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Mediators of Inflammation
Volume 2019, Article ID 3124745, 12 pages
The Ubiquitin-Specific Protease 18 Promotes Hepatitis C Virus Production by Increasing Viral Infectivity
1Institute of Blood Transfusion, Chinese Academy of Medical Sciences, Peking Union Medical College, Chengdu, Sichuan 610052, China
2Toronto General Research Institute, University of Toronto, Toronto, Ontario, Canada M5S 1A1
3Department of Infectious Diseases, The 1st Affiliated Hospital of Chongqing Medical University, Chongqing, China
4Department of Microbiology and Immunology, School of Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan
5Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University, Taipei, Taiwan
6Department of Microbiology & Immunology, Dalhousie University, Halifax, Nova Scotia, Canada B3H 4R2
7Department of Medicine, University of Toronto, Toronto, Canada M5S 1A1
8Department of Surgery, University of Toronto, Toronto, Canada M5S 1A1
Correspondence should be addressed to Ian D. McGilvray; firstname.lastname@example.com and Limin Chen; moc.oohay@99_nehc_nimil
Received 20 May 2019; Revised 13 September 2019; Accepted 3 October 2019; Published 18 November 2019
Academic Editor: Oleh Andrukhov
Copyright © 2019 Yujia Li et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background and Aims. Ubiquitin-specific protease 18 (USP18) is involved in immunoregulation and response to interferon- (IFN-) based treatment in patients chronically infected with hepatitis C virus (HCV). We investigated whether and how its upregulation alters HCV infection. Methods. Overexpression of wild-type (USP18 WT) or catalytically inactive mutant (USP18 C64S) USP18 was examined for effects on HCV replication in the absence and presence of IFNα or IFNλ using both the HCV-infective model and replicon cells. The IFN signaling pathway was assessed via STAT1 phosphorylation (western blot) and downstream ISG expression (real-time PCR). Mechanistic roles were sought by quantifying microRNA-122 levels and J6/JFH1 infectivity of Huh7.5 cells. Results. We found that overexpression of either USP18 WT or USP18 C64S stimulated HCV production and blunted the anti-HCV effect of IFNα and IFNλ in the infective model but not in the replicon system. Overexpressed USP18 showed no effect on Jak/STAT signaling nor on microRNA-122 expression. However, USP18 upregulation markedly increased J6/JFH1 infectivity and promoted the expression of the key HCV entry factor CD81 on Huh7.5 cells. Conclusions. USP18 stimulates HCV production and blunts the effect of both type I and III IFNs by fostering a cellular environment characterized by upregulation of CD81, promoting virus entry and infectivity.
1. Introduction
As one of the key effectors in the innate immune system, type I interferon (IFN) plays diverse roles in host defense against virus infection and has been recommended or studied as important/potential therapy in patients with virus infections such as hepatitis B virus (HBV) [1], hepatitis C virus (HCV) [2], hepatitis D virus (HDV) [3], and enterovirus 71 [4]. Unfortunately, type I IFN signaling is a "double-edged sword" [5], because it confers complicated action by regulating the expression of various interferon-stimulated genes (ISGs), which on the one hand control virus replication but on the other hand are involved in persistent viral infection. It is such an important target for which viruses, such as HCV, developed special survival strategies to evade host immune attack and benefit their replication. Thus, investigating how interferon signaling and effector mechanisms are altered in viral infection is critical to understand the intricate host-virus interaction.
Before 2011 when revolutionary direct-acting antivirals (DAAs) were developed, IFN-based therapy remained the most effective treatment for HCV infection. However, it is very challenging to choose an appropriate therapy strategy for every patient because their responses to the IFN-based treatment varied a lot. We have described a dichotomous hepatic gene expression that is linked to treatment response [6, 7]: patients with high expression of a subset of ISGs in hepatocytes were resistant to subsequent treatment with pegylated IFNα/ribavirin (PegIFN/Rib), while the patients with much lower expression of hepatic ISGs were very responsive to treatment with PegIFN/Rib. This nonresponder phenotype characterized by discrete patterns of "high ISG" expression has subsequently been confirmed by a number of laboratories [8, 9]. Three of the genes in the hepatocyte-expressed "high-ISG" subset are linked in the same ubiquitin-like biochemical pathway: interferon-stimulated gene 15 (ISG15), cyclin E-binding protein (Herc5/CEB1), and ubiquitin-specific protease 18 (USP18). In this pathway, ISG15 is covalently linked to target proteins by a tightly regulated series of E1/E2/E3 proteins: activating E1 enzyme (Ube1L), conjugating E2 enzyme (UbcH8), and E3 ligase (CEB1, Efp) [10]. ISG15 is cleaved from its targets by the USP18 cysteine protease. The consequences of protein ISGylation (the process of ISG15 conjugation to its target proteins) are currently under investigation, but the process clearly has implications for viral infection in a manner that is specific to the virus in question. ISG15 has antiviral activity for some viruses, such as influenza A and B viruses, herpesviruses, and Sindbis viruses, whereas for HIV, the ISGylation of the HIV gag protein is required for HIV viral egress from the cell [11]. ISGylation may also, in some circumstances, function as a negative regulator of the innate immune response by conjugating to intracellular viral sensor molecules, such as retinoic acid-inducible gene I (RIG-I) to promote viral replication [12].
The ISG15/USP18 pathway is likely to play a role in viral hepatitis, again in a virus-specific manner [13]. Kim et al. found that hepatitis B viral replication was not affected by loss of ISGylation in Ube1L-/- mice [14], while we and others found that ISGylation stimulates HCV replication in vitro [15, 16]. USP18's role in viral hepatitis may reflect an underlying effect in the innate immune response. For example, the CD169+ metallophilic macrophages with upregulated USP18 reduced IFN-induced capture of virus particles, allowing locally restricted replication of vesicular stomatitis virus (VSV) [17]. USP18-knockout mice experienced pronounced increases in protein ISGylation and were resistant to infection by lymphocytic choriomeningitis virus (LCMV), VSV, Sindbis virus, HIV, and other viral infection involving the ISGylation process (reviewed in [18]). We have previously shown that downregulation of USP18 augments the anti-HCV effect of IFNα [19]. Originally, we attributed this effect uniquely to an exaggeration of IFN signaling, but the data described above raised the possibility that the role of USP18 in HCV might be more complicated than previously thought.
