Drug Screening for Hepatitis A Virus (HAV): Nicotinamide Inhibits c-Jun Expression and HAV Replication

Reina Sasaki-Tanaka; Ryota Masuzaki; Hiroaki Okamoto; Toshikatsu Shibata; Mitsuhiko Moriyama; Hirofumi Kogure; Tatsuo Kanda

doi:10.1128/jvi.01987-22

. 2023 Feb 2;97(2):e01987-22. doi: 10.1128/jvi.01987-22

Drug Screening for Hepatitis A Virus (HAV): Nicotinamide Inhibits c-Jun Expression and HAV Replication

Reina Sasaki-Tanaka ^a,^✉, Ryota Masuzaki ^a, Hiroaki Okamoto ^b, Toshikatsu Shibata ^a, Mitsuhiko Moriyama ^a, Hirofumi Kogure ^a, Tatsuo Kanda ^a,^✉

Editor: Jae U Jung^c

PMCID: PMC9973044 PMID: 36728416

ABSTRACT

Hepatitis A virus (HAV) infection often causes acute hepatitis, which results in a case fatality rate of 0.2% and fulminant hepatitis in 0.5% of cases. However, no specific potent anti-HAV drug is available on the market to date. In the present study, we focused on inhibition of HAV internal ribosomal entry site (IRES)-mediated translation and investigated novel therapeutic drugs through drug repurposing by screening for inhibitors of HAV IRES-mediated translation and cell viability using a reporter assay and cell viability assay, respectively. The initial screening of 1,158 drugs resulted in 77 candidate drugs. Among them, nicotinamide significantly inhibited HAV HA11-1299 genotype IIIA replication in Huh7 cells. This promising drug also inhibited HAV HM175 genotype IB subgenomic replicon and HAV HA11-1299 genotype IIIA replication in a dose-dependent manner. In the present study, we found that nicotinamide inhibited the activation of activator protein 1 (AP-1) and that knockdown of c-Jun, which is one of the components of AP-1, inhibited HAV HM175 genotype IB IRES-mediated translation and HAV HA11-1299 genotype IIIA and HAV HM175 genotype IB replication. Taken together, the results showed that nicotinamide inhibited c-Jun, resulting in the suppression of HAV IRES-mediated translation and HAV replication, and therefore, it could be useful for the treatment of HAV infection.

IMPORTANCE Drug screening methods targeting HAV IRES-mediated translation with reporter assays are attractive and useful for drug repurposing. Nicotinamide (vitamin B3, niacin) has been shown to effectively inhibit HAV replication. Transcription complex activator protein 1 (AP-1) plays an important role in the transcriptional regulation of cellular immunity or viral replication. The results of this study provide evidence that AP-1 is involved in HAV replication and plays a role in the HAV life cycle. In addition, nicotinamide was shown to suppress HAV replication partly by inhibiting AP-1 activity and HAV IRES-mediated translation. Nicotinamide may be useful for the control of acute HAV infection by inhibiting cellular AP-1 activity during HAV infection processes.

KEYWORDS: hepatitis A virus, nicotinamide, IRES, AP-1, c-Jun, internal ribosome entry site

INTRODUCTION

Hepatitis A virus (HAV) is transmitted mainly through the fecal-oral route and is a major cause of acute viral hepatitis (1). HAV infection can lead to acute liver failure (ALF) and high mortality in some cases. In 2010, the global number of HAV-infected patients was ~1.4 million, with 27,731 deaths (2). Previous studies have demonstrated that risk factors for severe acute hepatitis A are an age over 40 years, preexisting chronic liver diseases with a limited hepatic functional reserve, and diabetes mellitus (1, 3). In Japan, where there is no universal program for vaccination against HAV infection and the number of people without HAV immunity is increasing due to improved hygienic conditions (4), there is an increasing number of patients who are infected with HAV at older ages, which might lead to more severe clinical manifestations. Therefore, more effective antiviral therapies for HAV infection are needed.

HAV is a single-stranded, positive-sense RNA virus of approximately 7.6 kb. The 5′ untranslated region (UTR) of the HAV genome forms an internal ribosome entry site (IRES), a structured RNA region with highly conserved stems and loops, which is responsible for the recruitment of the ribosome and translation factors required for the cap-independent initiation of translation and the promotion of translation of HAV proteins (5). A variety of animal viral IRESs have been classified according to their phylogenetic origin, secondary structure, and functionality. The Picornavirus IRESs have been divided into four classes: the HAV IRES is a class III IRES, while the hepatitis C virus (HCV) IRES is similar to the Picornavirus class IV IRESs (6).

Nicotinamide is a water-soluble and pyridine-3-carboxylic acid amide form of vitamin B3 (niacin) (7). Nicotinamide is obtained either by dietary intake through absorption almost completely in the small intestine or through synthesis by the conversion of nicotinic acid in the liver or hydrolysis of the coenzyme β-NAD (NAD⁺) in the body (8). Nicotinamide inhibits sirtuins (9, 10) and is also involved in cellular energy metabolism, DNA synthesis, and DNA repair (11 –13). Diets deficient in nicotinamide can result in fatigue, loss of appetite, oral ulcerations, pigmented rashes of the skin, and pellagra, which is characterized by diarrhea, dermatitis, dementia, and death (the 4Ds) (14). Nicotinamide has also been reported as a chemotherapeutic regimen for skin cancer, breast cancer, and hepatocellular carcinoma (HCC) (15, 16). There are several reports regarding the effect of nicotinamide as an oral antimicrobial agent against microbes such as hepatitis B virus (17), enterovirus (18), human immunodeficiency virus (HIV) (19, 20) and Mycobacterium tuberculosis (20).

The transcription factor AP-1 regulates the transcription of a wide variety of genes involved in cellular immunity and viral replication (21, 22). Significant activation of AP-1 and induction of transcription of its regulated genes have been observed in infections with various viruses, including coronaviruses (21). Although we and others have previously reported that HAV 2C and 3A proteins and HAV HM175/clone 1 infection activate the AP-1-associated signaling pathway (23, 24), there have been few reports about the association between HAV replication and the transcription factor AP-1 or c-Jun. In the present study, after identifying nicotinamide as showing inhibitory effects against HAV IRES-mediated translation, we examined the effects of nicotinamide on the replication of HAV HM175 genotype IB subgenomic replicon and HAV HA11-1299 genotype IIIA in cell culture infection systems. We also examined whether AP-1 is important for the mechanism of action of nicotinamide as a drug against HAV infection.

