the respective lanes. Mock cells served as a negative control. The western blots are representative of three independent experiments. The densitometry data corresponds to the representative blot. A B
A proposed mechanism of the HCV NS5A-induced lysosomal degradation of DGAT1 via eMI. HCV NS5A interacts with HSC70 and recruits HSC70 to DGAT1 protein through an interaction between NS5A and DGAT1. HSC70 interacts with DGAT1 at its KFERQ motif. Protein complexes are delivered to the late endosomes (MVB) by HSC70 and bind to phosphatidylserine (PS) of the endosomal membrane. HSC70 is internalized along with DGAT1 into microvesicles via the coordinating function of ESCRT I (TSG101), ESCRT III (VPS4B), and accessory protein (Alix). The degradation of microvesicles occurs in the lysosome via endosome-lysosome fusion. ESCRT: endosomal sorting complex required for transport, MVB: multivesicular bodies. NS5A interacts with HSC70 and HNF-1α. HSC70 is recognized by LAMP-2A, a lysosomal membrane protein. HNF-1α is degraded via CMA.
Discussion
We have reported that HCV NS5A protein promotes the lysosomal degradation of HNF-1α via CMA through an NS5A-mediated association of HNF-1α with cellular chaperone HSC70 protein [7,24,25]. To seek a novel target of HCV-induced CMA, we focused on DGAT1 among the NS5A-interacting proteins. However, to our surprise, we discovered that HCV NS5A protein promotes the lysosomal degradation of DGAT1 via eMI, but not CMA, using NS5A, HSC70, and KFERQ motif on DGAT1.
The immunoprecipitation analysis revealed that DGAT1 protein was co-immunoprecipitated with HCV NS5A protein. The immunofluorescent staining and the results of the PLA suggest that NS5A protein interacted with DGAT1 protein in Huh-7.5 cells (Fig. 1). We mapped the DGAT1-binding domain on NS5A protein and found that the region spanning from aa 1 to aa 213 is important for the interaction with DGAT1. We also mapped the NS5A-binding domain on DGAT1 protein and found that the region spanning from aa 157 to aa 399 is important for the interaction with NS5A (Fig. 2). Using the lysosomal protease inhibitor NH4Cl, we demonstrated that DGAT1 is degraded via an NS5A-dependent lysosomal degradation pathway. Treatment with 3-MA did not recover DGAT1 protein level, suggesting that macroautophagy is not involved in the reduction of DGAT1 protein (Fig. 3).
We demonstrated that DGAT1 protein contains a KFERQ motif in the region spanning from aa 149 to aa 153. The immunoprecipitation analysis revealed that the KFERQ motif is crucial for the interaction between DGAT1 and HSC70 (Fig. 4). Immunofluorescence staining showed that DGAT1 protein and NS5A protein was colocalized in the late endosome and the lysosome (Fig. 5). However, the HCV-induced degradation of DGAT1 protein was restored in shVPS4B Huh-7.5 cells, but not in shLAMP-2A Huh-7.5 cells (Fig. 6). Taken together, these results suggest that HCV NS5A protein interacts with HSC70 and DGAT1, thereby promoting the lysosomal degradation of DGAT1 via eMI in a VPS4B-dependent manner (Fig. 7). To our knowledge, this is the first report clarifying the molecular mechanism of eMI induced by HCV infection.
HCV NS5A is a large phosphoprotein (56–58 kDa) organized into three domains. NS5A domain I is essential for HCV RNA replication and is relatively conserved among HCV genotypes compared to domains II and III, suggesting that an NS5A-DGAT1 interaction is common to all HCV genotypes [26,27]. With the use of a series of NS5A deletion mutants (Fig. 2A), our findings demonstrated that domain I of NS5A is important for the association with DGAT1 protein (Fig. 2B). DGAT1 is an integral membrane protein synthesizing triacylglycerides from diacylglycerol and fatty acyl-CoA. DGAT1 has nine transmembrane helices, TM1–TM9, and three long loops [26]. With a series of DGAT1 deletion mutants (Fig. 2C), the results demonstrated that the region spanning from aa 157 to aa 399 of DGAT1 protein is important for the association with the NS5A protein (Fig. 2D–F).
The pentapeptide 149QVEKR153 of DGAT1 completely matches the rules for the KFERQ motif. A coimmunoprecipitation result showed that endogenous HSC70 interacted with wildtype DGAT1 protein, but not with DGAT1 Q149A mutant (Fig. 4B), emphasizing the importance of the KFERQ motif in target protein for the interaction with HSC70. We reported that the region spanning from aa 1 to aa 126 of NS5A protein is important for the interaction with HSC70 [7].