In this study, we used a human full-length USP18 wild type and an enzymatically inactive mutant to dissect the role of USP18 in the molecular pathogenesis of HCV. Our findings demonstrated that USP18 could create a cellular milieu that favored HCV and stimulated HCV production in a manner that was independent of ISG15.
2. Materials and Methods
2.1. Cell Line, J6/JFH Culture Model, and Plasmid DNA Transfection
Huh7.5 cells and the HCV infectious clone J6/JFH1, the full-length chimerical genome from the infectious JFH1 (genotype 2a) isolated from a Japanese patient with fulminant hepatitis [20], were kindly provided by Dr. Charles Rice (Rockefeller University, New York). Briefly, the J6/JFH1 RNA transcript was generated and electroporated into Huh7.5 cells. The culture supernatant was collected and applied to naive Huh7.5 cells for viral passage [21]. Because of abolished virus-induced IFN production resulting from RIG-I mutation, Huh7.5 cells can support complete and efficient HCV replication [22]. HCV genotype 1b (Con1b; AB12-A2) and genotype 2a (JFH1; sbJFH1-B2) subgenomic replicon-containing Huh-7 cells were also used [23]. The AB12-A2 cell line is a Huh-7.5 line carrying subgenomic full-length HCV genotype 1b replicon, and the sbJFH1-B2 cell line is a Huh-7 line cell population containing HCV JFH1 RNA. The replicon cell lines were maintained in DMEM supplemented with 10% fetal calf serum (FCS), 100 IU/ml penicillin and streptomycin, 100 μg/ml nonessential amino acid, and 1 mg/ml G418.
Plasmid DNA (prepared using Qiagen Maxiprep kit) transfection was performed with Lipofectamine 2000 as per the manufacturer's protocols (Invitrogen).
2.2. USP18 Plasmid Constructs
The human full-length USP18 gene was cloned into pcDNA-DEST53 fused to GFP at the N-terminus (Invitrogen) (USP18 wild type (USP18 WT)). Briefly, human USP18 ORF (in the pENTER221 entry vector, Invitrogen) was cloned into the destination vector (PcDNA-DEST53) by LR recombination. Positive clones were confirmed by sequencing across the junctions. USP18 protein expression was confirmed by western blot (anti-GFP and anti-USP18 antibodies). USP18 mutant forms C64S (referred to as USP18 C64S in the following experiments), C64/65S, and C65S were created by mutating cysteine to serine at point 64, both 64 and 65, or 65 by the GeneTailor site-directed mutagenesis kit (Invitrogen). The primers used were as follows: USP18 forward primer for C64S: 5-caacattggacagaccAgctgccttaactccttga-3; USP18 forward primer for C64/65S: 5-caacattggacagaccAgcAgccttaactccttga-3; USP18 forward primer for C65S: 5-caacattggacagacctgcAgccttaactccttga-3; and USP18 reverse primer for these mutant forms: 5-ggtctgtccaatgttgtgtaaaccaaccaggccat-3. After methylation, pENTER221-USP18 was used as a template for PCR reactions using the above mutant primer pairs, and the resulting mutant form of pENTER221-USP18 was screened on LB/agar plates containing 100 μg/ml kanamycin. pcDNA3.1-USP18 was constructed by cloning the wild-type human full-length USP18 gene into pcDNA3.1 (Invitrogen). All positive clones were sequence verified. Blank vectors pcDNA-DEST53 and pcDNA3.1 were used as controls as indicated in the manuscript.
2.3. Confirmation of USP18 Protease Activity: ISG15 Cleavage In Vitro and In Vivo
An ISG15/GST fusion protein construct was created by cloning the ISG15/GST fusion gene into the pcDNA4/HisMax vector (Invitrogen); the sequence was verified. ISG15/GST fusion protein expression in Huh7.5 cells was confirmed by western blot (polyclonal anti-human ISG15 antibody, Cedarlane) 2 days posttransfection. In vivo ISG15 cleavage activity of USP18 WT and USP18 mutant forms was analyzed by treating the USP18-transfected cells with IFNα (0-100 IU/ml) for 16 hours. ISG15 and ISGylation were detected by western blot as previously described [15], and the band densities were analyzed using ImageJ software.
2.4. Quantification of HCV RNA and Infectious HCV Virions
Total intracellular RNA was harvested and purified with 96-well RNA-easy columns as recommended by the manufacturer (Qiagen, Mississauga, ON, Canada). HCV-RNA and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) RNA were quantified by real-time PCR (SYBR Green, Qiagen) using an ABI Prism 7700 sequence detector (Applied Biosystems, Foster City, CA), and the results were analyzed with SDS 1.9 software from Applied Biosystems as described previously [15, 19]. Primers are listed in Table 1.
Virus titers were determined by limiting dilution analysis as described previously [24]. HCV-positive wells were counted, and the 50% infectious dose was calculated by the method of Reed and Muench [25].
2.5. Assessment of Jak/STAT Signaling and microRNA-122 Expression
Huh7.5 cells were seeded at , 2 ml per well in 6-well plates in antibiotic-free medium for 24 hours before either 4 μg USP18 WT or 4 μg USP18 mutant form (USP18 C64S) was transfected into each well. 36 hours posttransfection, 10 U/ml IFNα was added to each well. The cells were harvested at 0 min, 30 min, 2 hours, 4 hours, 8 hours, and 24 hours posttreatment. Total protein was extracted using lysis buffer and 1 mM EDTA with protease inhibitor cocktail (Sigma). Phospho-STAT1 (Tyr701) and total STAT1 were detected by western blot (Cell Signaling, USA), and the band densities were analyzed using ImageJ software. At each time point, total RNA was extracted by TRIzol (Thermo Fisher Scientific, USA), and ISG mRNAs were determined by real-time PCR described above with the primers listed in Table 1. All the primers were self-designed with the Primer3 program and were synthesized by a commercial company.
