Replication Of Subgenomic Replicon Research Articles

Evaluation of Potential Anti-Hepatitis A Virus 3C Protease Inhibitors Using Molecular Docking.

Hepatitis A virus (HAV) infection is a major cause of acute hepatitis worldwide and occasionally causes acute liver failure and can lead to death in the absence of liver transplantation. Although HAV vaccination is available, the prevalence of HAV vaccination is not adequate in some countries. Additionally, the improvements in public health reduced our immunity to HAV infection. These situations motivated us to develop potentially new anti-HAV therapeutic options. We carried out the in silico screening of anti-HAV compounds targeting the 3C protease enzyme using the Schrodinger Modeling software from the antiviral library of 25,000 compounds to evaluate anti-HAV 3C protease inhibitors. Additionally, in vitro studies were introduced to examine the inhibitory effects of HAV subgenomic replicon replication and HAV HA11-1299 genotype IIIA replication in hepatoma cell lines using luciferase assays and real-time RT-PCR. In silico studies enabled us to identify five lead candidates with optimal binding interactions in the active site of the target HAV 3C protease using the Schrodinger Glide program. In vitro studies substantiated our hypothesis from in silico findings. One of our lead compounds, Z10325150, showed 47% inhibitory effects on HAV genotype IB subgenomic replicon replication and 36% inhibitory effects on HAV genotype IIIA HA11-1299 replication in human hepatoma cell lines, with no cytotoxic effects at concentrations of 100 μg/mL. The effects of the combination therapy of Z10325150 and RNA-dependent RNA polymerase inhibitor, favipiravir on HAV genotype IB HM175 subgenomic replicon replication and HAV genotype IIIA HA11-1299 replication showed 64% and 48% inhibitory effects of HAV subgenomic replicon and HAV replication, respectively. We identified the HAV 3C protease inhibitor Z10325150 through in silico screening and confirmed the HAV replication inhibitory activity in human hepatocytes. Z10325150 may offer the potential for a useful HAV inhibitor in severe hepatitis A.

Functional and structural studies of Chikungunya Virus nsP1 Protein

Chikungunya virus (CHIKV) is a single-stranded, positive-sense RNA virus of the Alphavirus genus. CHIKV is an arbovirus, whose spread is mediated by Aedes species mosquitos and is associated with debilitating joint pain and febrile symptoms in infected humans. A lack of vaccine or specific antivirals, combined with increasing global spread, has facilitated the re-emergence of CHIKV in recent years. Our research focuses on the CHIKV encoded non-structural protein-1 (nsP1), which has methyltransferase activity and is essential for virus genome replication. Through a combination of structural, biochemical and reverse genetic approaches, we aim to investigate the relationship between the molecular structure and both canonical and non-canonical functions of nsP1, at different stages of CHIKV replication. We demonstrated that substitution of an in-frame methionine (M24), towards the N-terminal of nsP1, severely inhibits CHIKV replication in a host cell-dependent manner. Specifically, we demonstrated that an M24>A substitution had no significant effect on sub-genomic replicon replication but blocks production of infectious CHIKV virions – suggesting a role in later stages of virus replication, such as packaging or egress, rather than genome replication or translation. Utilising such a reverse genetic approach, analysis of a panel of M24 substitutions has improved our understanding on non-canonical yet essential functions of nsP1 during CHIKV replication. In order to further elucidate both the structural and biochemical basis for the observed mutant phenotypes, we developed a system for bacterial expression and purification of recombinant CHIKV nsP1 and have established crystallography trails, prior to analysis of the molecular structure by X-ray crystallography.