RESULTS

Assays designed to target the HAV IRES in COS7-HAV-IRES cells.

The HAV IRES, mainly located in the 5′ UTR, allows cap-independent translation of the HAV genome using the host ribosomal machinery (25). To identify drugs that inhibit HAV IRES-mediated translation, a drug screening assay was used following the optimization of multiple parameters, including HAV IRES-mediated translation and cell viability, using COS7-HAV-IRES cells. Both HAV IRES-mediated translation of <0.45-fold and cell viability of >90% were selected as criteria for further confirmation studies with HAV infection (Fig. 1). A total of 77 drugs (21 antiviral drugs, 20 stem cell differentiation drugs, 13 antidiabetic drugs, and 23 approved anticancer drugs) were selected (Table 1).

FIG 1 — Nicotinamide is a candidate drug for inhibition of HAV IRES-mediated transcription. Analyses of 1,158 drugs screened by luciferase assays and MTS assays. The results of the drug screening are shown. All drugs were plotted on a scattergram in which the cell viability of COS7-HAV-IRES cells and the relative luciferase activity of HAV IRES are indicated on the y axis and x axis, respectively. Nicotinamide is indicated with a red circle.

TABLE 1.

The 77 drugs that were initially selected with COS7-HAV-IRES cells

Drug category (no.)	Drug name
Antiviral (21)	Velpatasvir, AG-1478, dasabuvir, HZ-1157, 5436-1131, clevudine, cobicistat, E717-0652, 6321-0504, 4456-2665, 8014-9100, E565-0873, telaprevir, merimepodib, forsythin, enoxacin, dolutegravir, amprenavir, K780-0942, 6872-1644, 3370-3410
Stem cell differentiation (20)	Nicotinamide, cryptotanshinone, quercetin, myricitrin, PD0325901, miltefosine, SB202190, ruxolitinib phosphate (INCB-18424 phosphate), TTP 22, selumetinib, oridonin, honokiol, doramapimod, zebularine, emodin, SC514, Y-27632 dihydrochloride, palomid 529, INK 128, temsirolimus
Antidiabetic (13)	SNS032, 4-aminobutyric acid, metformin hydrochloride, 5,5-dimethyloxazolidine-2,4-dione, MBX 8025, HTH-01-015, IM12, PHA767491 HCl, AS1517499, ginkgolide C, MK3903, fenofibrate, C188-9
Approved anticancer (23)	Homoharringtonine, tanshinone I, gefitinib, hydrocortisone butyrate, cediranib, phenoxybenzamine hydrochloride, rifampicin, chloroquine diphosphate, nifedipine, sodium phenylbutyrate, beta-carotene, ethambutol dihydrochloride, thioproline, toremifene, enzalutamide, fluorocytosine, pemetrexed acid, DL-menthol, carboplatin, chlorambucil, temozolomide, cyclophosphamide monohydrate, uridine

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The 77 selected drugs were validated in HAV-infected cells, and the effects on cell viability were assessed using Huh7 cells. All 77 drugs were confirmed to have no cytotoxic activity at 1 μM. Nicotinamide significantly inhibited HAV HA11-1299 genotype IIIA replication in Huh7 cells (see Fig. S1 in the supplemental material). Further confirmation studies of nicotinamide were performed.

Nicotinamide significantly inhibited HAV HM175 genotype IB subgenomic replicon replication and HAV HA11-1299 genotype IIIA replication in human hepatoma cells.

We evaluated the replication kinetics of the HAV subgenomic replicon in HuhT7 cells. Nicotinamide treatment significantly inhibited HAV subgenomic replicon replication in a dose-dependent manner (Fig. 2A) with an estimated half maximal inhibitory concentration (IC₅₀) of 4,217 μM. We also evaluated the cytotoxic effects of nicotinamide on HuhT7 cells using dimethylthiazol carboxymethoxyphenyl sulfophenyl tetrazolium (MTS) assays. Cell viability was compared to that of dimethyl sulfoxide (DMSO)-treated controls. As depicted in Fig. 2B, nicotinamide was confirmed to have no cytotoxic activity in the range of 1 to 15,000 μM.

FIG 2 — Nicotinamide inhibits HAV HM175 genotype IB subgenomic replicon replication and HAV HA11-1299 genotype IIIA replication in a dose-dependent manner. HuhT7 cells were transfected with the HAV HM175 genotype IB subgenomic replicon and were then treated with nicotinamide (A and B) at the indicated concentrations for 48 h. Luciferase activity was measured 72 h after transfection (A), and cell viability was determined by an MTS assay (B). Huh7 and PLC/PRF/5 cells were infected with HAV HA11-1299 genotype IIIA and treated with nicotinamide (C to F) for 72 h. HAV RNA levels were measured by real-time RT-PCR (C and D), and cell viabilities were determined by MTS assay (E and F). Actin mRNA was used as an internal control. Data are presented means and standard deviations of triplicate determinations from at least three independent experiments. Statistical significance was analyzed using the two-tailed Student’s t test (*, P < 0.05; **, P < 0.01).

We measured HAV RNA levels using real-time reverse transcription-PCR (RT-PCR) in HAV HA11-1299 genotype IIIA-infected Huh7 or PLC/PRF/5 cells, which were treated with nicotinamide at 1 to 10,000 μM or 1,000 μM, respectively, for 72 h. As depicted in Fig. 2C, HAV RNA levels were significantly inhibited upon treatment with nicotinamide in Huh7 cells. Nicotinamide treatment at concentrations of 5,000 μM and 10,000 μM resulted in 47% and >60% reductions in HAV RNA levels in Huh7 cells, respectively (Fig. 2C). Similarly, 1,000 μM nicotinamide inhibited HAV RNA levels in PLC/PRF/5 cells (Fig. 2D). As depicted in Fig. 2E and F, nicotinamide did not impact cell viability at concentrations of 5,000 μM or lower in Huh7 and PLC/PRF/5 cells. These results showed that nicotinamide treatment leads to significant inhibition of HAV RNA levels in human hepatoma cell lines.

Nicotinamide significantly inhibited the activity of the transcription factor AP-1 and phosphorylated c-Jun.