MicroRNA-122 expression levels were also determined using the microRNA-122 kit (Applied Biosystems, USA) following the manufacturer's protocols and normalized to U6.
2.6. CD81 Expression Quantification
The CD81 expression on USP18 overexpressed cells and control cells was quantified by using the flow cytometry technique with specific antibodies. Briefly, the cells () for each acquisition of samples were washed twice in fluorescence-activated cell sorting (FACS) buffer (phosphate-buffered saline (PBS, pH 7.4) containing 2% FCS and 0.02% NaN3) and resuspended in 100 μl of FACS buffer and then stained with a PE-conjugated mouse monoclonal antibody against human CD81 (Santa Cruz Biotechnology) chilled on ice for 40 minutes. The cells were washed twice in FACS buffer and prepared with FIX & PERM reagents (Invitrogen Life Technologies) following the manufacturer's protocol, then stained with either a primary mouse monoclonal antibody against Flag (Invitrogen Life Technologies®) or a rabbit polyclonal antibody against USP18 incubated with the relevant antibody or relevant isotype matched control antibodies at 4°C for 40 minutes. The cells were washed twice in FACS buffer and resuspended in 100 μl of FCS for 30 minutes to prevent nonspecific antibody binding. This was followed by three washes in FACS buffer and incubation for 30 minutes at 4°C with the secondary goat anti-mouse IgG FITC (Santa Cruz Biotechnology) or goat anti-rabbit IgG Alexa 488. The cells were washed thrice and analyzed using the FACS Calibur flow cytometer (Becton Dickinson Immunocytometry Systems). Live cells were gated on the basis of forward and side scatter, and a minimum of 10,000 events were analyzed. FlowJo software (FlowJo, USA) was used to perform the data analysis.
2.7. Statistics
All the experiments were repeated at least three times, and where appropriate, Student's -test was used to compare categorical values. was considered statistically significant.
3. Results
3.1. Expression of Human Full-Length USP18 and Catalytic Activity of USP18 WT and USP18 C64S Forms in Huh7.5 Cells
Wild-type USP18-GFP fusion protein was expressed in Huh7.5 cells with an appropriate dose-response pattern (Figure 1(a)), and the transfection efficiency was shown by the GFP expression in the cells (Supplement Figure 1). USP18 is a cysteine protease, and cysteine 61 (C61) has been reported to be essential for its ability to cleave ISG15 from target proteins in murine cells [26]. There are two cysteine residues near this location in the human USP18 protein, at positions 64 and 65. In order to confirm which Cys is essential for the protease activity of human USP18, three different mutants were constructed: mutating C64 alone, both C64 and C65, or only C65 to serine. We then expressed an ISG15/GST fusion protein in the presence or absence of the various USP18 constructs. In these experiments, expression of wild-type USP18 led to the release of the ISG15 protein; USP18 C64S and C64/C65 mutants did not have this ability, while the C65S mutant did (Figure 1(b)). Thus, in our model system, C64 but not C65 of human USP18 is critical for USP18 protease activity. In order to test the ISG15 protease activity of USP18 WT and USP18 C64S under more physiologically relevant conditions, these constructs were overexpressed prior to exposing the cells to different amount of IFNα stimulation. Increased USP18 cleavage activity was observed in IFNα-stimulated cells (up to 100 IU/ml) as shown by decreased ISG15 conjugates (Figure 1(c)). As expected, USP18 C64S, the enzymatically inactive mutant form of USP18, did not have this effect.
3.2. USP18 Stimulates HCV Production and Blunts Anti-HCV Activity Induced by IFNα or IFNλ
We next asked whether the USP18 protein and its protease activity directly affect HCV production in the presence and absence of IFNα or IFNλ. In the absence of IFNα, overexpression of either USP18 WT or USP18 C64S increased HCV RNA (Figure 2(a)) and HCV virion titers (Figure 2(b)) by 10-25-fold, respectively. In the presence of IFNα, overexpression of USP18 WT blunted IFNα anti-HCV activity (Figures 2(a) and 2(b)). Taken together, these data demonstrate that USP18 can promote HCV production and modulate IFNα anti-HCV activity independent of its ISG15 protease activity in the J6/JFH1 HCV infectious culture system.
IFNλ is a member of the relatively new type III IFN family [27], which signals through a different receptor other than the type I IFNs. The effects of USP18 on type I IFN signaling may in part be mediated via binding of USP18 to IFNAR2 (type 1 IFN receptor subunit 2, part of the type I IFN receptor) [28]. Although USP18 deficiency resulted in hypersensitivity of mouse mammary epithelial cells to IFNλ which could be restored by USP18 overexpression [29], the study [30] in human cells demonstrated that IFN-induced USP18 expression specifically suppresses the response to IFNα, but not to IFNβ or IFNλ. The contradictory findings indicated that USP18 might regulate IFN signaling in various pathways depending on the cell type. Thus, we also investigated whether the anti-HCV effect of IFN was influenced by USP18 or not. In the present cell model, overexpression of either USP18 WT or USP18 C64S blunted the IFNλ anti-HCV activity as shown by the upregulated intracellular HCV RNA (Figure 2(c)) and increased HCV virion secretion into medium (Figure 2(d)). These data suggest that the pro-HCV production and blunting effect of USP18 are not dependent on specific IFNs.
3.3. Overexpression of USP18 Has Little Effect on Type I IFN Jak/STAT Signaling
As noted above, a previous study reported that murine USP18 expressed in human cells could block the Jak1-IFNAR2 interaction independent of its protease activity [28]. We then asked whether IFNα signaling was altered in the presence of USP18 overexpression in our model system. We considered upstream STAT1 activation (phosphorylated STAT1 levels), downstream ISG mRNA expression, and STAT1 protein levels. Overexpression of either USP18 WT or USP18 C64S had little effect on STAT1 phosphorylation at early time points (30 min) following IFNα treatment but did slightly decrease STAT1 phosphorylation at later time points (2 hours and 4 hours, Figure 3(a), upper). However, when p-STAT1 was normalized to STAT1 (p-STAT1/STAT1), no statistically difference was observed (Figure 3(a), bottom). Consistent with this, the Jak/STAT signaling pathway was not altered as shown by the similar levels of downstream ISG mRNA expression (Figure 3(b)). These data suggest that the pro-HCV production activity of USP18 is not mediated through changes in Jak/STAT signaling.