Mosaic gene network modelling identified new regulatory mechanisms in HCV infection

Modelling of gene networks is widely used in systems biology to study the functioning of complex biological systems. Most of the existing mathematical modelling techniques are useful for analysis of well-studied biological processes, for which information on rates of reactions is available. However, complex biological processes such as those determining the phenotypic traits of organisms or pathological disease processes, including pathogen-host interactions, involve complicated cross-talk between interacting networks. Furthermore, the intrinsic details of the interactions between these networks are often missing. In this study, we developed an approach, which we call mosaic network modelling, that allows the combination of independent mathematical models of gene regulatory networks and, thereby, description of complex biological systems. The advantage of this approach is that it allows us to generate the integrated model despite the fact that information on molecular interactions between parts of the model (so-called mosaic fragments) might be missing. To generate a mosaic mathematical model, we used control theory and mathematical models, written in the form of a system of ordinary differential equations (ODEs). In the present study, we investigated the efficiency of this method in modelling the dynamics of more than 10,000 simulated mosaic regulatory networks consisting of two pieces. Analysis revealed that this approach was highly efficient, as the mean deviation of the dynamics of mosaic network elements from the behaviour of the initial parts of the model was less than 10%. It turned out that for construction of the control functional, data on perturbation of one or two vertices of the mosaic piece are sufficient. Further, we used the developed method to construct a mosaic gene regulatory network including hepatitis C virus (HCV) as the first piece and the tumour necrosis factor (TNF)-induced apoptosis and NF-κB induction pathways as the second piece. Thus, the mosaic model integrates the model of HCV subgenomic replicon replication with the model of TNF-induced apoptosis and NF-κB induction. Analysis of the mosaic model revealed that the regulation of TNF-induced signaling by the HCV network is crucially dependent on the RIP1, TRADD, TRAF2, FADD, IKK, IκBα, c-FLIP, and BAR genes. Overall, the developed mosaic gene network modelling approach demonstrated good predictive power and allowed the prediction of new regulatory nodes in HCV action on apoptosis and the NF-κB pathway. Those theoretical predictions could be a basis for further experimental verification.

Intracellular Effects of the Hepatitis C Virus Nucleoside Polymerase Inhibitor RO5855 (Mericitabine Parent) and Ribavirin in Combination

Mericitabine (RG7128) is the prodrug of a highly selective cytidine nucleoside analog inhibitor (RO5855) of the hepatitis C virus (HCV) NS5B RNA-dependent RNA polymerase. This study evaluated the effects of combining RO5855 and ribavirin on HCV replication in the HCV subgenomic replicon by using two drug-drug interaction models. The effects of RO5855 and ribavirin on the intracellular metabolism of each compound, on interferon-stimulated gene (ISG) expression, and on the viability of hepatocyte-derived cells were also investigated. RO5855 and ribavirin had additive inhibitory activities against HCV subgenomic replicon replication in drug-drug interaction analyses. RO5855 did not affect the uptake or phosphorylation of ribavirin in primary human hepatocytes, human peripheral blood mononuclear cells, or genotype 1b (G1b) replicon cells. Similarly, ribavirin did not affect the concentrations of intracellular species derived from RO5855 in primary human hepatocytes or the formation of the triphosphorylated metabolites of RO5855. Ribavirin at concentrations of >40 μM significantly reduced the viability of primary hepatocytes but not of Huh7, the G1b replicon, or interferon-cured Huh7 cells. RO5855 alone or with ribavirin did not significantly alter the viability of Huh7 or G1b replicon cells, and it did not significantly affect the viability of primary hepatocytes when it was administered alone. The viability of primary hepatocytes was reduced when they were incubated with RO5855 and ribavirin, similar to the effects of ribavirin alone. RO5855 alone or with ribavirin had no effect on ISG mRNA levels in any of the cells tested. In conclusion, RO5855 did not show any unfavorable interactions with ribavirin in human hepatocytes or an HCV subgenomic replicon system.