In eMI, substrate proteins in vesicles are degraded in the late endosome or through the fusion with the lysosomes (endolysosome) [20, 21]. Therefore, the HCV NS5A may be colocalized with DGAT1 protein in both the endosome and the lysosome. It is still unclear whether the entire ESCRT machinery is necessary for the eMI pathway [19-21]. The substrate protein’s intrinsic properties may be responsible for switching between CMA and eMI pathways. Because these two pathways require distinct receptors to transport the target protein to the lysosome, knocking down each receptor aids in the analysis of protein degradation; for example, knocking down the LAMP-2A membrane protein increases the amount of target protein in the CMA pathway. On the other hand, when the VPS4A/B protein is knocked down, the substrate protein level increases in the eMI pathway [14,21]. In our present study, the DGAT1 protein expression level was not recovered in HCV-infected shLAMP-2A cells, suggesting that the CMA pathway is not involved in the degradation of DGAT1 protein. The DGAT1 protein expression level was recovered only in shVPS4B-Huh-7.5 cells, suggesting that HCV NS5A induced the lysosomal degradation of DGAT1 protein via eMI in a VPS4B-dependent manner. However, further experiments are needed to understand how substrate specificity is determined between CMA and eMI.
DGAT1 interacts with HCV core protein and is required for the trafficking of the core to lipid droplets [22]. DGAT1 localizes NS5A protein to lipid droplets and enhances the NS5A-core interaction to promote the production of viral particles [23]. Our present findings provide evidence suggesting that HCV promotes the lysosomal degradation of DGAT1 protein via eMI. HCV infection paradoxically downregulates DGAT1 protein in the cells. We are currently seeking to determine the physiological significance of HCV-induced DGAT1 degradation in HCV life cycle. In our preliminary result, we observed that overexpression of DGAT1 protein resulted in decrease of extracellular HCV infectivity titers, although there were no significant differences in intracellular and extracellular HCV RNA levels and intracellular HCV infectivity titers between control cells and DGAT1-overexpressed cells (data not shown). We speculate that removal of too much DGAT1 protein or quality control of DGAT1 protein via eMI may be important for efficient production of infectious HCV particles. Further research is necessary to elucidate a pathophysiological role of the HCV-induced lysosomal degradation of DGAT1 protein via eMI.
DGAT1 plays an important role in the final step of triglyceride synthesis. Human DGAT1 is expressed primarily in the small intestine and liver. A study using DGAT1-deficient Huh-7.5 cell lines demonstrated dedifferentiated and stem cell-like characteristics in stem cell culture medium without serum. The complete and long-term silencing of DGAT1 decreased E-cadherin and integrin β1, an adhesion molecule that contributes to the cell-extracellular matrix or cell-substrate adhesion, similar to the process of liver cirrhosis without fatty degeneration [28,29]. It may be informative to investigate the pathogenesis of HCV-induced DGAT1 downregulation in terms of liver cirrhosis.
In conclusion, we propose that HCV NS5A interacts with HSC70 and recruits HSC70 to DGAT1, thereby promoting the lysosomal degradation of DGAT1 via eMI. Further investigations of the HCV-induced selective degradation of host proteins via CMA and eMI may contribute to our understanding of the pathogenesis of HCV.
Materials And Methods
Cell culture and viruses. A human hepatoma cell line, Huh-7.5 cells, was provided by Dr. Charles M. Rice (The Rockefeller University, New York, NY)[30]. The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (high glucose) with L-glutamine and phenol red (Fuji Film Wako Pure Chemical Industries, Ltd., 044-29765) and supplemented with 50 IU/ml penicillin, 50 μg/ml streptomycin (Gibco, 15-140-122), 10% heat-inactivated fetal bovine serum (Biowest, S1760-500), and 0.1 mM nonessential amino acids (Invitrogen, 11140050) at 37°C in a 5% CO2 incubator. Cells were washed using PBS (-) solution (Nissui Pharmaceutical Co., Ltd., 05913). Cells were transfected with plasmid DNA using FuGENE 6 transfection reagents (Promega, E269A). The pFL-J6/JFH1 plasmid, which encodes the entire viral genome of a chimeric strain of HCV-2a, J6/JFH1 was provided by Dr. C.M. Rice [5]. The HCV genome RNA was synthesized in vitro using pFL-J6/JFH1 as a template and was transfected into Huh-7.5 cells by electroporation [4,5,31,32]. The virus produced in the culture supernatant was used for infection experiments [31].