3.4. USP18 Overexpression Has No Effect on HCV Replicon
If the stimulatory effect of USP18 on HCV production was mediated at the level of viral replication, then HCV replication in a noninfectious model (replicon system) should also be affected by USP18 overexpression. In addition, we might also predict that expression of cofactors necessary for HCV RNA replication, such as microRNA-122, which is directly interacting with HCV RNA [31], might be sensitive to the manipulation of USP18. Surprisingly, overexpression of both USP18 WT or USP18 C64S had no effect on HCV RNA replication in either genotype 1b replicon (Con1b; AB12-A2, Figure 4(a)) or genotype 2a subgenomic replicon (JFH1; sbJFH1-B2, Figure 4(b)) cells in the presence or absence of IFNα. Moreover, USP18 overexpression also had no effect on microRNA-122 levels (Figure 4(c)). These data argue that USP18 does not directly affect intracellular HCV replication.
3.5. Overexpression of USP18 Increases HCV Infectivity in Huh7.5 Cells
If USP18 does not contribute to HCV RNA replication but does promote HCV production, it must alter the cellular milieu in a manner that favors the HCV life cycle at steps other than RNA replication. One possibility is that USP18 increases the susceptibility of the cell to infection by HCV. To examine this possibility, we asked whether USP18 overexpression alters HCV infectivity. As shown in Figure 2, increased USP18 expression led to markedly increased HCV infection of Huh7.5 cells by 5-6-fold. Like most other viruses, the infectivity of HCV is mainly determined by the interaction between the viral glycoproteins and a series of attachment factors and entry factors which are involved in the initiation of infection [32]. Therefore, we analyze the expression of the entry factor CD81, which is in the most essential position of the HCV entry factor complex, in Huh7.5 cells. As expected, there was a USP18 concentration-dependent upregulation in CD81 mRNA expression (Figure 5(a)). FACS also revealed a significant upregulation of CD81 expression in parallel with elevated USP18 level (Figure 5(b)).
4. Discussion
USP18 is a cysteine protease with specific ISG15 cleavage (deconjugating) activity [33]. Originally cloned from leukemia fusion protein AML1-ETO-expressing mice [34], USP18 is an ISG whose expression level is inducible by type I IFN and is degraded by proteolysis through the SCFSkp2 ubiquitin ligase [35]. The roles of the USP18 pathway in viral infection differ depending on the virus involved and can be mediated through multiple different routes. Although originally attributed to the effects of ISGylation, subsequently it has been shown that USP18 has ISG15-independent effects. Although Usp18-/- mice exhibited less replication of LCMV and VSV, ISG15- or Ube1L-knockout mice had the same sensitivity to LCMV and VSV infection as wild-type mice, indicating that USP18 may behave in an ISG15-independent manner [36]. Specifically, murine USP18 can bind to human IFNAR2 and block type I IFN signaling by competitively interfering with Jak1 binding to the receptor [28]. Thus, at least some of its effects may be mediated through its effects on IFN signaling and not through ISG15 directly. And USP18 also has the ability to affect cellular pathways (and expression of surface proteins), as demonstrated by its ability to (1) regulate the expression of the EGF receptor in carcinoma cells [37, 38], (2) inhibit tumor necrosis factor- (TNF-) related apoptosis-inducing ligand- (TRAIL-) induced apoptosis [39], and (3) employ USP20 to promote deubiquitination of the mitochondrial adaptor protein STING [40]. Taken together, these data illustrate that the effects of USP18 in any given viral infection can be mediated by a number of routes and may or may not be dependent on the USP18 deconjugase function or on a direct inhibition of IFN signaling.
The results of our study suggested that the deISGylation (cleaving ISG15 from its target proteins) process was not involved in the effect of USP18 on HCV. We synthesized a mutant USP18 with no ability to cleave ISG15 from an ISG15/GFP fusion protein. Overexpression of this mutant form of USP18 led to a similar degree of increased HCV production and blunting of the anti-HCV effect of IFNα. Furthermore, the work from our laboratory has shown that ISGylation is required for efficient HCV production, in that inhibition of ISGylation by knockdown of the E1 Ube1L enzyme reduces HCV viral titers and RNA [15]. If the effect of USP18 on HCV production were dependent on ISG15 and ISGylation, the decrease in ISGylation seen with the overexpression of USP18 would be expected to inhibit HCV production. In fact, despite a decrease in cellular ISGylation, there is a consistent increase in HCV titers and RNA following the overexpression of wild-type USP18. These data are consistent with an ISG15-independent ability of USP18 to stimulate HCV production.
In earlier work, we found that knockdown of USP18 enhanced the anti-HCV effect of IFNα, in concert with increased cellular protein ISGylation and increased activation of the Jak/STAT signaling pathway [19]. It has also been reported that exposure of HLLR1-1.4 cells or primary hepatocytes to either type I or type III IFNs interfered with the cells' ability to further respond to IFNα subtypes (desensitization), but that the response to IFNβ or IFNλ is not affected [30]. Although no mechanistic details were given, the authors speculated on a threshold effect linking USP18 expression and type I IFN receptor inhibition. The article figured out that IFN treatment-induced USP18 was sufficient to induce a "refractory" state to IFNα because IFNα has lower affinity for the receptor compared with IFNβ. By contrast, in the current study, we found that overexpression of USP18 had little to no effect on IFNα-induced ISG expression, only a mild effect on IFNα Jak/STAT signaling, and no effect on IFNα-induced STAT1 protein expression and yet markedly increased HCV production in the absence of IFNα and blunted the anti-HCV effect of IFNα. The fact that IFN signaling was enhanced when USP18 expression had been decreased and showed little change when USP18 expression had been increased was more in keeping with its effect on IFN signaling being mediated through an intermediary than through direct binding to the receptor of type I IFN. We did ask whether steric effects from the relatively large GFP protein tagged to the USP18 construct could interfere with binding to the IFN receptor. But similar results were obtained (data not shown) when we created a separate USP18 expression construct with a much smaller His-tag (pDEST26). These data, taken together, argue that binding of USP18 to the IFNAR2 receptor cannot fully explain the effect of USP18 in the present infectious HCV model system. Furthermore, since USP18 is involved in regulating various signaling pathways including the IFNλ pathway [18], new evidence will be needed to elucidate whether USP18 could promote HCV production by inhibiting the signaling of type III IFN.