Artemisinin Analogues as Potent Inhibitors of In Vitro Hepatitis C Virus Replication

We reported previously that Artemisinin (ART), a widely used anti-malarial drug, is an inhibitor of in vitro HCV subgenomic replicon replication. We here demonstrate that ART exerts its antiviral activity also in hepatoma cells infected with full length infectious HCV JFH-1. We identified a number of ART analogues that are up to 10-fold more potent and selective as in vitro inhibitors of HCV replication than ART. The iron donor Hemin only marginally potentiates the anti-HCV activity of ART in HCV-infected cultures. Carbon-centered radicals have been shown to be critical for the anti-malarial activity of ART. We demonstrate that carbon-centered radicals-trapping (the so-called TEMPO) compounds only marginally affect the anti-HCV activity of ART. This provides evidence that carbon-centered radicals are not the main effectors of the anti-HCV activity of the Artemisinin. ART and analogues may possibly exert their anti-HCV activity by the induction of reactive oxygen species (ROS). The combined anti-HCV activity of ART or its analogues with L-N-Acetylcysteine (L-NAC) [a molecule that inhibits ROS generation] was studied. L-NAC significantly reduced the in vitro anti-HCV activity of ART and derivatives. Taken together, the in vitro anti-HCV activity of ART and analogues can, at least in part, be explained by the induction of ROS; carbon-centered radicals may not be important in the anti-HCV effect of these molecules.

Analysis of hepatitis C virus resistance to silibinin in vitro and in vivo points to a novel mechanism involving nonstructural protein 4B

Intravenous silibinin (SIL) is an approved therapeutic that has recently been applied to patients with chronic hepatitis C, successfully clearing hepatitis C virus (HCV) infection in some patients even in monotherapy. Previous studies suggested multiple antiviral mechanisms of SIL; however, the dominant mode of action has not been determined. We first analyzed the impact of SIL on replication of subgenomic replicons from different HCV genotypes in vitro and found a strong inhibition of RNA replication for genotype 1a and genotype 1b. In contrast, RNA replication and infection of genotype 2a were minimally affected by SIL. To identify the viral target of SIL we analyzed resistance to SIL in vitro and in vivo. Selection for drug resistance in cell culture identified a mutation in HCV nonstructural protein (NS) 4B conferring partial resistance to SIL. This was corroborated by sequence analyses of HCV from a liver transplant recipient experiencing viral breakthrough under SIL monotherapy. Again, we identified distinct mutations affecting highly conserved amino acid residues within NS4B, which mediated phenotypic SIL resistance also in vitro. Analyses of chimeric viral genomes suggest that SIL might target an interaction between NS4B and NS3/4A. Ultrastructural studies revealed changes in the morphology of viral membrane alterations upon SIL treatment of a susceptible genotype 1b isolate, but not of a resistant NS4B mutant or genotype 2a, indicating that SIL might interfere with the formation of HCV replication sites. Mutations conferring partial resistance to SIL treatment in vivo and in cell culture argue for a mechanism involving NS4B. This novel mode of action renders SIL an attractive candidate for combination therapies with other directly acting antiviral drugs, particularly in difficult-to-treat patient cohorts.

Development of New Sulfur-Containing Conjugated Compounds as Anti-HCV Agents

Various conjugated compounds containing a coumarin moiety and a heterocyclic nucleus were synthesized. Their activity against hepatitis C virus was tested on subgenomic replicon replication in Huh 5-2 cells. Some heterobicycle–coumarin conjugates with the ‒S‒CH2‒ linker were found to possess appealing antiviral activities. The sulfur atom in these conjugated compounds was found to be an essential element to their antiviral activity.

Double-stranded RNA-activated protein kinase inhibits hepatitis C virus replication but may be not essential in interferon treatment

Double-stranded RNA-activated protein kinase (PKR), an interferon (IFN)-stimulated gene, is activated by binding with double-stranded RNA, a putative replicative intermediate of the hepatitis C virus (HCV). Activated PKR phosphorylates the alpha subunit of eukaryotic initiation factor-2 to inhibit the translation of viral protein. We established stable PKR knockdown Huh7 cells using RNA interference and investigated the effect of PKR against HCV replication using a subgenomic replicon that expressed luciferase reporter protein and the JFH1 full-length HCV genome. In stable PKR knockdown cells that harboured a subgenomic replicon, luciferase activity was approximately three times higher than that of control cells, indicating that the subgenomic replicon replicated with a higher efficiency in stable PKR knockdown cells than that in control cells. Furthermore, stable PKR knockdown cells secreted significantly more HCV particles than did control cells after transfection with the full-length HCV genome. The replication of the subgenomic replicon was suppressed by the addition of IFN-alpha in both cells. Although the extent of suppression was significantly lower in stable PKR knockdown than control cells using a low concentration (2.5-5 U/ml) of IFN-alpha, even 10 U/ml IFN-alpha suppressed the replication of subgenomic replicon by >98% in both cells. Double-stranded RNA-activated protein kinase plays an important role in suppressing HCV replication in an innate state, but may not be essential in IFN therapy.

