Hepatitis A Virus Internal Ribosomal Entry Site Research Articles

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

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.

The sirtuin inhibitor sirtinol inhibits hepatitis A virus (HAV) replication by inhibiting HAV internal ribosomal entry site activity

Epigenetics plays a role in the regulation of gene expression. Epigenetic changes control gene expression at the transcriptional level. Our previous study suggested that the La protein, which is mainly localized in the nucleus, was associated with hepatitis A virus (HAV) internal ribosomal entry site (IRES)-mediated translation and HAV replication. The aim of this study was to investigate whether epigenetic compounds have effects on HAV IRES-mediated translation and HAV replication. Sirtinol, a sirtuin inhibitor, inhibited HAV IRES-mediated translation in COS7-HAV-IRES cells. Treatment with 10 μM sirtinol resulted in a significant reduction in the intracellular RNA levels of HAV HA11-1299 genotype IIIA in Huh7 cells. Epigenetic treatment with a sirtuin inhibitor may represent a new treatment option for HAV infection. In conclusion, epigenetic control was involved in HAV IRES-dependent translation and HAV replication. Special attention should also be paid to underlying viral diseases in the clinical use of epigenetic treatments for malignancies.

Direct-acting Antivirals and Host-targeting Agents against the Hepatitis A Virus.

Hepatitis A virus (HAV) infection is a major cause of acute hepatitis and occasionally leads to acute liver failure in both developing and developed countries. Although effective vaccines for HAV are available, the development of new antivirals against HAV may be important for the control of HAV infection in developed countries where no universal vaccination program against HAV exists, such as Japan. There are two forms of antiviral agents against HAV: direct-acting antivirals (DAAs) and host-targeting agents (HTAs). Studies using small interfering ribonucleic acid (siRNA) have suggested that the HAV internal ribosomal entry site (IRES) is an attractive target for the control of HAV replication and infection. Among the HTAs, amantadine and interferon-lambda 1 (IL-29) inhibit HAV IRES-mediated translation and HAV replication. Janus kinase (JAK) inhibitors inhibit La protein expression, HAV IRES activity, and HAV replication. Based on this review, both DAAs and HTAs may be needed to control effectively HAV infection, and their use should continue to be explored.

The JAK2 inhibitor AZD1480 inhibits hepatitis A virus replication in Huh7 cells

The JAK2 inhibitor AZD1480 has been reported to inhibit La protein expression. We previously demonstrated that the inhibition of La expression could inhibit hepatitis A virus (HAV) internal ribosomal entry-site (IRES)-mediated translation and HAV replication in vitro. In this study, we analyzed the effects of AZD1480 on HAV IRES-mediated translation and replication. HAV IRES-mediated translation in COS7-HAV-IRES cells was inhibited by 0.1–1 μM AZD1480, a dosage that did not affect cell viability. Results showed a significant reduction in intracellular HAV HA11-1299 genotype IIIA RNA levels in Huh7 cells treated with AZD1480. Furthermore, AZD1480 inhibited the expression of phosphorylated-(Tyr-705)-signal transducer and activator of transcription 3 (STAT3) and La in Huh7 cells. Therefore, we propose that AZD1480 can inhibit HAV IRES activity and HAV replication through the inhibition of the La protein.

Suppression of La antigen exerts potential antiviral effects against hepatitis A virus.

BackgroundDespite the development and availability of hepatitis A virus (HAV) vaccine, HAV infection is still a major cause of acute hepatitis that occasionally leads to fatal liver disease. HAV internal ribosomal entry-site (IRES) is one of the attractive targets of antiviral agents against HAV. The aim of the present study is to evaluate the impact of La, one of the cellular proteins, on HAV IRES-mediated translation and HAV replication.Methods and FindingsWe investigated the therapeutic feasibility of siRNAs specific for cellular cofactors for HAV IRES-mediated translation in cell culture. It was revealed that siRNA against La could inhibit HAV IRES activities as well as HAV subgenomic replication. We also found that the Janus kinase (JAK) inhibitors SD-1029 and AG490, which reduce La expression, could inhibit HAV IRES activities as well as HAV replication.ConclusionsInhibition of La by siRNAs and chemical agents could lead to the efficient inhibition of HAV IRES-mediated translation and HAV replication in cell culture models. La might play important roles in HAV replication and is being exploited as one of the therapeutic targets of host-targeting antivirals.