Expression plasmids. The expression plasmids for NS5A and a series of NS5A deletion mutants constructed as HA-tagged or Myc-His6-tagged proteins have been described [7,24,25,33]. The expression plasmids pEF-FLAG-NS5A [34], pDsRed-Monomer-Rab7-C3 [35], and pLEF-GFP-LC3 [36] were kindly provided by Dr. Y. Matsuura (Osaka University, Osaka, Japan). The expression plasmid for HSC70 has been described [7]. The cDNA fragment of DGAT1 was amplified by PCR using pCMV6-XL4-DGAT1 as a template. The specific primers used for PCR were as follows: sense primer 5’-TCGAGCTCAGCGGCCATGGGCGACCGCGGCAGC-3’ and anti-sense primer 5’-AGTGAATTCGCGGCCTCAGGCCTCTGCCGCTGG-3’.
The amplified PCR product was purified and inserted into NotI site of pCAG-FLAG using an In-Fusion HD cloning kit (Takara Bio USA, Inc., 639649). The Q149A point mutant of DGAT1 was constructed by overlap extension PCR using pCAG-FLAG-DGAT1 as a template. The specific primer used for PCR were as follows: sense primer (Q149A) 5’-GTGGCTGCATTCCGGGTTGAGAAGCGCCTG-3’ and anti-sense primer (Q149A), 5’CAGGCGCTTCTCAACCCGGAATGCAGCCAC-3’. The sequences of the inserts were extensively verified by sequencing (Eurofins Genomics, Tokyo).
Antibodies and reagents. The mouse monoclonal antibodies (mAbs) used in this study were anti-FLAG (M2) mAb (Sigma-Aldrich, F-3165), anti-HSC70 (B-6) mAb (Santa Cruz Biotechnology, sc-7298), anti-c-Myc (9E10) mAb (Santa Cruz Biotechnology, sc-40), anti-ß-actin mAb (Sigma-Aldrich, A-5441), anti-DDDDK-tag mAb (MBL International Corporation, M185-3L), anti-VPS4B (A-11) mAb (Santa Cruz Biotechnology, sc-377162) and anti DGAT1 (A-5) mAb (Santa Cruz Biotechnology, sc-271934).
The rabbit polyclonal antibodies (pAbs) used in this study were anti-HA pAb (Sigma-Aldrich, H-6908), anti-LAMP2A pAb (Abcam, ab18528), anti-DDDDK-tag pAb (MBL International Corporation, PM020), anti-VPS4A (UT289) pAb (Millipore, ABS1646), anti-NS5A (2914-1) pAb (a kind gift from T. Wakita, National Institute of Infectious Diseases, Tokyo), anti-LC3 (PM036) pAb (MBL Life Science, PM036), and anti-p62 (Cell Signaling Technology, 5114). Horseradish peroxidase (HRP)-conjugated anti-mouse IgG (Cell Signaling Technology, 7076S) and HRP-conjugated anti-rabbit IgG (Cell Signaling Technology, 7074S) were used as secondary antibodies. MG132 was purchased from Fuji Film Wako Pure Chemical Industries, Ltd., 135-18453. Ammonium chloride (NH4Cl) was purchased from Fuji Film Wako Pure Chemical Industries, Ltd., 017-02995. The macroautophagy inhibitor 3-MA was purchased from Sigma-Aldrich Co., LLC., M9281.
Immunoblot analysis. The immunoblot (IB) analysis was performed essentially as described [24,37-39]. The cell lysates were separated by 10% or 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a 0.45 μm Immobilon-P polyvinylidene difluoride membrane (PVDF) (Millipore, IPVH00010). The membranes were incubated with a primary antibody, followed by incubation with a peroxidase-conjugated secondary antibody. The positive bands were visualized using Amersham enhanced chemiluminescence (ECL) western blotting detection reagents (Cytiva, RPN2106). The intensity of bands was quantified using ImageJ 1.53r software.
Immunoprecipitation. Cultured cells were lysed with a buffer containing 150 mM NaCl, 50 mM Tris-HCl (pH 7.5), 1% NP-40, 1 mM EDTA, 100% glycerol, and cOmplete™, EDTA-free, protease inhibitor cocktail (Roche Diagnostics, 05056489001). The lysate was centrifuged at 12,500 g for 15 min at 4°C, and the supernatant was immunoprecipitated with appropriate antibodies. Immunoprecipitation (IP) was performed as described [7,37]. Briefly, the cell lysates were immunoprecipitated with anti-FLAG M2 affinity gel (Sigma-Aldrich Co., A2220) or Protein A-Sepharose 4 Fast Flow (GE Healthcare, GE17-5280-04) incubated with appropriate antibodies at 4°C for 4 h. After being washed with the lysis buffer five times, the immunoprecipitants were analyzed by immunoblotting.