From a mechanistic standpoint, the positive effect of USP18 on HCV production is not mediated at the level of RNA replication per se. Overexpression of either USP18 WT or protease-inactive USP18 C64S had no effect in HCV model systems that require only RNA replication (HCV replicon-containing cells). Neither the wild type nor the protease-inactive variant of USP18 altered HCV RNA in two distinct replicon systems. There was also no change in levels of cellular cofactors that have been shown to be important for HCV replication, such as microRNA-122.
Although USP18 upregulation does not seem to influence HCV RNA replication, increased expression of wild-type or mutant USP18 stimulates HCV infectivity in Huh7.5 cells. We have further demonstrated that USP18 upregulation leads to increased surface expression of CD81 which could form a receptor complex for HCV internalization into hepatocytes with other proteins such as calpain-5 (CAPN5) and the ubiquitin ligase casitas B-lineage lymphoma proto-oncogene B (CBLB) [41]. This elevated CD81 expression might be responsible for the increased HCV infectivity and subsequent viral production in Huh7.5 cells. Thus, our work pointed out that the role of USP18 in innate immunity and in particular in a cell's susceptibility and response to viral infection is considerably more complex than previously thought although the precise mechanism for this effect remains to be investigated.
In conclusion, our present study demonstrates that USP18 contributes to the viral/host interplay, creating a hepatocellular environment that is more favorable to viral production. The fact that USP18 expression augments markedly increased CD81 expression and HCV infectivity provided some mechanistic insight into the effect and may illustrate a novel means by which HCV can subvert the host innate immune response to its benefit. USP18 is an important modulator of the host innate response and clearly plays an important role in clinical HCV. Further studies investigating the effects of USP18 on other viral infections should be done to reveal its potentiality as a biomarker of diseases and as a therapeutic target.
|Hepatitis C virus
|Interferon-stimulated gene
|Interferon-stimulated gene 15
|Ubiquitin-specific protease 18
|Janus kinase/signal transducer and activator of transcription
|Pegylated interferon/ribavirin
|Fluorescence-activated cell sorting
|Glyceraldehyde-3-phosphate dehydrogenase.
Data Availability
All the data used to support the findings of this study are included in the article.
Part of the present manuscript has been presented as a poster in the "Canadian Symposium on Hepatitis C Virus 2012."
Conflicts of Interest
The authors declare that there is no conflict of interest regarding the publication of this paper.
The authors would like to thank Dr. Charles Rice for the J6/JFH1 HCV plasmid and Huh7.5 cell line. Technical support from Larry Meng (University of Toronto) and Tram NQ Pham (Dalhousie University) is greatly appreciated. This work was funded by grants from the CAMS Initiative for Innovative Medicine (CAMS-2016-I2M-3-025 and CAMS-2017-I2M-B&R-15), the National Key Research and Development Program from Ministry of Science and Technology of the People's Republic of China (2018YFE0107500 to LC), and the Canadian Institute of Health Research (No. 62488 to IDM). YL and BQ were supported by China Scholarship Council (CSC).
Supplementary Materials
Supplemental materials and methods: transfection efficiency evaluation pmaxGFP (Lonza, Switzerland) was used to evaluate the transfection efficiency. Huh7.5 cells were seeded at , 2 ml per well in 6-well plates in antibiotic-free medium for 24 hours before 1 μg, 2 μg, and 4 μg (C) GFP plasmid DNA or 4 μg empty vector (D) was transfected into each well. Fluorescent microscopy images were taken 48 hours post-transfection. An anti-CD81 antibody ([M38], Abcam) was used to block CD81. Briefly, Huh7.5 cells were preincubated for one hour with 1 μg/ml or 20 μg/ml anti-CD81 ([M38], Abcam). The cells were washed 3 times with PBS before the J6/JFH1 virus was added (). After 4 hours of incubation, the cells were washed and left 24 hours before collecting total intracellular RNA. J6/JFH RNA was determined by real-time PCR. Effect of USP18 on other entry or attachment factors in HCV infection. Huh7.5 cells were seeded at , 2 ml per well in 6-well plates in antibiotic-free medium for 24 hours before 1 μg, 2 μg, 4 μg, and 6 μg USP18 WT or 4 μg empty vector pcDNA-DEST53 was transfected into each well. 48 hours posttransfection, total RNA was extracted; SR-BI, occludin, and claudin-1 were quantified by real-time PCR. The primers (purchased from Sangon Biotech, China) are as follows: CD81, forward primer: 5-GTGATCCTGGGTGCCCT-3 and reverse primer: 5-CATCATCCACCACGGCCTGC-3; SR-BI, forward primer: 5-ACGACACCGTGTCCTTCCTCG-3 and reverse primer: 5-CGGGCTGTAGAACTCCAGCGA-3; occludin, forward primer: 5-AGTGTGATAATAGTGAGTGCTATCC-3 and reverse primer: 5-TGTCATACCTGTCCATCTTTCTTC-3; and claudin-1, forward primer: 5-TTCTCGCCTTCCTGGGATG-3 and reverse primer: 5-CTTGAACGATTCTATTGCCATACC-3. (Supplementary Materials)
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Mae Matte Silk Blouse
Fluid matte silk blouse with slight cap sleeves. Roundneck. Cap sleeves. Curved hem. Pullover style. About 26 from shoulder to hem. Silk. Dry clean. Made in Italy. Model shown is 5'10 (177cm) wearing US size 4.