Interleukin-29 suppresses hepatitis A and C viral internal ribosomal entry site-mediated translation.

Our aim was to investigate the effects of interferons (IFNs)-λ (interleukin-29 [IL-29], IL-28A, and IL-28B) on hepatitis C virus (HCV) and hepatitis A virus (HAV) internal ribosomal entry site (IRES)-mediated translation. The effects of these IFNs on HCV/HAV translation from HAV/HCV IRES were investigated using bicistronic reporter constructs. We transfected HCV/HAV IRES constructs into these IFN-expressing cell lines. IL-29 showed stronger inhibition of their IRES-mediated translation. Combining IL-29 with IFN-α or amantadine resulted in stronger inhibition of HAV IRES activity. Our findings demonstrated a novel antiviral effect of IFNs-λ against HAV and HCV through the suppression of IRES-mediated translation.

Internal ribosomal entry‐site activities of clinical isolate‐derived hepatitis A virus and inhibitory effects of amantadine

Little is known about specific naturally-occurring internal ribosomal entry site (IRES) activities of hepatitis A virus (HAV). We examined these activities using the bicistronic reporter assay and the effects of antiviral amantadine against their activities. Six HAV IRES clones from three patients with fulminant hepatitis and three with self-limited acute hepatitis were obtained. The activities of their IRES were analyzed using bicistronic reporter assay in hepatocyte- and non-hepatocyte-derived cell lines, and the potential efficaciousness of the amantadine was examined. One clone from fulminant hepatitis had a deletion in domains III-IV of HAV IRES had higher IRES activities than HM175 in HLE and Huh-7 cells. In Huh-7 cells, amantadine is effective for inhibiting HAV IRES activities, and especially fulminant hepatitis-derived ones. HAV IRES derived from clinical isolates have various activities. Bicistronic reporter assay using clinical isolates may be another useful tool for testing antiviral activities like those of amantadine and the new acridines and hydrazones recently reported.

Hepatitis A virus (HAV) proteinase 3C inhibits HAV IRES-dependent translation and cleaves the polypyrimidine tract-binding protein

Hepatitis A virus (HAV) infection is still an important issue worldwide. A distinct set of viruses encode proteins that enhance viral cap-independent translation initiation driven by an internal ribosome entry site (IRES) and suppress cap-dependent host translation. Unlike cytolytic picornaviruses, replication of HAV does not cause host cell shut off, and it has been questioned whether HAV proteins interfere with its own and/or host translation. HAV proteins were coexpressed in Huh-7 cells with reporter genes whose translation was initiated by either cap-dependent or cap-independent mechanisms. Among the proteins tested, HAV proteinase 3C suppressed viral IRES-dependent translation. Furthermore, 3C cleaved the polypyrimidine tract-binding protein (PTB) whose interaction with the HAV IRES had been demonstrated previously. The combined results suggest that 3C-mediated cleavage of PTB might be involved in down-regulation of viral translation to give way to subsequent viral genome replication.