RNA interference and stable knockdown cells. The short hairpin RNA (shRNA) target sequences for LAMP-2A, VPS4A, VPS4B, and scramble were as follows:
shLAMP-2A, 5’- GGCAGGAGUACUUAUUCUA-3’,
shVPS4A, 5’-GCUGAAGGAUUAUUUACGA-3’,
shVPS4B, 5’-AGCGAUAGAUCUGGCUAGCAA-3’,
and shScramble, 5’-GGACAUCGACGGCUUUAUA-3’. The shVPS4A and shVPS4B were inserted into the pSilencer 2.1 U6 puro vector (Ambion, AM5762). The shLAMP2A was inserted into the pSilencer 2.1 U6 hygro vector (Ambion, AM5760). Huh-7.5 cells were transfected with the plasmids and drug-resistant clones were selected by 1 μg/ml puromycin (Sigma-Aldrich, P9620) or 200 μg/ml hygromycin B (Nacalai Tesque, 09287-84) to establish the stable knockdown cells for LAMP-2A, VPS4A, and VPS4B.
Immunofluorescence staining. Huh-7.5 cells cultured on glass coverslips were incubated with LysoTracker™ Deep Red (Invitrogen, DND-99) for 2 h at 37°C. The cells were fixed with 4% paraformaldehyde at room temperature (RT) for 15 min. After being washed with phosphate-buffered saline (PBS) (Nissui Pharmaceutical Co., Ltd., 05913), the cells were permeabilized for 15 min at RT with PBS containing 0.1% Triton X-100 and incubated in PBS containing 1% bovine serum albumin (BSA) (Nacalai Tesque, 01859-47) to block nonspecific reaction for 60 min. The cells were incubated with 1% BSA in PBS containing mouse anti-DGAT1 antibody and rabbit anti-NS5A antibody at RT for 60 min. The cells were washed three times with PBS and incubated with 1% BSA in PBS containing Alexa Fluor™ 488-conjugated anti-mouse IgG (Invitrogen, A11001), Alexa Fluor™ 488-conjugated anti-rabbit IgG (Invitrogen, A11008), Alexa Fluor™ 594-conjugated anti-rabbit IgG (Invitrogen, A11012), or Alexa Fluor™ 405-conjugated anti-mouse IgG (Invitrogen, A31553) at RT for 60 min. The cells were washed four times with PBS, mounted on glass slides, and examined with a confocal microscope (LSM 700) (Zeiss, Germany).
Proximity ligation assay (PLA). In situ PLA was performed using a Duolink In Situ PLA kit (Sigma-Aldrich, DUO92008) as described [33]. Briefly, Huh-7.5 cells were transfected with plasmid pEF1A-NS5A-Myc-His6 together with pCAG-FLAG-DGAT1 and cultured. At 48 h after transfection, cells grown on poly-D-lysine-coated glass coverslips were fixed with 4% paraformaldehyde for 15 min at RT and then permeabilized with PBS containing 0.1% Triton X-100 for 15 min at RT.
The coverslips were incubated with anti-c-Myc mouse mAb and anti-FLAG rabbit pAb. The samples were washed three times with the wash buffer from the kit. The PLA probes anti-Mouse PLUS (Sigma-Aldrich, DUO92001) and anti-Rabbit MINUS (Sigma-Aldrich, DUO92005), were diluted in the antibody diluent provided with the kit. The samples were incubated for 1 h at 37°C in a humidity chamber. The samples were washed and processed according to the manufacturer’s instructions for probe ligation, signal amplification, and mounting. The samples were examined with a confocal microscope (LSM 700) (Zeiss, Germany).
Real-time quantitative reverse transcription-PCR (RT-PCR). The total cellular RNA was isolated using a ReliaPrep RNA cell miniprep system (Promega, Z6012) according to the manufacturer’s instructions. The cDNA was generated using a GoScript reverse transcription system (Promega, A5001). The real-time quantitative RT-PCR was performed using TB Green Premix Ex Taq II (Tli RNaseH Plus) (Takara Bio, RR820A) with SYBR green chemistry on the StepOnePlus real-time PCR system (Applied Biosystems, USA) as described previously [24]. The primer sequences were as follows: DGAT1, 5’-GGACTACTCACGCATCATCG-3’ and 5’-GGTCTCCAAACTGCATGAGC-3’. As an internal control, human GAPDH gene expression levels were measured using the primer 5’-GCCATCAATGACCCCTTCATT-3’ and 5’-TCTCGCTCCTGGAAGATGG-3’.