A Conserved Proline between Domains II and III of Hepatitis C Virus NS5A Influences both RNA Replication and Virus Assembly

We previously demonstrated that two closely spaced polyproline motifs, with the consensus sequence Pro-X-X-Pro-X-Lys/Arg, located between residues 343 to 356 of NS5A, mediated interactions with cellular SH3 domains. The N-terminal motif (termed PP2.1) is only conserved in genotype 1 isolates, whereas the C-terminal motif (PP2.2) is conserved throughout all hepatitis C virus (HCV) isolates, although this motif was shown to be dispensable for replication of the genotype 1b subgenomic replicon. In order to investigate the potential role of these motifs in the viral life cycle, we have undertaken a detailed mutagenic analysis of these proline residues in the context of both genotype 1b (FK5.1) or 2a subgenomic replicons and the genotype 2a infectious clone, JFH-1. We show that the PP2.2 motif is dispensable for RNA replication of all subgenomic replicons and, furthermore, is not required for virus production in JFH-1. In contrast, the PP2.1 motif is only required for genotype 1b RNA replication. Mutation of proline 346 within PP2.1 to alanine dramatically attenuated genotype 1b replicon replication in three distinct genetic backgrounds, but the corresponding proline 342 was not required for replication of the JFH-1 subgenomic replicon. However, the P342A mutation resulted in both a delay to virus release and a modest (up to 10-fold) reduction in virus production. These data point to critical roles for these proline residues at multiple stages in the HCV life cycle; however, they also caution against extrapolation of data from culture-adapted replicons to infectious virus.

Slow Binding Inhibition and Mechanism of Resistance of Non-nucleoside Polymerase Inhibitors of Hepatitis C Virus

The binding affinity of four palm and thumb site representative non-nucleoside inhibitors (NNIs) of HCV polymerase NS5B to wild-type and resistant NS5B polymerase proteins was determined, and the influence of RNA binding on NNI binding affinity was investigated. NNIs with high binding affinity potently inhibited HCV RNA polymerase activity and replicon replication. Among the compounds tested, HCV-796 showed slow binding kinetics to NS5B. The binding affinity of HCV-796 to NS5B increased 27-fold over a 3-h incubation period with an equilibrium Kd of 71 +/- 2 nm. Slow binding kinetics of HCV-796 was driven by slow dissociation from NS5B with a k(off) of 4.9 +/- 0.5 x 10(-4) s(-1). NS5B bound a long, 378-nucleotide HCV RNA oligonucleotide with high affinity (Kd = 6.9 +/- 0.3 nm), whereas the binding affinity was significantly lower for a short, 21-nucleotide RNA (Kd = 155.1 +/- 16.2 nm). The formation of the NS5B-HCV RNA complex did not affect the slow binding kinetics profile and only slightly reduced NS5B binding affinity of HCV-796. The magnitude of reduction of NNI binding affinity for the NS5B proteins with various resistance mutations in the palm and thumb binding sites correlated well with resistance -fold shifts in NS5B polymerase activity and replicon assays. Co-crystal structures of NS5B-Con1 and NS5B-BK with HCV-796 revealed a deep hydrophobic binding pocket at the palm region of NS5B. HCV-796 interaction with the induced binding pocket on NS5B is consistent with slow binding kinetics and loss of binding affinity with mutations at amino acid position 316.

