Po-Yuan Ke

Activation of the unfolded protein response and autophagy after hepatitis C virus infection suppresses innate antiviral immunity in vitro.

Autophagy, a process for catabolizing cytoplasmic components, has been implicated in the modulation of interactions between RNA viruses and their host. However, the mechanism underlying the functional role of autophagy in the viral life cycle still remains unclear. Hepatitis C virus (HCV) is a single-stranded, positive-sense, membrane-enveloped RNA virus that can cause chronic liver disease. Here we report that HCV induces the unfolded protein response (UPR), which in turn activates the autophagic pathway to promote HCV RNA replication in human hepatoma cells. Further analysis revealed that the entire autophagic process through to complete autolysosome maturation was required to promote HCV RNA replication and that it did so by suppressing innate antiviral immunity. Gene silencing or activation of the UPR-autophagy pathway activated or repressed, respectively, IFN-β activation mediated by an HCV-derived pathogen-associated molecular pattern (PAMP). Similar results were achieved with a PAMP derived from Dengue virus (DEV), indicating that HCV and DEV may both exploit the UPR-autophagy pathway to escape the innate immune response. Taken together, these results not only define the physiological significance of HCV-induced autophagy, but also shed light on the knowledge of host cellular responses upon HCV infection as well as on exploration of therapeutic targets for controlling HCV infection.

Mitotic degradation of human thymidine kinase 1 is dependent on the anaphase-promoting complex/cyclosome-Cdh1-mediated pathway

ABSTRACT The expression of human thymidine kinase 1 (hTK1) is highly dependent on the growth states and cell cycle stages in mammalian cells. The amount of hTK1 is significantly increased in the cells during progression to the S and M phases, and becomes barely detectable in the early G1 phase by a proteolytic control during mitotic exit. This tight regulation is important for providing the correct pool of dTTP for DNA synthesis at the right time in the cell cycle. Here, we investigated the mechanism responsible for mitotic degradation of hTK1. We show that hTK1 is degraded via a ubiquitin-proteasome pathway in mammalian cells and that anaphase-promoting complex/cyclosome (APC/C) activator Cdh1 is not only a necessary but also a rate-limiting factor for mitotic degradation of hTK1. Furthermore, a KEN box sequence located in the C-terminal region of hTK1 is required for its mitotic degradation and interaction capability with Cdh1. By in vitro ubiquitinylation assays, we demonstrated that hTK1 is targeted for degradation by the APC/C-Cdh1 ubiquitin ligase dependent on this KEN box motif. Taken together, we concluded that activation of the APC/C-Cdh1 complex during mitotic exit controls timing of hTK1 destruction, thus effectively minimizing dTTP formation from the salvage pathway in the early G1 phase of the cell cycle in mammalian cells.

Hepatitis C Virus and Cellular Stress Response: Implications to Molecular Pathogenesis of Liver Diseases

Infection with hepatitis C virus (HCV) is a leading risk factor for chronic liver disease progression, including steatosis, cirrhosis, and hepatocellular carcinoma. With approximately 3% of the human population infected worldwide, HCV infection remains a global public health challenge. The efficacy of current therapy is still limited in many patients infected with HCV, thus a greater understanding of pathogenesis in HCV infection is desperately needed. Emerging lines of evidence indicate that HCV triggers a wide range of cellular stress responses, including cell cycle arrest, apoptosis, endoplasmic reticulum (ER) stress/unfolded protein response (UPR), and autophagy. Also, recent studies suggest that these HCV-induced cellular responses may contribute to chronic liver diseases by modulating cell proliferation, altering lipid metabolism, and potentiating oncogenic pathways. However, the molecular mechanism underlying HCV infection in the pathogenesis of chronic liver diseases still remains to be determined. Here, we review the known stress response activation in HCV infection in vitro and in vivo, and also explore the possible relationship of a variety of cellular responses with the pathogenicity of HCV-associated diseases. Comprehensive knowledge of HCV-mediated disease progression shall shed new insights into the discovery of novel therapeutic targets and the development of new intervention strategy.

Autophagy: A novel guardian of HCV against innate immune response

Autophagy is an evolutionarily conserved process that catabolizes intracellular components and maintains cellular homeostasis. Autophagy involves the sequestration of cytoplasmic content within a double-membraned autophagosome, and the fusion of the autophagosome with a lysosome to form an autolysosome for subsequent degradation (Fig. 1A). Autophagy plays a pivotal role in various aspects of cellular responses to stresses, such as nutrient deprivation, damaged organelles, aggregated proteins, exposure to endoplasmic reticulum (ER) stress and pathogen infections. Virus infection often leads to ER stress and induction of the unfolded protein response (UPR). Recent studies reveal that virus-induced UPR may activate autophagy to support the virus life cycle. However, the exact roles of the UPR and autophagy in host cell-virus interactions are still enigmatic.