The Picornavirus Avian Encephalomyelitis Virus Possesses a Hepatitis C Virus-Like Internal Ribosome Entry Site Element

Avian encephalomyelitis virus (AEV) is a picornavirus that causes disease in poultry worldwide, and flocks must be vaccinated for protection. AEV is currently classified within the hepatovirus genus, since its proteins are most closely related to those of hepatitis A virus (HAV). We now provide evidence that the 494-nucleotide-long 5' untranslated region of the AEV genome contains an internal ribosome entry site (IRES) element that functions efficiently in vitro and in mammalian cells. Unlike the HAV IRES, the AEV IRES is relatively short and functions in the presence of cleaved eIF4G and it is also resistant to an inhibitor of eIF4A. These properties are reminiscent of the recently discovered class of IRES elements within certain other picornaviruses, such as porcine teschovirus 1 (PTV-1). Like the PTV-1 IRES, the AEV IRES shows significant similarity to the hepatitis C virus (HCV) IRES in sequence, function, and predicted secondary structure. Furthermore, mutational analysis of the predicted pseudoknot structure at the 3' end of the AEV IRES lends support to the secondary structure we present. AEV is therefore another example of a picornavirus harboring an HCV-like IRES element within its genome, and thus, its classification within the hepatovirus genus may need to be reassessed in light of these findings.

Suppression of hepatitis A virus genome translation and replication by siRNAs targeting the internal ribosomal entry site

Small interfering RNAs (siRNAs) targeting the coding region of hepatitis A virus (HAV) were shown to specifically inhibit viral genome replication. Compared to the coding region, the HAV internal ribosomal entry site (IRES) in the 5′ non-coding region is highly sequence-conserved and folds into stable secondary structures. Here, we report efficient and sustained RNA interference mediated by both RNase III-prepared siRNA (esiRNA) and vector-derived short hairpin RNAs (shRNAs) that are targeted to various domains of the HAV IRES. Using reporter constructs, and the DNA-based HAV replicon system, we found that shRNAs targeting the HAV IRES domains IIIc and V sustainably suppressed genome translation and replication whereas the IRES domains IIIa and IV were resistant to RNA interference. Our study suggests that some HAV IRES domains might be used as a universal and effective target for specific inhibition of HAV infection.

A 25 kDa cleavage product of polypyrimidine tract binding protein (PTB) present in mouse tissues prevents PTB binding to the 5′ untranslated region and inhibits translation of hepatitis A virus RNA

The 5′ untranslated region (5′UTR) of the hepatitis A virus (HAV) genomic RNA contains an internal ribosome entry site (IRES) which interacts with various cellular proteins and facilitates cap-independent translation. We report the interaction of a 25 kDa protein (p25), present in certain murine tissues and most abundantly in mouse kidney, with the HAV 5′UTR. This protein was found to be a cleavage product of the polypyrimidine tract-binding protein (PTB) and competed with it for binding to the HAV 5′UTR RNA. The binding site of p25 overlapped with the reported binding site of PTB. Exogenous addition of partially purified p25 to in vitro translation reactions resulted in the inhibition of HAV IRES-mediated translation, which could be rescued by the addition of purified PTB. These results suggest that p25 is a cleavage product of PTB which binds to the HAV IRES and antagonizes the translation-stimulating activity of PTB. The presence of the 25 kDa cleavage product of PTB may therefore play a role in the inhibition of HAV IRES-mediated translation in mouse tissues.

Activity of the hepatitis A virus IRES requires association between the cap-binding translation initiation factor (eIF4E) and eIF4G.

The question of whether translation initiation factor eIF4E and the complete eIF4G polypeptide are required for initiation dependent on the IRES (internal ribosome entry site) of hepatitis A virus (HAV) has been examined using in vitro translation in standard and eIF4G-depleted rabbit reticulocyte lysates. In agreement with previous publications, the HAV IRES is unique among all picornavirus IRESs in that it was inhibited if translation initiation factor eIF4G was cleaved by foot-and-mouth disease L-proteases. In addition, the HAV IRES was inhibited by addition of eIF4E-binding protein 1, which binds tightly to eIF4E and sequesters it, thus preventing its association with eIF4G. The HAV IRES was also inhibited by addition of m(7)GpppG cap analogue, irrespective of whether the RNA tested was capped or not. Thus, initiation on the HAV IRES requires that eIF4E be associated with eIF4G and that the cap-binding pocket of eIF4E be empty and unoccupied. This suggests two alternative models: (i) initiation requires a direct interaction between an internal site in the IRES and eIF4E/4G, an interaction which involves the cap-binding pocket of eIF4E in addition to any direct eIF4G-RNA interactions; or (ii) it requires eIF4G in a particular conformation which can be attained only if eIF4E is bound to it, with the cap-binding pocket of the eIF4E unoccupied.