To examine the intracellular mechanisms modulated by nicotinamide in hepatoma cells, we investigated whether 20,398 human proteins interacted with nicotinamide using artificial intelligence (AI)-based reverse LIGHTHOUSE (lead identification with a graph-ensemble network for arbitrary targets by harnessing only underlying primary sequence) analysis. There was no protein with an interaction score higher than 5.5 and a direct relationship between the protein and the IRES (Table 2). Adenosine receptor A3 (ADORA3) had the highest interaction score (6.15), indicating a strong bonding force of >6.0, and a higher confidence score (0.88), indicating a strong reliability of >0.8 (Table 2). ADORA3 is the only protein that has an interaction score greater than 6.0. Matot et al. reported that ADORA3 attenuated indices of injury and apoptosis and increased phosphorylated c-Jun amino-terminal protein kinase (JNK) and extracellular signal-regulated kinase 1/2 (ERK1/2) levels (26).

TABLE 2.

Selected in silico screening data for the 20,398 human proteins binding to nicotinamide using reverse LIGHTHOUSE analysis

Protein ID^a	Protein name	Confidence score	Interaction score
P0DMS8	Adenosine receptor A3	0.877780571	6.149171524
Q9H2K2	Poly(ADP-ribose) polymerase tankyrase-2	0.812458765	5.878513312
P19099	Cytochrome P450 11B2, mitochondrial	0.806174599	5.848099454
Q8TDS4	Hydroxycarboxylic acid receptor 2	0.785626032	5.787384103
O43193	Motilin receptor	0.825488792	5.770445917
Q969F8	KiSS-1 receptor	0.71792964	5.687440897
Q16790	Carbonic anhydrase 9	0.769611007	5.662385462
P16473	Thyrotropin receptor	0.872089937	5.645568509
P15086	Carboxypeptidase B	0.845349137	5.629515171
O95271	Poly(ADP-ribose) polymerase tankyrase-1	0.872812972	5.623320568
Q96IY4	Carboxypeptidase B2	0.845858602	5.535316351
Q9BZJ3	Tryptase delta	0.402899298	5.513584428
P55201	Peregrin	0.555773551	5.51349062
Q9P055	JNK1/MAPK8-associated membrane protein	0.593854041	4.517494312
Q9UQF2	c-Jun-amino-terminal kinase-interacting protein 1	0.826267542	4.471963235
Q9Y228	TRAF3-interacting JNK-activating modulator	0.60724404	4.373996297
Q13387	c-Jun-amino-terminal kinase-interacting protein 2	0.574289127	4.327412491
Q9UPT6	c-Jun-amino-terminal kinase-interacting protein 3	0.58371957	4.31537217
P27361	Mitogen-activated protein kinase 3	0.803732835	4.292801051
O60271	c-Jun-amino-terminal kinase-interacting protein 4	0.791630873	3.924898823

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UniProt accession ID (https://www.uniprot.org; accessed 13 December 2022).

In the present study, we focused on the transcription factor AP-1 and phosphorylated c-Jun, a member of the AP-1 transcription complex, as AP-1 regulates the transcription of a wide variety of genes involved in cellular immunity and viral replication (21, 22). We investigated the effect of nicotinamide on AP-1 and phosphorylated c-Jun using a luciferase assay and Western blot analysis, respectively. Nicotinamide inhibited AP-1 activity compared to the control in Huh7 and PLC/PRF/5 cells (Fig. 3A and B). We also observed that nicotinamide inhibited the expression of phosphorylated c-Jun and total c-Jun by Western blot analysis (Fig. 3C and D).

FIG 3 — Nicotinamide inhibits AP-1 activity and c-Jun expression in hepatoma cells. Huh7 and PLC/PRF/5 cells were transfected with the pAP-1-luc plasmid and then treated with nicotinamide (A and B) for 48 h. Luciferase activity for the activation of AP-1 was measured at 72 h after transfection. Western blot analysis of total and phosphorylated c-Jun was performed, and Huh7 cells were treated with nicotinamide at the indicated concentrations (C and D). GAPDH was used as an internal control. (E) Immunofluorescence staining analysis of HAV HM175-18f genotype IB-infected Huh7 cells. Green represents HAV VP1, red represents phosphorylated c-Jun, and blue represents the nuclei stained with Hoechst. Positive immunofluorescence staining for HAV VP1 was observed in HAV-infected cells but not in control cells. HAV HM175-18f genotype IB-infected Huh7 cells treated with 5,000 μM nicotinamide showed reduced HAV VP1 and phosphorylated c-Jun staining. HAV HM175-18f genotype IB-infected Huh7 cells treated with 0.1 μg/mL IFN-α 2a stained with an antibody against HAV VP1 served as the negative control. Statistical significance was analyzed using the two-tailed Student’s t test (*, P < 0.05; **, P < 0.01).

An immunofluorescence study revealed that 5,000 μM nicotinamide inhibited the protein levels of both HAV VP1 and phosphorylated c-Jun in HAV HM175 genotype IB-infected Huh7 cells and that phosphorylated c-Jun and HAV HM175 genotype IB existed in the nucleus and cytoplasm of HAV-infected cells, respectively (Fig. 3E). It is possible that phosphorylated c-Jun may indirectly activate HAV IRES-mediated translation.

The expression of phosphorylated ERK and phosphorylated JNK, which regulate c-Jun transcription activities (27, 28), was inhibited by nicotinamide treatment, as shown by Western blot analysis (Fig. 4A to D). Reverse LIGHTHOUSE analysis demonstrated no protein with an interaction score higher than 4.6 and a direct relationship between nicotinamide and c-Jun, ERK, or JNK (Table 2).

FIG 4 — Nicotinamide inhibits phosphorylated ERK and phosphorylated JNK expression in hepatoma cells. Huh7 cells were treated with nicotinamide for 72 h. Western blot analysis of total and phosphorylated ERK and total and phosphorylated JNK was performed (A and C). GAPDH was used as an internal control. Densitometric scanning results are shown on the right (B and D).

Knockdown of c-Jun inhibited HAV replication in hepatoma cells.

To further confirm the association between c-Jun and HAV HA11-1299 genotype IIIA replication, after the transfection of small interfering RNAs (siRNAs) against c-Jun siRNA (si-c-Jun) or control siRNA (si-C) into Huh7 and PLC/PRF/5 cells, cells were infected with HAV, and HAV RNA levels were measured at 72 h postinfection (Fig. 5A and B). Knockdown of c-Jun significantly inhibited HAV RNA levels compared with si-C transfection in Huh7 cells (Fig. 5C). Similar results were also observed in PLC/PRF/5 cells (Fig. 5D). Thus, we confirmed that c-Jun is involved in the regulation of HAV replication.