Chromosome 19 open reading frame 80 is upregulated by thyroid hormone and modulates autophagy and lipid metabolism

The thyroid hormone, T3, regulates cell growth, differentiation and development through binding to the nuclear thyroid hormone receptor (THR), a member of the steroid/TR superfamily of ligand-dependent transcriptional factors. T3 modulates lipid metabolism in liver, although the detailed molecular mechanisms are unclear at present. Here, by a microarray analysis, we identified a novel chromosome 19 open reading frame 80 (C19orf80) which was activated by T3. T3 stimulation led to upregulation of both mRNA and protein levels of C19orf80. Immunofluorescence analysis revealed a vesicle-like pattern of C19orf80 around lipid droplets or within the lysosome-associated compartment in cells. Furthermore, T3 treatment as well as C19orf80 overexpression specifically activated the autophagic response and lipid metabolism, as observed from lipidated LC3 (LC3-II) and levels of oxygen consumption rate, respectively. Reciprocally, knockdown of C19orf80 obstructed T3-activated autophagy and lipolysis. Moreover, treatment with autolysosome maturation inhibitors, ammonium chloride and chloroquine, not only suppressed the T3-activated autophagic process but also lipid metabolism. Our results collectively suggested that T3 regulates lipid metabolism through a C19orf80-activated autophagic process.

Identification of the LWYIK Motif Located in the Human Immunodeficiency Virus Type 1 Transmembrane gp41 Protein as a Distinct Determinant for Viral Infection

ABSTRACT The highly conserved LWYIK motif located immediately proximal to the membrane-spanning domain of the gp41 transmembrane protein of human immunodeficiency virus type 1 has been proposed as being important for the surface envelope (Env) glycoproteins association with lipid rafts and gp41-mediated membrane fusion. Here we employed substitution and deletion mutagenesis to understand the role of this motif in the virus life cycle. None of the mutants examined affected the synthesis, precursor processing, CD4 binding, oligomerization, or cell surface expression of the Env, nor did they alter Env incorporation into the virus. All of the mutants, particularly the ΔYI, ΔIK, and ΔLWYIK mutants, in which the indicated residues were deleted, exhibited greatly reduced one-cycle viral replication and the Env trans-complementation ability. All of these deletion mutant proteins were still localized in the lipid rafts. With the exception of the Trp-to-Ala (WA) mutant, which exhibited reduced viral infectivity albeit with normal membrane fusion, all mutants displayed loss of some or almost all of the membrane fusion ability. Although these deletion mutants partially inhibited in trans wild-type (WT) Env-mediated fusion, they were more effective in dominantly interfering with WT Env-mediated viral entry when coexpressed with the WT Env, implying a role of this motif in postfusion events as well. Both T20 and L43L peptides derived from the two gp41 extracellular C- and N-terminal α-helical heptad repeats, respectively, inhibited WT and ΔLWYIK Env-mediated viral entry with comparable efficacies. Biotin-tagged T20 effectively captured both the fusion-active, prehairpin intermediates of WT and mutant gp41 upon CD4 activation. Env without the deletion of the LWYIK motif still effectively mediated lipid mixing but inhibited content mixing. Our study demonstrates that the immediate membrane-proximal LWYIK motif acts as a unique and distinct determinant located in the gp41 C-terminal ectodomain by promoting enlargement of fusion pores and postfusion activities.

Autophagy in hepatitis C virus-host interactions: Potential roles and therapeutic targets for liver-associated diseases

Autophagy is a lysosome-associated, degradative process that catabolizes cytosolic components to recycle nutrients for further use and maintain cell homeostasis. Hepatitis C virus (HCV) is a major cause of chronic hepatitis, which often leads to end-stage liver-associated diseases and is a significant burden on worldwide public health. Emerging lines of evidence indicate that autophagy plays an important role in promoting the HCV life cycle in host cells. Moreover, the diverse impacts of autophagy on a variety of signaling pathways in HCV-infected cells suggest that the autophagic process is required for balancing HCV-host cell interactions and involved in the pathogenesis of HCV-related liver diseases. However, the detailed molecular mechanism underlying how HCV activates autophagy to benefit viral growth is still enigmatic. Additionally, how the autophagic response contributes to disease progression in HCV-infected cells remains largely unknown. Hence, in this review, we overview the interplay between autophagy and the HCV life cycle and propose possible mechanisms by which autophagy may promote the pathogenesis of HCV-associated chronic liver diseases. Moreover, we outline the related studies on how autophagy interplays with HCV replication and discuss the possible implications of autophagy and viral replication in the progression of HCV-induced liver diseases, e.g., steatosis and hepatocellular carcinoma. Finally, we explore the potential therapeutics that target autophagy to cure HCV infection and its related liver diseases.