Hepatitis A Virus Translation Is Rate-Limiting for Virus Replication in MRC-5 Cells

Translation of hepatitis A virus (HAV) RNA is controlled by an internal ribosome entry site (IRES) located within the 5′ untranslated region (UTR). In some cell types, the characteristically slow growth of HAV may be due to inefficient viral translation. We investigated whether this is true in MRC-5 cells, which are used for vaccine production. We measured the impact of two clusters of mutations in the 5′ UTR on virus translation and replication: the AG group was selected during passage in African green monkey kidney cells, and the MR group was selected during subsequent passage in MRC-5 cells. The efficiency of cap-independent translation was assessed by inserting cDNA encoding an HAV IRES upstream of the chloramphenicol acetyl transferase gene and transcription was drivenin vivoby a hybrid T7/vaccinia virus system. A luciferase gene was inserted upstream of the IRES to serve as an internal control. Each HAV UTR was also inserted into an infectious cDNA clone; the average rate of viral RNA accumulation was determined for each mutant virus. In MRC-5 cells, the rate of virus replication was highly correlated with the efficiency of cap-independent translation (P= 0.006). The MR but not the AG mutations significantly increased both translation and viral RNA accumulation. Reversion of just one MR mutation (687 G to A) eliminated all of the replication-stimulating and translation-enhancing effects of the MR mutations. In the control BS-C-1 cells, there was no discernible correlation between the rate of virus replication and the efficiency of cap-independent translation (P= 0.136): the AG and MR groups combined had a small impact on translation, but no detectable impact on virus replication. We conclude that in MRC-5 cells viral translation is rate-limiting for HAV replication.

Characterization of replication-competent hepatitis A virus constructs containing insertions at the N terminus of the polyprotein.

To determine whether hepatitis A virus (HAV) could tolerate the insertion of exogenous sequences, we constructed full-length HAV cDNAs containing in-frame insertions at the N terminus of the polyprotein and transfected the derived T7 RNA polymerase in vitro transcripts into FRhK-4 cells. Replication of HAVvec1, a construct containing an insertion of 60 nucleotides coding for a polylinker, a 2B/2C cleavage site for HAV protease 3Cpro, and two initiation codons that restored the sequence of the N terminus of the polyprotein, was detected 2 weeks after transfection by indirect immunofluorescence analysis using anti-HAV monoclonal antibodies. Western blot analysis of HAVvec1-infected cells using anti-VP2 and anti-VP4 antibodies failed to detect the expression of the inserted sequences. Insertion of a 24-mer oligonucleotide coding for a FLAG epitope into HAVvec1 resulted in its HAV-mediated expression which was retained upon deletion of a Gln residue from the inserted 2B/2C cleavage site. Western blot analysis using anti-FLAG and anti-VP2 antibodies showed that the FLAG epitope accumulated in infected cells fused to VP0. Replacement of the FLAG epitope with an epitope of the circumsporozoite protein (CSP) of Plasmodium falciparum resulted in its stable HAV-mediated expression for at least six serial passages in FRhK-4 cells. Sedimentation analysis in sucrose density gradients showed that the CSP epitope accumulated in infected cells fused to VP0, forming 80S empty capsids which also contained native VP0. Our data suggest that the HAV internal ribosome entry site can efficiently direct dual initiation of translation of the polyprotein from AUG codons separated by 66 to 78 nucleotides and show that HAV can tolerate insertions at the N terminus of the polyprotein.