In general, AP-1 directly binds to the AP-1-binding DNA sites of AP-1-associated gene promoters and leads to the enhancement of AP-1-associated gene expression. As the HAV genome is RNA, AP-1 is not expected to directly bind to the HAV genome. To explore the mechanism of suppression of HAV IRES-mediated translation through the inhibition of AP-1 activation, we cotransfected si-c-Jun or si-C with pT7-HAV-IRES (Fig. 5E), pSV40-HAV-IRES (Fig. 5F), pGL3-control vector (Fig. 5G) or pSV40-HCV-IRES (Fig. 5H) in HuhT7 or Huh7 cells (Fig. 5E to H). In both cell lines, we observed the inhibition of HAV IRES-mediated translation by the transfection of si-c-Jun compared to that of si-C (Fig. 5E and F).

We also cotransfected Huh7 cells with si-c-Jun or si-C and the pGL3-control vector (Fig. 5G). We did not observe any inhibition of firefly luciferase activity in Huh7 cells transfected with si-c-Jun or si-C. These results showed that c-Jun did not have any impact on SV40 promoter activity (Fig. 5G). Notably, HCV IRES-mediated translation was also inhibited by the transfection of si-c-Jun compared to that of si-C (Fig. 5H). Taken together, these results showed that AP-1 has a positive effect on HAV IRES-mediated translation and that suppression of c-Jun expression can lead to the inhibition of HAV IRES-mediated translation.

Nicotinamide and knockdown of c-Jun significantly inhibited the activity of phosphorylated eIF4G and phosphorylated eIF4E.

To clarify how nicotinamide and knockdown of c-Jun inhibited HAV IRES activity, we investigated the protein expression levels of total and phosphorylated eukaryotic translation initiation factor 4E (phospho-eIF4E), total and phospho-eIF4G, poly (rC)-binding protein 2 (PCBP2/hnRNP-E2), La/SSB, polypyrimidine tract-binding protein (PTB/hnRNP1), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by Western blot analysis. The expression of phosphorylated eIF4E and phosphorylated eIF4G was inhibited in nicotinamide treatment and knockdown of c-Jun as determined by Western blot analysis (Fig. 6A to D). However, the expression levels of total eIF4E, total eIF4G, PCBP2, La/SSB, PTB, and GAPDH were not significantly changed during nicotinamide treatment, as shown by Western blot analysis (Fig. S3A and B).

FIG 6 — Nicotinamide and knockdown of c-Jun inhibits phosphorylated eIF4G and phosphorylated eIF4E expression in hepatoma cells. Huh7 cells were treated with nicotinamide for 72 h. Western blot analysis of phosphorylated eIF4G and phosphorylated eIF4E was performed (A). Transfection of 50 nM c-Jun siRNA (si-c-Jun) or control siRNA (si-C) into Huh7 cells was performed. Western blot analysis of phosphorylated eIF4G and phosphorylated eIF4E was performed (C). GAPDH was used as an internal loading control. Densitometric scanning results are shown on the right (B and D).

Additive effects of nicotinamide on the inhibition of HAV HM175 genotype IB subgenomic replicon replication and HAV HA11-1299 genotype IIIA replication by ribavirin or favipiravir in human hepatoma cell lines.

The polymerase inhibitors ribavirin and favipiravir play a critical role in the inhibition of HAV subgenomic replicon replication (29, 30). First, we examined the additive effects of ribavirin on nicotinamide of HAV subgenomic replicon replication in HuhT7 cells. HAV HM175 genotype IB subgenomic replicon replication of HuhT7 cells treated with 1 μM ribavirin alone or with a combination of 100 μM nicotinamide or 500 μM nicotinamide with 1 μM ribavirin was 100%, 72%, or 63%, respectively (Fig. 7A). The cell viabilities were 100%, 111%, or 112%, respectively (Fig. 7B).

FIG 7 — Additive effects of ribavirin on the inhibition of HAV HM175 genotype IB subgenomic replicon replication and HAV HA11-1299 genotype IIIA replication by nicotinamide. (A) Additive effects of ribavirin on the inhibition of HAV subgenomic replicon replication by nicotinamide in HuhT7 cells. HuhT7 cells were transfected with the HAV HM175 genotype IB subgenomic replicon; after 24 h of transfection, cells were treated with 0 or 1 μM ribavirin in combination with 0, 100, and 500 μM nicotinamide for 48 h, and luciferase activity was measured after 72 h of transfection. (B) Cell viabilities of HuhT7 cells treated with 0 or 1 μM ribavirin in combination with 0, 100, and 500 μM nicotinamide. Huh7 and PLC/PRF/5 cells infected with HAV HA11-1299 genotype IIIA were treated with 0 or 1 μM ribavirin in combination with 0, 100, or 500 μM nicotinamide (C) or 0 or 500 μM nicotinamide (D), respectively, for 72 h, and HAV RNA levels were measured by real-time RT-PCR (C and D). The cell viabilities of Huh7 and PLC/PRF/5 cells treated with 0 or 1 μM ribavirin in combination with 0, 100, and 500 μM nicotinamide (E) or 0 or 500 μM nicotinamide (F) are shown. Statistical significance was analyzed using the two-tailed Student’s t test (*, P < 0.05).

Next, the additive effects of ribavirin on nicotinamide of HAV HA11-1299 genotype IIIA replication in Huh7 and PLC/PRF/5 cells were examined. Compared to the effects of 1 μM ribavirin alone on HAV RNA levels (100%), those of addition of 500 μM nicotinamide to 1 μM ribavirin were 81% and 86%, respectively, in Huh7 and PLC/PRF/5 cells (Fig. 7C and D) and cell viabilities were 98% and 112%, respectively, in Huh7 and PLC/PRF/5 cells (Fig. 7E and F).

Similarly, 10 μM favipiravir alone or the combination of 500 μM nicotinamide and 10 μM favipiravir resulted in HAV subgenomic replicon replication that was 100% and 82% (P = 0.1), respectively, and HuhT7 cell viability that was 100% and 105%, respectively. In addition, HAV RNA levels in Huh7 cells treated with 10 μM favipiravir alone or the combination of 500 μM nicotinamide with 10 μM favipiravir resulted in 100% and 67% (P < 0.01), respectively, and Huh7 cell viability that was 100% and 102%, respectively.

Thus, these results showed that the combination of these drugs, which have different mechanisms of HAV inhibition, induces a dramatic decrease in HAV subgenomic replicon replication in HuhT7 and HAV replication in Huh7 cells compared to nicotinamide alone.