Studies of antiviral activity and cytotoxicity ofWrightia tinctoriaandMorinda citrifolia

Different extracts of leaf parts of Wrightia tinctoria and fruit powder of Morinda citrifolia have been studied against replication of HIV-1(IIIB) in MT-4 cells and HCV in Huh 5.2 cells. Chloroform extract of Wrightia tinctoria exhibited a maximum protection of 48% against the cytopathic effect of HIV-1(IIIB) in MT-4 cells. Fruit juice of Morinda citrifolia exhibited a displayed marked cytotoxic activity in lymphocyte (MT-4) cells (CC50: 0.19 mg/ml). The 50% effective concentration for inhibition of HCV subgenomic replicon replication in Huh 5-2 cells by Morinda citrifolia was 0.98 μg/ml and by chloroform extract of Wrightia tinctoria was 10 μg/ml. The concentration that reduced the growth of exponentially proliferating Huh 5-2 cells by 50% was greater than 50 μg/ml.

Hemin potentiates the anti-hepatitis C virus activity of the antimalarial drug artemisinin

We report that the antimalarial drug artemisinin inhibits hepatitis C virus (HCV) replicon replication in a dose-dependent manner in two replicon constructs at concentrations that have no effect on the proliferation of the exponentially growing host cells. The 50% effective concentration (EC 50) for inhibition of HCV subgenomic replicon replication in Huh 5-2 cells (luciferase assay) by artemisinin was 78 ± 21 μM. Hemin, an iron donor, was recently reported to inhibit HCV replicon replication [mediated by inhibition of the viral polymerase (C. Fillebeen, A.M. Rivas-Estilla, M. Bisaillon, P. Ponka, M. Muckenthaler, M.W. Hentze, A.E. Koromilas, K. Pantopoulos, Iron inactivates the RNA polymerase NS5B and suppresses subgenomic replication of hepatitis C virus, J. Biol. Chem. 280 (2005) 9049–9057.)] at a concentration that had no adverse effect on the host cells. When combined, artemisinin and hemin resulted, over a broad concentration range, in a pronounced synergistic antiviral activity. Also at a concentration (2 μM) that alone had no effect on HCV replication, hemin still potentiated the anti-HCV activity of artemisinin.

408 RNA interference against the regulatory 3′-untranslated region efficiently inhibits HCV subgenomic replicon replication

408 RNA interference against the regulatory 3′-untranslated region efficiently inhibits HCV subgenomic replicon replication

P.177 Repression of interferon regulatory factor 1 by hepatitis C virus core protein results in inhibition of antiviral and immunomodulatory genes

The hepatitis C virus (HCV) 3′nontranslated region (3′NTR) is important for virus infection and replicon replication. Here, we constructed a panel of chimera replicons containing non-structural (NS) and 3′NTR sequences from different HCV strains or types, and examined the requirements for stable replication. A subgenomic replicon chimera comprising the polymerase and 3′NTR from HCV strain Con1, and other non-structural genes from type 1a strain H77, supported stable colony formation and replication in Huh7 cells. However, extending the type 1a sequence to include 132 amino acids of NS5B resulted in a defective HCV replicon. In contrast, a similar chimera containing HCV strain J4 sequences linked in cis to Con1 NS5B and 3′NTR supported stable replication suggesting that the interaction between the NS proteins and the 3′NTR may represent a critical determinant. Lastly, the type 1a 3′NTR from pCV-J4L6S was unable to confer replication when paired with non-structural coding sequences from BB7 or J4 and the 3′NTR from Con1 was unable to confer replication when paired with J4 or H77 sequences. These results highlighted the importance of sequence specific interaction among 3′NTR and two distinct subdomains of the NS coding region as a determinant in supporting stable replication of subgenomic replicons. The results underscore the importance of directly cloning 3′NTR sequences from relevant clinical samples.