Reticulon 3 interacts with NS4B of the hepatitis C virus and negatively regulates viral replication by disrupting NS4B self-interaction

The non‐structural protein 4B (NS4B) of the hepatitis C virus (HCV) is an endoplasmic reticulum (ER) membrane protein comprising two consecutive amphipathic α‐helical domains (AH1 and AH2). Its self‐oligomerization via the AH2 domain is required for the formation of the membranous web that is necessary for viral replication. Previously, we reported that the host‐encoded ER‐associated reticulon 3 (RTN3) protein is involved in the formation of the replication‐associated membranes of (+)RNA enteroviruses during viral replication. In this study, we demonstrated that the second transmembrane region of RTN3 competed for, and bound to, the AH2 domain of NS4B, thus abolishing NS4B self‐interaction and leading to the downregulation of viral replication. This interaction was mediated by two crucial residues, lysine 52 and tyrosine 63, of AH2, and was regulated by the AH1 domain. The silencing of RTN3 in Huh7 and AVA5 cells harbouring an HCV replicon enhanced the replication of HCV, which was counteracted by the overexpression of recombinant RTN3. The synthesis of viral RNA was also increased in siRNA‐transfected human primary hepatocytes infected with HCV derived from cell culture. Our results demonstrated that RTN3 acted as a restriction factor to limit the replication of HCV.

Active RNA Replication of Hepatitis C Virus Downregulates CD81 Expression

So far how hepatitis C virus (HCV) replication modulates subsequent virus growth and propagation still remains largely unknown. Here we determine the impact of HCV replication status on the consequential virus growth by comparing normal and high levels of HCV RNA expression. We first engineered a full-length, HCV genotype 2a JFH1 genome containing a blasticidin-resistant cassette inserted at amino acid residue of 420 in nonstructural (NS) protein 5A, which allowed selection of human hepatoma Huh7 cells stably-expressing HCV. Short-term establishment of HCV stable cells attained a highly-replicating status, judged by higher expressions of viral RNA and protein as well as higher titer of viral infectivity as opposed to cells harboring the same genome without selection. Interestingly, maintenance of highly-replicating HCV stable cells led to decreased susceptibility to HCV pseudotyped particle (HCVpp) infection and downregulated cell surface level of CD81, a critical HCV entry (co)receptor. The decreased CD81 cell surface expression occurred through reduced total expression and cytoplasmic retention of CD81 within an endoplasmic reticulum -associated compartment. Moreover, productive viral RNA replication in cells harboring a JFH1 subgenomic replicon containing a similar blasticidin resistance gene cassette in NS5A and in cells robustly replicating full-length infectious genome also reduced permissiveness to HCVpp infection through decreasing the surface expression of CD81. The downregulation of CD81 surface level in HCV RNA highly-replicating cells thus interfered with reinfection and led to attenuated viral amplification. These findings together indicate that the HCV RNA replication status plays a crucial determinant in HCV growth by modulating the expression and intracellular localization of CD81.

RacGTPase-activating protein 1 interacts with hepatitis C virus polymerase NS5B to regulate viral replication

Hepatitis C virus (HCV) is a positive-strand RNA virus responsible for chronic liver disease and hepatocellular carcinoma (HCC). RacGTPase-activating protein 1 (RacGAP1) plays an important role during GTP hydrolysis to GDP in Rac1 and CDC42 protein and has been demonstrated to be upregulated in several cancers, including HCC. However, the molecular mechanism leading to the upregulation of RacGAP1 remains poorly understood. Here, we showed that RacGAP1 levels were enhanced in HCV cell-culture-derived (HCVcc) infection. More importantly, we illustrated that RacGAP1 interacts with the viral protein NS5B in mammalian cells. The small interfering RNA (siRNA)-mediated knockdown of RacGAP1 in human hepatoma cell lines inhibited replication of HCV RNA, protein, and production of infectious particles of HCV genotype 2a strain JFH1. Conversely, these were reversed by the expression of a siRNA-resistant RacGAP1 recombinant protein. In addition, viral protein NS5B polymerase activity was significantly reduced by silencing RacGAP1 and, vice versa, was increased by overexpression of RacGAP1 in a cell-based reporter assay. Our results suggest that RacGAP1 plays a crucial role in HCV replication by affecting viral protein NS5B polymerase activity and holds importance for antiviral drug development.