DISCUSSION

We screened 1,158 drugs for their efficacy in inhibiting HAV IRES-mediated translation and identified the efficacy of nicotinamide with respect to its inhibition of HAV HA11-1299 genotype IIIA replication. Our study demonstrated several important findings: (i) HAV IRES-mediated translation was inhibited by nicotinamide; (ii) nicotinamide inhibited AP-1 activation; (iii) total and phosphorylated c-Jun, phosphorylated ERK, and phosphorylated JNK were inhibited by nicotinamide; (iv) the knockdown of c-Jun inhibited HAV replication in human hepatoma cells; (v) nicotinamide and the knockdown of c-Jun inhibited phosphorylated eIF4G and phosphorylated eIF4E; and (vi) the combination of ribavirin or favipiravir with nicotinamide induced an additive inhibition of HAV replication compared to nicotinamide alone. Because no medications are currently available for HAV infection in our country, nicotinamide represents a promising anti-HAV drug.

Some of the 77 selected drugs that had inhibitory effects on HAV IRES-mediated translation showed enhancement of HAV replication (Fig. S1). Similar findings were also observed in previous studies (25). It is possible that some drugs may enhance HAV replication in another way, such as an inhibitory effect on antiviral pathways (31).

Murray and Srinivasan showed that there was 27% and 55% inhibition at 5 mM and 10 mM nicotinamide, respectively, against HIV in vitro (19). Although the IC₅₀ of nicotinamide was calculated to be 4,217 μM in HuhT7 cells in the present study, it does not seem to be a higher concentration than what has been previously observed (32 –34). Nicotinamide administration at adult human doses of up to 3 g/day is well tolerated (34).

HAV HM175 genotype IB IRES-mediated translation was inhibited 0.4-fold by 1 μM nicotinamide, whereas 0.5-fold inhibition of HAV HM175 genotype IB subgenomic replicon replication and HAV HA11-1299 genotype IIIA replication required approximately 5,000 μM nicotinamide. This discrepancy may be due to these cell lines, which have different c-Jun expression levels, and the stable expression system and transient-transfection system in COS7-HAV-IRES and HuhT7 cells transfected with the HAV subgenomic replicon, respectively.

Nicotinamide has been reported to be an oral antimicrobial agent against HBV (17), to block interferon gamma inducible protein-10 (IP-10) and monocyte chemoattractant protein-1 (MCP-1) secretion in enterovirus infections (18), to act as a poly(ADP-ribose) polymerase (PARP) inhibitor in HIV infections (19, 20), and to play an immunomodulatory role in Mycobacterium tuberculosis infections (20). Nicotinamide inhibits HBV promoter activity, possibly by targeting the transcription factors AP-1, C/EBPα, and PPARα (17). We and others have previously reported that HAV 2C and 3A proteins activate the AP-1-associated signaling pathway and that c-Jun genes are induced in HAV HM175/clone 1-infected FrhK4 monkey kidney cells (23, 24), and in this study, HAV HA11-1299 genotype IIIA infection was shown to stimulate phosphorylated c-Jun protein levels compared to uninfected Huh7 cells (Fig. S2A and B).

AP-1 is a dimer composed of members of the c-Jun, c-Fos, ATF, and MAF protein families (35). Initially, AP-1 was discovered as a driver of oncogenic transformation and has since been implicated in a variety of transcriptional mechanisms related to development, cell homeostasis, inflammation, and environmental responses (35, 36). In fact, AP-1 may play important roles in the pathogenesis of hepatitis-associated tumorigenesis and hepatocellular carcinoma (HCC) (37, 38). As shown in Fig. 3A to E, nicotinamide inhibited AP-1 activity and the activation of c-Jun. Phosphorylated ERK and phosphorylated JNK regulate c-Jun transcription activities through the phosphorylation of c-Jun (27, 28). Histone deacetylase (HDAC) inhibitors also suppress c-Jun activities through phosphorylated ERK and phosphorylated JNK inhibition (39). We also revealed by Western blot analysis that the HDAC inhibitor nicotinamide inhibited c-Jun activity by suppressing the expression levels of phosphorylated ERK and phosphorylated JNK proteins (Fig. 4A to D).

si-c-Jun inhibited HAV RNA levels (Fig. 5C and D) and HAV IRES-mediated translation in human hepatoma cell lines (Fig. 5E and F). Moreover, the proteasome inhibitor MG132, which activates JNK and c-Jun (40), significantly upregulated HAV HM175 genotype IB subgenomic replicon replication and HAV HA11-1299 genotype IIIA replication (Fig. S3A and B) and tended to upregulate HAV IRES activities in COS7-HAV-IRES cells (Fig. S3C). Cell viabilities were not significantly changed at the concentrations of MG132 we used (Fig. S3D to F). While we and others reported that La/SSB (41), GAPDH (42), PTB (43), PCBP2 (44), eIF4E (45), and eIF4G (45) could interact with HAV IRES and be associated with HAV replication, there were no significant changes observed between the cell lysates treated with or without nicotinamide (Fig. S4A and B). We observed the inhibition of phosphorylated eIF4G and phosphorylated eIF4E as well as phosphorylated c-Jun in Huh7 cells treated with nicotinamide (Fig. 6A to D). He et al. showed that phosphorylation of eIF4E is regulated by the phosphorylation of ERK (46). While HCV IRES-mediated translation was inhibited, consistent with the phosphorylation levels of both eIF2α and eIF4E (46), phosphorylated eIF4G slightly enhanced c-myc IRES-driven translation (47). It is possible that AP-1 may be involved in the regulation of HAV IRES activity and HAV replication via the inhibition of eIF4G and eIF4E phosphorylation.

We also demonstrated that combination therapy with compounds that have distinct mechanisms of HAV inhibition may be a useful treatment against HAV infection compared to a single drug alone (Fig. 6A, C, and D). Nicotinamide, a sirtuin inhibitor, can also lead to epigenetic changes. Epigenetic changes are involved in HAV IRES-mediated translation, and sirtinol, an inhibitor of sirtuin, can also inhibit HAV replication (25). Further identification of their molecular mechanism and in vivo studies are needed.

Conclusion.

Nicotinamide effectively inhibited HAV IRES-mediated translation and HAV HA11-1299 genotype IIIA replication through the inhibition of c-Jun expression; therefore, nicotinamide could be useful for the control of HAV infection.

MATERIALS AND METHODS

Cell lines and reagents.