The non-immunosuppressive cyclosporin DEBIO-025 is a potent inhibitor of hepatitis C virus replicationin vitro

Cyclosporin A (CsA) inhibits the in vitro replication of HCV subgenomic replicons. We here report on the potent anti-HCV activity of the non-immunosuppressive cyclosporin DEBIO-025. The 50% effective concentration for inhibition of HCV subgenomic replicon replication in Huh 5-2 cells (luciferase assay) by DEBIO-025 was 0.27 +/- 0.03 microg/mL and for CsA 2.8 +/- 0.4 microg/mL. The concentration that reduced the growth of exponentially proliferating Huh 5-2 cells by 50% was greater than 27 microg/mL for DEBIO-025 and 12 +/- 6 microg/mL for CsA, resulting in a selectivity index of approximately 900 for DEBIO-025 and 40 for CsA. The superior activity of DEBIO-025, as compared with CsA, was corroborated by monitoring HCV RNA levels in Huh 5-2, two other HCV subgenomic replicon-containing cell lines, and by monitoring the luciferase signal and viral antigen production in hepatoma cells that had been infected with an infectious full-length chimeric HCV construct. The combination of interferon alpha 2a with either CsA or DEBIO-025 resulted in an additive to slightly synergistic antiviral activity. DEBIO-025, at concentrations of 0.5 and 1 microg/mL, was able to clear cells from their HCV replicon within three to four passages, whereas treatment with CsA at the same concentrations for seven consecutive passages did not result in clearance of the HCV replicon. In conclusion, DEBIO-025, a compound that is also endowed with potent anti-HIV activity and is well tolerated in animals and humans, may form an attractive new option for the therapy of HCV infections, particularly in HCV/HIV co-infected patients.

Sequence requirements for the development of a chimeric HCV replicon system

The hepatitis C virus (HCV) 3′nontranslated region (3′NTR) is important for virus infection and replicon replication. Here, we constructed a panel of chimera replicons containing non-structural (NS) and 3′NTR sequences from different HCV strains or types, and examined the requirements for stable replication. A subgenomic replicon chimera comprising the polymerase and 3′NTR from HCV strain Con1, and other non-structural genes from type 1a strain H77, supported stable colony formation and replication in Huh7 cells. However, extending the type 1a sequence to include 132 amino acids of NS5B resulted in a defective HCV replicon. In contrast, a similar chimera containing HCV strain J4 sequences linked in cis to Con1 NS5B and 3′NTR supported stable replication suggesting that the interaction between the NS proteins and the 3′NTR may represent a critical determinant. Lastly, the type 1a 3′NTR from pCV-J4L6S was unable to confer replication when paired with non-structural coding sequences from BB7 or J4 and the 3′NTR from Con1 was unable to confer replication when paired with J4 or H77 sequences. These results highlighted the importance of sequence specific interaction among 3′NTR and two distinct subdomains of the NS coding region as a determinant in supporting stable replication of subgenomic replicons. The results underscore the importance of directly cloning 3′NTR sequences from relevant clinical samples.

Analysis of hepatitis C virus/classical swine fever virus chimeric 5'NTRs: sequences within the hepatitis C virus IRES are required for viral RNA replication.

Hepatitis C virus (HCV) is classified in the genus Hepacivirus of the family Flaviviridae, whose members have a single-stranded RNA genome of positive polarity, which encodes a single polyprotein. Within this family, HCV is closely related to viruses of the genus Pestivirus, which includes classical swine fever virus (CSFV). Translation of the hepaci- and pestiviral polyprotein is initiated by internal entry of ribosomes, promoted by the 5'NTR. The secondary and tertiary RNA structures of the HCV and pestivirus 5'NTRs are well conserved, despite the fact that their sequences differ significantly from one another. By analogy with other positive-stranded RNA viruses, the 5'NTR of HCV is likely to contain cis-acting determinants for replication as well as the determinants for translation. Studies on both signals could be complicated, as these signals might overlap. In this study, this problem was addressed by constructing chimeric HCV/CSFV 5'NTRs. A two-step analysis of these 5'NTRs was performed: (a) in a translation assay, which provided the possibility to study translation independently of the possible effects on replication; and (b) in a replication assay, in which were studied only the chimeric 5'NTRs for which IRES-dependent translation was demonstrated. An overlap was observed between HCV RNA elements involved in these processes. Exchange of domain II had a minor effect on the translation efficiency of the chimeric 5'NTRs, while replication of subgenomic replicons with these chimeric 5'NTRs was abolished. Exchange of domain III subdomains severely decreased translation activity, while replication was maintained.