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Hi and welcome to my page. My name is Carrie and I have been growing my own food since 2012 in my parents back garden. In 2016 I got a allotment but sadly had to give it up in October 2019. In 2020 I am hoping to be starting to grow from my back garden.
Hope you all want to stick around to see what I have been up to and what I am growing.


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Head Cashier | Commonwealth
CW Home Depot
- Quezon City, PhilippinesCommonwealth Avenue , Quezon City, Metro Manila, PhilippinesQuezon CityMetro ManilaPhilippines
- Full time
Job Description
- Oversee the cashiering functions of Treasury department
- Makes required reports daily such as the Daily Consolidated Sales Report, redeemed Credit Memo Reports, daily deposits of leasing collections, cancelled Official Receipts, cancelled Sales Invoices, invalid Credit Memos, items not found, and cleared checks to be submitted to Treasury Department.
- Prepares cash boxes, cash change and other requirements needed by cashiers for operational functions.
- Prepares and deposits all sales whether in cash or in checks to the bank.
- Prepares petty cash replenishment weekly
- Accepts and makes incoming payment of all transactions in the branch other than those received by the cashiers.
- Deposits POS/SAP cash, credit card and checks in the SAP B1 System.
- Files and safeguards records of sales, credit card slips, credit card settlements, bank deposit slips, tenant invoices which are submitted daily by cashiers.
- Prepares other reports as needed by Treasury department and other concerned departments
- Performs other related tasks assigned from time to time.
Minimum Qualifications
- Must have at least 2 years of supervising cashiering activities
- Knowledgeable in retail store operations, accounting process and bank procedures
- Knowledgeable in cash management and funding
- Background in using SAP B1 Banking and Finance module is an advantage
- Strong written and verbal communication skills
- With leadership qualities to manage the team
- With high analytical and strong arithmetic skills
Perks and Benefits
- Paid Holidays
- Paid Vacation Leave
- Paid Sick Leave
- Maternity & Paternity Leave
- Single Parent Leave
- Special Leave Benefits for Women
Jobs Summary
- Job level
- Associate / Supervisor
- Job category
- Accounting and Finance
- Educational requirement
- Bachelor's degree graduate
- Recruiter response to application
- Sometimes
- Office Address
- Commonwealth Avenue, Barangay Matandang Balara, Quezon City, Metro Manila, Philippines
- Industry
- Retail Industry
- Vacancy
- 1 opening
- Website
- http://www.cwhomedepot.com
Feel secure when applying: look for the verified icon and always do your research on a company. Avoid and report situations when employers require payment or work without compensation as part of their application process.
About CW Home Depot
Founded in 2003, CW Marketing & Development Corporation has introduced the first store-within-store retail concept wherein everything you need to build or renovate your home is all under one roof. The said unique platform offers a variety of building, finishing & furnishing products in the convenience of a one-stop-shop. Similar, to a shopping mall, CW Home Depot has its own department stores segmented into Home Builders Superstore or DIY Section, Home Interior Superstore, Home Appliance Superstore with a wide range of specialty stores which cater to a more specific requirement.
It has 8 existing branches located in Ortigas, Alabang , Manila Bay, Balintawak, Pampanga, Commonwealth, Imus, and Sta. Rosa.


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Northcash helps you get the online loan you need when you're in a hurry with our express application and quick approvals. Apply Today, Cash Tomorrow!
If you have a relatively new car, it probably has a USB port in the dashboard, glove box, or center console. So naturally, you should just use them to charge your devices, right? Not so fast: if you want speedy charging, those built-in ports just don't cut it.
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Learning how to make money online can seem like a black box filled with bad ideas. Well, not here. Start with these 65 legitimate ways of how to make money online, of which I've personally done almost all myself (or have interviewed others with money making experience to share).
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Unusual Ways to Get Money Fast. Posted by Emily Guy Birken Last updated on May 29, 2019 | Make Money, Side Hustle Advertiser Disclosure: Opinions, reviews, analyses & recommendations are the author's alone, and have not been reviewed, endorsed or approved by any other entity.
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Harry back stage at the Anderson Paak concert tonight via ruthkoleva
justimagine_it: @Harry_Styles once said "Treat People With Kindness" and guys he did just that. THANK YOU KING!!!💋💋😍 FINALLY! My Birthday was made 😭❤
I love having pictures of fans with Taylor all over my dash and blog. This is life.
👋😍☀ Happy Friday!
#repost and thanks to Mark Faulkner fb
"Walked into this charming fruit and veg shop and cafe, and who should walk in, but Tom Hardy! :)😎"


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Gets Cancelled Comic Strips - Page 4
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189 Results for Gets Cancelled
View 31 - 40 results for gets cancelled comic strips. Discover the best "Gets Cancelled" comics from Dilbert.com.
Saturday July 04,
Tags #Dilbert, #baby, #office, #new, #sneeze, #achooo, #prune
A woman hands Dilbert a baby and says, "Everybody in the office gets a turn holding my new baby." Ted says, "Next." Dilbert holds the baby and thinks, "Uh-oh . . . Sneeze coming." Dilbert sneezes. Dilbert looks at the baby and says, "Ooh! Look what he does when you sneeze on him." Ted says, "He looks like a prune!" The mother looks shocked.
Wednesday July 15,
Tags #Dilbert, #stock market, #Dogbert, #bad luck, #tragedy, #fortune, #joy, #pain, #compensate, #good luck, #friend
Dilbert sits in his chair and Dogbert sits on the hassock. Dilbert's head is bandaged and his arm is in a sling. Dilbert says, "I've had nothing but tragedy since making a fortune in the stock market." Dilbert continues, "Sometimes, Dogbert, it seems like our lives have preset balances of joy and pain; when one gets too high the other kicks in to compensate." Dilbert continues, "But through it all, I always have you, my friend." Dogbert replies, "At least until my good luck kicks in."