Human hepatoma cell lines Huh7, PLC/PRF/5, and HuhT7, a stably transformed derivative of Huh7 expressing T7 RNA polymerase in the cytoplasm (48), were cultured in Roswell Park Memorial Institute medium (RPMI; Sigma-Aldrich, St. Louis, MO, USA) containing 10% heat-inactivated fetal bovine serum (FBS; Sigma-Aldrich), 100 U/mL penicillin, and 100 μg/mL streptomycin (Sigma-Aldrich) in 5% CO₂ at 37°C (29). The African green monkey kidney cell line COS7-HAV-IRES stably expresses the simian virus 40 (SV40) promoter plasmid pSV40-HAV-IRES (25). This HAV IRES was derived from pHM175 genotype IB (kindly provided by Suzanne U. Emerson, National Institutes of Health, Maryland, USA), and firefly luciferase (Fluc) is translated by HAV IRES-mediated translation initiation. COS7-HAV-IRES cells were also cultured in RPMI with 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin (Sigma-Aldrich) in 5% CO₂ at 37°C as described previously (25, 49).

Huh7 and HuhT7 cells were kindly provided by R. Bartenschlager and V. Gauss-Müller, respectively (48, 50). PLC/PRF/5 and COS7 cells were purchased from the National Institutes of Biomedical Innovation, Health and Nutrition JCRB Cell Bank (Ibaraki, Osaka, Japan) (51). The HAV HA11-1299 genotype IIIA strain was used for HAV infection in the present study (29). The HAV polyprotein (HAV strain HM175 18f) was produced by the replication-competent HAV subgenomic replicon pT7-18f-LUC, which contains Fluc and an open reading frame flanked by the first four amino acids of the HAV polyprotein and by the C-terminal 12 amino acids of VP1 (29).

A total of 1,158 antiviral, stem cell differentiation, antidiabetic, and anticancer drugs were purchased from TargetMol (Wellesley Hills, MA, USA). Drugs were solubilized at 100 μM in DMSO and diluted in assay medium to a final concentration of 1 μM for drug screening. Ribavirin and favipiravir (T-705) were purchased from Sigma-Aldrich and Selleck Biotech (Tokyo, Japan), respectively. The concentration of DMSO in each assay well, including all control wells, was 1%. Primary antibodies against phospho-c-Jun (Ser63) (54B3), c-Jun (60A8), and hnRNP E2/PCBP2 (D1S5E) were purchased from Cell Signaling Technology (Danvers, MA, USA), those against eIF4E (A19044), eIF4G1 (A7552), phospho-eIF4E (A19044), phospho-eIF4G1 (A7552), and SSB (A0630) were purchased from ABclonal Technology (Tokyo, Japan), and those against hnRNP1 and tubulin were purchased from Abcam (Cambridge, MA, USA). An antibody against GAPDH (FL-335) was purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Horseradish peroxidase (HRP)-conjugated anti-rabbit IgG secondary antibody (no. 7074) was purchased from Cell Signaling Technology. si-c-Jun (sc-29223) and si-C were purchased from Santa Cruz Biotechnology.

The plasmid pAP-1-luc containing the firefly luciferase reporter gene driven by a promoter element (TATA box) plus the inducible cis enhancer element of 7 repeats of AP-1 (Pathdetect; Stratagene, La Jolla, CA, USA) was used for the determination of AP-1 activation (23). The plasmids pSV40-HAV-IRES and pT7-HAV-IRES were previously described (30). The plasmid pSV40-HCV-IRES, which carries the entire HCV core gene under the translational control of the HCV 5′ UTR and the Fluc gene, followed by the 3′ UTR of HCV genotype 1a, was provided by Martin Krüger (52). The pGL3-control vector, which expresses the Fluc gene under the control of the SV40 promoter, was purchased from Promega (Madison, WI, USA).

Drug screening in COS7 cells stably expressing HAV IRES-luc.

Drug screening was performed in 96-well plates (MS-8096 W; Sumitomo Bakelite Company Limited, Tokyo, Japan). COS7-HAV-IRES cells were plated at a density of 2 × 10⁴ to 2.5 × 10⁴ cells/well. After 24 h, the cells were treated with a 1 μM concentration of each of the 1,158 drugs or DMSO alone. After 24 h, HAV IRES-mediated translation was determined by the luciferase reporter assay (49), and cell viability was determined by an MTS assay (29).

Luciferase reporter assays.

The cells were harvested using reporter lysis buffer (Promega), and luciferase activities were determined with a PicaGene luminescence kit (Toyo Ink, Tokyo, Japan) using a luminometer (AB-2200-R; ATTO, Tokyo, Japan). The luciferase activity fraction (percent) was calculated as follows: [(test compound − blank)/(DMSO − blank)] × 100. Luciferase activities presented are averages from three independent experiments.

Cell viability assays.

For the evaluation of cell viability, an MTS assay was performed using the CellTiter 96 Aqueous One-Solution cell proliferation assay (Promega). Enzyme activity was measured with an iMark microplate reader (Bio-Rad, Hercules, CA, USA) at a wavelength of 490 nm. The cell viability fraction (%) was calculated as follows: [(OD490 nm −test compound)/(OD490 nm −DMSO)] × 100. Data presented are averages from three independent experiments.

Examination of candidate drugs for anti-HAV activity in HAV HA11-1299 genotype IIIA-infected Huh7 cells.

Huh7 cells were seeded 24 h prior to infection at a density of 1 × 10⁵ cells/well in 12-well plates (Iwaki Glass, Tokyo, Japan). The cells were washed twice with phosphate-buffered saline (PBS) (Fujifilm Wako Pure Chemical Corporation, Osaka, Japan) and infected with the HAV HA11-1299 genotype IIIA strain at a multiplicity of infection (MOI) of 0.1 in serum-free RPMI. The HAV inoculum was incubated with hepatoma cells for 6 h, and 0.5 mL of RPMI containing 2% FBS was added. After 24 h of incubation, the cells were washed once with PBS, followed by the addition of 0.5 mL of RPMI containing 5% FBS. Then, 77 selected drugs (1 μM) were individually added to HAV-infected Huh7 cells. After 72 h of incubation, the HAV RNA levels were determined using real-time RT-PCR as outlined below.

RNA extraction and quantification of HAV RNA.