Mutations within the 5' nontranslated RNA of cell culture-adapted hepatitis A virus which enhance cap-independent translation in cultured African green monkey kidney cells

Mutations in the 5' nontranslated RNA (5'NTR) of an attenuated, cell culture-adapted hepatitis A virus (HAV), HM175/P16, enhance growth in cultured African green monkey kidney (BS-C-1) cells but not in fetal rhesus monkey kidney (FRhK-4) cells (S. P. Day, P. Murphy, E. A. Brown, and S. M. Lemon, J. Virol. 66: 6533-6540, 1992). To determine whether these mutations enhance cap-independent translation directed by the HAV internal ribosomal entry site (IRES), we compared the translational activities of the 5'NTRs of wild-type and HM175/P16 viruses in two stably transformed cell lines (BT7-H and FRhK-T7) which constitutively express cytoplasmic bacteriophage T7 RNA polymerase and which are derived from BS-C-1 and FRhK-4 cells, respectively. Translational activity was assessed by monitoring expression of a reporter protein, chloramphenicol acetyltransferase (CAT), following transfection with plasmid DNAs containing bicistronic T7 transcriptional units of the form luciferase-5'NTR-CAT. In both cell types, transcripts containing the 5'NTR of HM175/P16 expressed CAT at levels that were 50- to 100-fold lower than transcripts containing the IRES elements of Sabin type 1 poliovirus or encephalomyocarditis virus, confirming the low activity of the HAV IRES. However, in BT7-H cells, transcripts containing the 5'NTR of wild-type virus. This translational enhancement was due to additive effects of a UU deletion at nucleotides 203 and 204 and a U-to-G substitution at nucleotide 687 of HM175/P16. These mutations did not enhance translation in FRhK-T7 or Huh-T7 cells (a T7 polymerase-expressing cell line derived from human hepatoblastoma cells) or in vitro in rabbit reticulocyte lysates. These results demonstrate that mutations in the 5'NTR of a cell culture-adapted HAV enhance viral replication by facilitating cap-independent translation in a cell-type-specific fashion and support the concept that picornaviral host range is determined in part by differences in cellular translation initiation factors.

Low efficiency of the 5' nontranslated region of hepatitis A virus RNA in directing cap-independent translation in permissive monkey kidney cells.

To characterize in vivo the translational control elements present in the 5' nontranslated region (5'NTR) of hepatitis A virus (HAV) RNA, we created an HAV-permissive monkey kidney cell line (BT7-H) that stably expresses T7 RNA polymerase and carries out cytoplasmic transcription of uncapped RNA from transfected DNA containing the T7 promoter. The presence of an internal ribosomal entry site (IRES) within the 5'NTR of HAV was confirmed by using BT7-H cells transcribing bicistronic RNAs in which the 5'NTR was placed within the intercistronic space, controlling translation of a downstream reporter protein (bacterial chloramphenicol acetyltransferase). However, translation directed by the 5'NTR in these bicistronic transcripts and in monocistronic T7 transcripts in which the HAV 5'NTR was placed upstream of the chloramphenicol acetyltransferase coding sequence was very inefficient compared with the translation of monocistronic transcripts containing either the IRES of encephalomyocarditis (EMC) virus or a short nonpicornavirus 5' nontranslated leader sequence. A large deletion within the HAV IRES (delta 355-532) eliminated IRES activity in bicistronic transcripts. In contrast, larger deletions within the IRES in monocistronic transcripts (delta 1-354, delta 1-532, delta 1-633, and delta 158-633) resulted in 4- to 14-fold increases in translation. In the latter case, this was most probably due to a shift from IRES-directed translation to translation initiation by 5'-end-dependent scanning. Translation of RNAs containing either the EMC virus IRES or the nonpicornavirus leader was significantly enhanced by cotransfection of the reporter constructs with pEP2A, which directs transcription of RNA containing the EMC virus IRES fused to the poliovirus 2Apro coding region. This 2Apro enhancement of cap-independent translation suggests a greater availability of limiting cellular translation factors following 2Apro-mediated cleavage of the p220 subunit of the eukaryotic initiation factor eIF-4F and subsequent shutdown of 5' cap-dependent translation. In contrast, pEP2A cotransfection resulted in severe inhibition of translation directed by the HAV IRES in either monocistronic or bicistronic transcripts. This inhibition was due to competition from the EMC virus IRES present in pEP-2A transcripts, as well as the expression of proteolytically active 2Apro. 2Apro-mediated suppression of HAV translation was not seen with transcripts containing large deletions in the HAV IRES (delta 158-633, delta 1-532, or delta 1-633). These data suggest that the HAV IRES may have a unique requirement for intact p220 or that it may be dependent on active expression of another cellular translation factor which is normally present in severely limiting quantities.(ABSTRACT TRUNCATED AT 400 WORDS)