Monday October 11,
Tags #business meeting, #Promotion, #Dogbert, #executive man, #management
Dogbert and several executives sit at a conference table. Dogbert says, "Thank you all for coming to this emergency board meeting." Dogbert continues, "As you know, all promotions to senior management are based on hair. I think we all agree this is the best system." Dogbert lifts the president's toupee with a pointer and continues, "But have you noticed that I have a rich lustrous coat, whereas our current president gets a little outside help?"
Monday December 20,
Tags #Dilbert, #Dogbert, #christmas, #dog
Dilbert sits on the couch and Dogbert stands on the armrest. Dogbert hands Dilbert a list and says, "Here's a list of my Christmas demands." Dogbert says, "Follow the instructions and nobody gets hurt." Dilbert says, "You're bluffing. You wouldn't hurt my plastic window Santa." Dogbert holds the electric cord to the decoration and says, "One yank and he's off life support!"
Friday March 11,
Tags #no raise, #engineers wuit, #the goal, #reduce headcount, #smart people leave, #organize pot luck
Dilbert: "I'm telling you - if nobody gets a raise, half the engineers will quit!" The Boss: "That's the goal. We're trying to reduce headcount by fifty percent." Dilbert: "But all the smart people will leave!" Dilbert: "Would you mind organizing a goodbye potluck lunch for them?"
Saturday July 30,
Tags #exaggerating accomplishments, #failing, #monkey, #more humiliating, #stupid monkey, #typical, #zimbu, #animals
The Boss: Our CEO cancelled his visit, He's sending his top aid, ZIMBU the monkey in his place. Dilbert: Isn't that typical? I spent a week exaggerating my accomplishments for this now he sends a stupid monkey! what could be more humiliating than trying to suck up to a monkey? Monkey: Failing at it?
Tuesday October 11,
Tags #future trends, #guess, #more valid, #published, #share vision, #people are dumb
Dogbert: I'm writing a book of my guesses about future trends. If it gets published then my guesses will seem more valid than other peoples. I'll charge huge fees to share my 'vision" with audiences. Dilbert: Why would people pay huge fees for guesses? Dogbert: Trend number one is that people aren't getting any smarter.
Friday October 14,
Tags #2004, #cheaper than dating, #date at door, #Dogbert, #future, #holodeck, #virtual reality, #writing article, #money, #male hormones
Dogbert: "I can predict the future by assuming that money and male hormones are the driving forces for new technology." "Therefore, when virtual reality gets cheaper than dating, society is doomed." "Year 2004" Date: "Is Dilbert available?" DOgbert: "He's been in the holodeck since March."
Friday November 04,
Tags #physical intimidation, #smack face, #stupid, #manage by intimidation, #mob menatlity, #gets smacked
The Boss says to Dilbert, "I've decided to manage by physical intimidation. If somebody says something stupid I'll just smack them." Dilbert says, "That's the stupidest thing I've ever heard." The Boss hits himself in the head. Dilbert thinks, "On the other hand, maybe I should give it a chance."
Tuesday November 22,
Tags #dogbert the consultant, #warranty replacements, #customers, #repeat customers
DOGBERT THE CONSULTANT Dogbert and the Boss sit at a conference table. Dogbert says, "You can gauge your success by the number of repeat customers you have." The Boss says, "I'm proud to say that virtually every customer gets another unit within three months of buying the first one!" Dogbert asks, "What if you don't count warranty replacements?" The Boss replies, "Ooh . . . Then we don't look so good."


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The ongoing drive to improve power conversion performance while reducing system size and thermal dissipation is at the heart of today's MOSFET development. And to achieve this, semiconductor manufacturers must consider both the underlying semiconductor processes and the packaging technology.
The image here shows the improvements in electrical performance brought about by some of the recent advances in device and package technology. As MOSFETs in the 5mm x 6mm footprint have evolved from UMOS VIII to UMOS IX-H technology, the new silicon has reduced RDS(ON) by 42%, while the lower DFPR of the DSOP Advance package is shown to be responsible for an additional 25% reduction. This diagram, of course, is concerned only with electrical performance and does not express the increased thermal capability of the DSOP Advance package.
Toshiba's white paper on the latest silicon and package advances for power MOSFETs provides more information on the capabilities of these new technologies.


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ISO 14001 Environmental Certification
Consultancy and Training
Environmental Friendly Business is More Profit Friendly
Know More
ISO 14001 Environmental Certification
What is ISO 14001 Environmental Certification?
Universally acceptable and highly acknowledged ISO 14001 is known well for its contribution to the environment. It is for all organizations regardless size, capital and volume or scale of business. Increasing environmental regulations and support of general public for environmental well being has encouraged organizations to embrace Environmental Certification ISO 14001.
ISO 14001 encourages organizations to protect and safeguard environment from man and machine made hazards. It is a symbol of organizational social responsibility. Organizations with concern for society and environment welcome this certification and compliance required by ISO 14001. It creates a strong positive image for the organization, its products and people working with is. Getting into ISO 14001 Certification is a good investment.
Objectives of ISO 14001 Certification
Environmental Management System initiated through ISO 14001 in any organization aims at providing a comprehensive prevention to further environmental damage by protecting the spread of hazardous content in air, water and soil. ISO 14001 further defines social, individual and organizational responsibilities to safeguard mother earth and its environment. It makes organizations, and stakeholders more concerned, responsible and proactive for environmental protection. Better business and organization is not possible without complying with ISO 14001 in near future.
Who can apply ISO 14001?
It is for all types, sizes, volume and nature of organizations regardless their nature of business and location. The organizations that produce, discharge, emit or leak harmful content in liquid, solid, gas or radiation form are the main organizations that ISO 14001 aims for. Environmental certification helps the organizations control the wastes and dispose these wastes off in a practical and non-harmful way.