Total cellular RNA was extracted from harvested cells using the RNeasy minikit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. cDNA was synthesized using PrimeScript RT reagent (Perfect Real Time; TaKaRa, Otsu, Japan). The RT reaction was performed at 37°C for 15 min, followed by 95°C for 5 s. For HAV RNA quantification, the following primers were used: sense primer, 5′-AGGCTACGGGTGAAACCTCTTAG-3′, and antisense primer, 5′-GCCGCTGTTACCCTATCCAA-3′ (53). The primers for the quantification of actin mRNA were as follows: sense primer, 5′-CAGCCATGTACGTTGCTATCCAGG-3′, and antisense primer, 5′-AGGTCCAGACGCAGGATGGCATG-3′. Real-time PCR was performed using Power SYBR green master mix (Thermo Fisher Scientific, Tokyo, Japan) with a 7500 fast real-time PCR system (Applied Biosystems, Tokyo, Japan). PCR was performed as follows: 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. Actin was used for normalization, and the data were analyzed by the comparative threshold cycle (C_T) method. Relative quantification of gene expression using the ΔΔC_T method correlated with absolute gene quantification obtained from the standard curve. Each real-time PCR assay was performed in triplicate.

Transfection of the HAV HM175 genotype IB subgenomic replicon into HuhT7 cells and luciferase reporter assays.

Briefly, HuhT7 cells were plated at a density of 1.6 × 10⁵ to 2 × 10⁵ cells/well in a 12-well plate. After 24 h, transfection of 0.2 μg of pT7-18f-luc plasmid (HAV strain HM175 18f) (54) or control plasmid into HuhT7 cells was performed using Effectene transfection reagent (Qiagen) following the manufacturer’s protocol. Twenty-four hours later, the cells were treated with or without 1 to 15,000 μM nicotinamide (Selleckchem, Houston, TX, USA) or 1 μM ribavirin and 100 μM or 500 μM nicotinamide. After 48 h, the cells were harvested using reporter lysis buffer, and luciferase activities were determined.

Infection of Huh7 or PLC/PRF/5 cells with HAV HA11-1299 genotype IIIA.

Huh7 or PLC/PRF/5 cells were seeded 24 h prior to infection at a density of 1 × 10⁵ cells/well in 12-well plates. The cells were washed twice with PBS and infected with HAV HA11-1299 genotype IIIA at an MOI of 0.1 in serum-free RPMI. The HAV inoculum was incubated with the hepatoma cells for 6 h, and 0.5 mL of RPMI containing 2% FBS was added. After 24 h of incubation, the cells were washed once with PBS, followed by the addition of 0.5 mL of RPMI containing 5% FBS. Then, various concentrations of nicotinamide (100 μM and 500 μM) and 1 μM ribavirin were added to HAV-infected Huh7 cells, or 1,000 μM or 500 μM nicotinamide with or without 1 μM ribavirin or 10 μM favipiravir was added to HAV-infected cells. After 72 h of incubation, the HAV RNA levels in the inoculated cells were determined using real-time RT-PCR.

In silico screening for protein binding to nicotinamide using reverse LIGHTHOUSE.

A new AI-based drug discovery platform, lead identification with a graph-ensemble network for arbitrary targets by harnessing only underlying primary sequence (LIGHTHOUSE), was developed by a graph-based deep learning approach for discovery of the hidden principles underlying the association of small-molecule compounds with target proteins (55). We investigated the interaction between nicotinamide [NSC 13128; SMILES: C1 = CC(=CN=C1)C(=O)N] and 20,398 human proteins using reverse LIGHTHOUSE (Qinnovation, Fukuoka, Japan) analysis. Protein ID and names of these proteins are available on the following website: https://docs.google.com/spreadsheets/d/1-0VqKJDzVEIvhAE-5SpzZEBA4tqjHWy-/edit?usp=share_link&ouid=106064632755172112432&rtpof=true&sd=true (accessed 15 December 2022).

Transfection of the plasmid containing AP-1-luc into Huh7 and PLC/PRF/5 cells.

The plasmid pAP-1-luc was transfected into Huh7 and PLC/PRF/5 cells using Effectene transfection reagent following the manufacturer’s protocol. Briefly, cells were plated at a density of 1.6 × 10⁵ to 2 × 10⁵ cells/well in a 12-well plate and transfected with plasmid pAP-1-luc. Twenty-four hours later, the cells were treated with or without 1, 10, 100, 1,000, or 5,000 μM nicotinamide. Forty-eight hours later, the cells were harvested using reporter lysis buffer, and luciferase activities were determined using a luminometer.

Western blot analysis.

Cells were lysed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample loading buffer. The lysates were subjected to SDS-PAGE and transferred onto a nitrocellulose membrane. The membranes were blocked with 5% nonfat dried milk in PBS with Tween 20 (Sigma-Aldrich) or 5% bovine serum albumin (BSA) in TBS with Tween 20 and probed with specific primary antibodies. After washing, the blots were incubated with secondary antibody for 1 h. Proteins were detected by using an enhanced chemiluminescence (ECL) Western blot substrate (Prime Western blotting system; Merck, Tokyo, Japan). Membranes were reprobed with an antibody against GAPDH or tubulin as an internal control for normalization of the protein load. ImageJ software (National Institutes of Health [NIH]) was used for densitometric scanning of Western blot images.

Immunofluorescence staining.

Huh7 cells were seeded 24 h prior to infection at a density of 5 × 10⁴ cells/well on coverslips in 24-well plates. The cells were washed twice with PBS (Fujifilm Wako Pure Chemical Corporation) and infected with the HAV HM175 genotype IB strain at an MOI of 0.1 in serum-free RPMI. Then, 5,000 μM nicotinamide or 0.1 μg/mL alpha interferon 2a (IFN-α; Sigma-Aldrich) was added to HAV-infected Huh7 cells. After 24 h of incubation, the cells were washed once with PBS, followed by the addition of 500 μL of RPMI containing 5% FBS with 5,000 μM nicotinamide or 0.1 μg/mL IFN-α 2a. After 72 h of infection, cells were fixed with 4% paraformaldehyde for 10 min at room temperature. After the removal of paraformaldehyde, the cells were washed with PBS twice, followed by cell membrane permeabilization with Triton X-100. After the removal of Triton X-100, the cells were washed with PBS twice. Subsequently, the cells were blocked using a blocking solution (3% BSA in PBS) for 30 min at room temperature and then incubated for 24 h with primary antibodies against HAV VP1 (anti-HAV VP1 antibody [amino acids 7 to 143]; 1:50) (LS-C137674; LifeSpan BioSciences, Washington, USA) and phospho-c-Jun (Ser63) (54B3) (1:150; Cell Signaling Technology), followed by incubation with a secondary antibody, Alexa Fluor 488 F(ab′)₂ fragment of goat anti-mouse IgG (1:500; A-11017; Thermo Fisher Scientific). The nuclei were stained with 5 μg/mL Hoechst 33342 (Sigma-Aldrich) in PBS for 10 min, and the coverslip was mounted using an anti-fluorescent quencher (SlowFade Gold antifade mountant; Thermo Fisher Scientific). Immunofluorescence images were captured under a Keyence BZ-X710 fluorescence microscope (Keyence Corp., Osaka, Japan) using magnification with a 40× objective.