Analysis of hepatitis A virus translation in a T7 polymerase-expressing cell line.

Hepatitis A virus (HAV) exhibits several characteristics which distinguish it from other picornaviruses, including slow growth in cell culture even after adaptation, and lack of host-cell protein synthesis shut-down. Like other picornaviruses, HAV contains a long 5' nontranslated region (NTR) incorporating an internal ribosomal entry site (IRES), which directs cap-independent translation. We compared HAV IRES-initiated translation with translation initiated by the structurally similar encephalomyocarditis virus (EMCV) IRES, using plasmids in which each of the 5'NTRs is linked in-frame with the chloramphenicol acetyltransferase (CAT) gene. Translation was assessed in an HAV-permissive cell line which constitutively expresses T7 RNA polymerase and transcribes high levels of uncapped RNA from these plasmids following transfection. RNAs containing the EMCV IRES were efficiently translated in these cells, while those containing the HAV IRES were translated very poorly. Analysis of translation of these RNAs in the presence of poliovirus protein 2A, which shuts down cap-dependent translation, demonstrated that their translation was cap independent. Our results suggest that the HAV IRES may function poorly in these cells, and that inefficient translation may contribute to the exceptionally slow replication cycle characteristic of cell culture-adapted HAV.

Identification of the Hepatitis A Virus Internal Ribosome Entry Site: In Vivo and in Vitro Analysis of Bicistronic RNAs Containing the HAV 5′ Noncoding Region

Hepatitis A virus (HAV), a RNA virus of positive polarity, contains a long 5′ noncoding region (5′NCR) that lacks the characteristic m7GpppN cap group of most eukaryotic messages. By creating bicistronic constructs that contain the bacterial chloramphenicol acetyltransferase gone followed by the HAV 5′NCR and the luciferase gene we have demonstrated by assaying in vitro and in vivo that ribosome entry for translation initiation occurs via binding to sequences within the HAV 5′NCR. Using mutations created within this region we have identified that the HAV internal ribosome entry site (IRES) is located downstream of nucleotide 45 and including sequences up to nucleotide 734 of the HAV 5′NCR. Translation of a number of mutant constructs both in vitro in a rabbit reticulocyte lysate and in vivo by transfection of the cDNAs into BS-C-1 cells in the presence of the recombinant vaccinia virus, vTF7-3, gave similar results. However, a 4-nucleotide insertion at base 628 showed an increased activity over wild-type when transfected into BS-C-1 cells that was not seen in vitro. This increase in activity correlated with an increase in luciferase gene product as assayed by immunoprecipitations of [ 35S]methionine radiolabeled cells. Comparison of mono- and bicistronic RNAs that were synthesized with or without a m7GpppG cap group showed a competition for ribosome binding when translated in a rabbit reticulocyte lysate system. The presence of the cap group on the RNA 5′ terminus of the RNA led to a greater ability of this RNA to translate than the RNA containing the HAV IRES.