Benefits of ISO 14001
- Improved business, corporate image and social acceptance
- Prestigious status among competitor
- Strong advocacy of organizational social responsibility
- Enhanced business productivity and capacity building
- Improved cost of production, more savings, higher profits
- Easy compliance for other ISO standards e.g., ISO 9001, ISO 45001 et.
- Improved machine and human performance
- Reduced operational health and well-being of organization


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Here in Citibank, you will be part of a
high-performing organization that will set you up for success!
Role: Customer Service Representative (Voice Account for an In-House Contact Center)
• Provide world class service excellence to clients by offering a consistent telephone banking experience
• Attend to inbound and outbound telephone calls in accordance with standard operating procedures
• Process online banking transactions which include financial entries and data maintenance
• Identify opportunities to further enhance relationships with our existing clients
CBPS may assign candidates to different businesses based on candidate profile, available openings & business need. Applicants must be willing to work on a shifting schedule.
Apply on the following dates from 9AM - 5PM and look for Magnus. For interested, text CT_NAME and send to 09161337456 / 09102950866
-Graduate of any 4/ 5 year course
-Graduate of any 4/ 5 year course
2017 Magnus Eventus Inc. ventured in Millennial Technology. This year we softly launch our Magnus Job Fair mobile application and it can now be downloaded via Google Play Store for Android Users and App Store for IOS Apple users.
Employers and Applicants can now have an up to date access to Magnus Eventus Events and job opportunities. Applicants can create their profile and choose which event they can attend to and also apply directly to your company once you subscribe to this application. Contact us to know more about this product!


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New BCCI sees first departure among old guard as CFO quits
MUMBAI: As expected, winds of change have begun to blow in the BCCI headquarters in Mumbai and along with it. The first wicket amongst the Board's professionally-hired staff, has fallen after the BCCI elections held last month. The Board's first Chief Financial Officer, Santosh Rangnekar, put in his papers on Thursday . Rangnekar refused to comment on the development, but it's reliably learnt that that he has chosen to resign.
Rangnekar was hired in 2016 as the Lodha reforms mandated the recruitment of a CFO. The BCCI will now have to float an ad for the job of a new CFO in the Board. However, it could not be separately confirmed if Rangnekar will be quitting the position with immediate effect or serve his notice period. In July this year, the CFO was handed a fancy appraisal by the Committee of Administrators (COA) that became a talking point.
During his stint as CFO, his allegations against then treasurer Anirudh Chaudhry, the trips to the United States, his bringing up of the Champions League T20 payment history and more — all of these remained a subject of discussion. Whether Rangnekar serves the notice period or walks away with a severance package or none of it will have to be seen.
BCCI AGM on Dec 1: The Annual General Meeting (AGM) of the BCCI is expected to be held in Mumbai on December 1 and the notice for the same is expected to be sent out any time before Monday evening. Those in the know say that the agenda for the AGM is being drafted on the lines of what is mentioned under the Board's constitution.


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Child care
Hey everyone! I wanted to get some opinions on what y'all have had success with for childcare? We have looked at a few daycares, but I am still considering other options and am pretty nervous (FTM)!
I always had my mom at home growing up, but being a SAHM doesn't make sense for us financially right now. We live near downtown and I work in the medical center so care near that area is ideal!
Let me know your thoughts on daycares, nannies, nanny shares, in Home day cares, etc :)


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|Carsten Strotmann
|Re: [gforth] Suitable terminal for Windows
|Tue, 24 Apr 2018 17:50:38 +0200
|mu4e 1.0; emacs 25.3.1
Hi Ethan, Ethan A. Gardener <address@hidden> writes:
Under Windows, Gforth's default terminal window has text a bit too small and too dim for my aging eyes. Under a MINGW terminal it runs, but history doesn't work; up-arrow moves the cursor. Does anyone have any sort of solution for Gforth to run under Windows with a terminal window of adjustable font and colors, please? Or even fixed 14pt font in high contrast would do.
you can try 'mlterm' (<http://mlterm.sourceforge.net/>). I use 'mlterm' on MacOS, Linux and xBSD. There are Windows binaries on the webpage.
You can also use Putty as a terminal together with a local telnet server: <https://superuser.com/questions/270300/is-it-possible-to-use-putty-as-the-ui-for-the-windows-shell> Greetings Carsten
|[Prev in Thread]
|Current Thread
|[Next in Thread]


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Nahalchand Nl Himalaya is located in Dahisar East, Mumbai Western Suburbs in Mumbai. Nahalchand Nl Himalaya is currently a Ready to Move project and is available at approximate price of 10350 Per Sq. Ft. Onwards.Nahalchand Nl Himalaya is offering 4 towers of 2, 3 BHK Apartment in size ranging from 1100 Sq. Ft. to 1400 Sq. Ft.(Saleable) Nahalchand Nl Himalaya is approximately at 1.0 Kms from Western Express Highway and ~1.0 Kms from Western Express Highway. Project has a connectivity rating of 10, livability rating of 5.3, lifestyle rating of 6 and value for money rating of 6.5 out of 10. Project has Central AC.
2 BHK 1100 Sq. Ft. Apartment
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Q: How can i get a brochure of this project?
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This website is only for the purpose of providing information regarding real estate projects in different geographies. Any information which is being provided on this website is not an advertisement or a solicitation. The company has not verified the information and the compliances of the projects. Further, the company has not checked the RERA* registration status of the real estate projects listed herein. The company does not make any representation in regards to the compliances done against these projects. Please note that you should make yourself aware about the RERA* registration status of the listed real estate projects.
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|h.g. muller
|Re: [XBoard-devel] promotion issue with translation
|Sun, 04 Dec 2011 21:07:48 +0100
There is only one reason I can think of why we should keep the promo popup,and that is that blind people cannot use the new mechanism because they cannot use a mouse. But it is actually a bad reason, as there currently is no special XBoard version for the blind as there is for WinBoard. And to handle the promo popup through key-strokes + audio feedback is actually more cumbersome than typing the move using auto-popup of the move type-in dialog.
So my guess is that even blind people would not miss the promo-popup very much.
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