Transfection of siRNAs into Huh7 cells.

The siRNAs si-c-Jun and si-C were transfected into Huh7 cells using Effectene transfection reagent (Qiagen). Huh7 cells were plated at a density of approximately 3 × 10⁵ cells/well in 6-well plates and transfected with 50 nM si-c-Jun or si-C. After 24 h, the cells were infected with the HAV HA11-1299 genotype IIIA strain at an MOI of 0.1 in serum-free RPMI. The HAV inoculum was incubated with hepatoma cells for 6 h, and 0.5 mL of RPMI containing 2% FBS was added. After 24 h of incubation, the cells were washed once with PBS, followed by the addition of 0.5 mL of RPMI containing 5% FBS. After 72 h of transfection, HAV RNA levels in the inoculated cells were determined using real-time RT-PCR, and the levels of c-Jun protein in the inoculated cells were determined using Western blot analysis.

Transfection of pSV40-HAV-IRES with si-c-Jun or si-C into Huh7 cells.

Huh7 cells and HuhT7 cells were cotransfected with si-c-Jun or si-C and pSV40-HAV-IRES, pT7-HAV-IRES, pGL3-control or pSV40-HCV IRES using the transfection reagent Effectene (Qiagen). After 48 h, luciferase activities were determined using a luminometer.

Calculation of IC₅₀.

IC₅₀s are the concentrations of each drug that produce 50% of the maximal inhibitory effect against HAV, which were obtained using the formula 10^{log(A/B) × (50 − C)/(D − C) + log(B)}, where A is the higher concentration at which there was more than 50% inhibition, B is the lower concentration at which there was less than 50% inhibition, C is the HAV RNA level (in percent) at B, and D represents the HAV RNA level (in percent) at A (29).

Statistical analysis.

Data are expressed as means and standard deviations (SD). Statistical analysis was performed using Student’s t test. A P value of <0.05 was considered significant.

ACKNOWLEDGMENTS

We thank Ratna Ray (Saint Louis University, Missouri, USA), Ranjit Ray (Saint Louis University, Missouri, USA), Stanley M. Lemon (University of North Carolina at Chapel Hill, North Carolina, USA), Asuka Hirai-Yuki (National Institute of Infectious Diseases, Tokyo, Japan), Suzanne U. Emerson (National Institute of Allergy and Infectious Diseases, NIH, Maryland, USA), Ralf Bartenschlager (Heidelberg University, Heidelberg, Germany), Verena Gauss-Müller (University of Lübeck, Lübeck, Germany), and Martin Krüger (Medizinische Hochschule Hannover, Hannover, Germany) for generously providing the plasmids and cell lines.

This research was funded by the Japan Agency for Medical Research and Development (AMED), grant JP22fk0210075.

Footnotes

Supplemental material is available online only.

Supplemental file 1

Fig. S1 to S4. Download jvi.01987-22-s0001.pdf, PDF file, 0.5 MB^{(465.8KB, pdf)}

Contributor Information

Reina Sasaki-Tanaka, Email: sasaki.reina@nihon-u.ac.jp.

Tatsuo Kanda, Email: kanda.tatsuo@nihon-u.ac.jp.

Jae U. Jung, Lerner Research Institute, Cleveland Clinic

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Associated Data

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Supplementary Materials

Supplemental file 1

Fig. S1 to S4. Download jvi.01987-22-s0001.pdf, PDF file, 0.5 MB^{(465.8KB, pdf)}

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PERMALINK

Drug Screening for Hepatitis A Virus (HAV): Nicotinamide Inhibits c-Jun Expression and HAV Replication

Reina Sasaki-Tanaka

Ryota Masuzaki

Hiroaki Okamoto

Toshikatsu Shibata

Mitsuhiko Moriyama

Hirofumi Kogure

Tatsuo Kanda

Roles

ABSTRACT

INTRODUCTION

RESULTS

Assays designed to target the HAV IRES in COS7-HAV-IRES cells.

FIG 1.

TABLE 1.

Nicotinamide significantly inhibited HAV HM175 genotype IB subgenomic replicon replication and HAV HA11-1299 genotype IIIA replication in human hepatoma cells.

FIG 2.

Nicotinamide significantly inhibited the activity of the transcription factor AP-1 and phosphorylated c-Jun.

TABLE 2.

FIG 3.

FIG 4.

Knockdown of c-Jun inhibited HAV replication in hepatoma cells.

FIG 5.

Nicotinamide and knockdown of c-Jun significantly inhibited the activity of phosphorylated eIF4G and phosphorylated eIF4E.

FIG 6.

Additive effects of nicotinamide on the inhibition of HAV HM175 genotype IB subgenomic replicon replication and HAV HA11-1299 genotype IIIA replication by ribavirin or favipiravir in human hepatoma cell lines.

FIG 7.

DISCUSSION

Conclusion.

MATERIALS AND METHODS

Cell lines and reagents.

Drug screening in COS7 cells stably expressing HAV IRES-luc.

Luciferase reporter assays.

Cell viability assays.

Examination of candidate drugs for anti-HAV activity in HAV HA11-1299 genotype IIIA-infected Huh7 cells.

RNA extraction and quantification of HAV RNA.

Transfection of the HAV HM175 genotype IB subgenomic replicon into HuhT7 cells and luciferase reporter assays.

Infection of Huh7 or PLC/PRF/5 cells with HAV HA11-1299 genotype IIIA.

In silico screening for protein binding to nicotinamide using reverse LIGHTHOUSE.

Transfection of the plasmid containing AP-1-luc into Huh7 and PLC/PRF/5 cells.

Western blot analysis.

Immunofluorescence staining.

Transfection of siRNAs into Huh7 cells.

Transfection of pSV40-HAV-IRES with si-c-Jun or si-C into Huh7 cells.

Calculation of IC50.

Statistical analysis.

ACKNOWLEDGMENTS

Footnotes

Contributor Information

REFERENCES

Associated Data

Supplementary Materials

ACTIONS

PERMALINK

RESOURCES

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Links to NCBI Databases

Calculation of IC₅₀.