PPP2R5D promotes hepatitis C virus infection by binding to viral NS5B and enhancing viral RNA replication.

BACKGROUND: Hepatitis C virus (HCV) infection increased the risk of hepatocellular carcinoma. Identification of host factors required for HCV infection will help to unveil the HCV pathogenesis. Adaptive mutations that enable the replication of HCV infectious clones could provide hints that the mutation-carrying viral protein may specifically interact with some cellular factors essential for the HCV life cycle. Previously, we identified D559G mutation in HCV NS5B (RNA dependent RNA polymerase) important for replication of different genotype clones. Here, we searched for the factors that potentially interacted with NS5B and investigated its roles in HCV infection. METHODS: Wild-type-NS5B and D559G-NS5B of HCV genotype 2a clone, J6cc, were ectopically expressed in hepatoma Huh7.5 cells, and NS5B-binding proteins were pulled down and identified by mass spectrometry. The necessity and mode of action of the selected cellular protein for HCV infection were explored by experiments including gene knockout or knockdown, complementation, co-immunoprecipitation (Co-IP), colocalization, virus infection and replication, and enzymatic activity, etc. RESULTS: Mass spectrometry identified a number of cellular proteins, of which protein phosphatase 2 regulatory subunit B'delta (PPP2R5D, the PP2A regulatory B subunit) was one of D559G-NS5B-pulled down proteins and selected for further investigation. Co-IP confirmed that PPP2R5D specifically interacted with HCV NS5B but not HCV Core and NS3 proteins, and D559G slightly enhanced the interaction. NS5B also colocalized with PPP2R5D in the endoplasmic reticulum. Knockdown and knockout of PPP2R5D decreased and abrogated HCV infection in Huh7.5 cells, respectively, while transient and stable expression of PPP2R5D in PPP2R5D-knockout cells restored HCV infection to a level close to that in wild-type Huh7.5 cells. Replicon assay revealed that PPP2R5D promoted HCV replication, but the phosphatase activity and catalytic subunit of PP2A were not affected by NS5B. CONCLUSIONS: PPP2R5D interactes with HCV NS5B and is required for HCV infection in cultured hepatoma cells through facilitating HCV replication.

Accounting for strain variations and resistance mutations in the characterization of hepatitis C NS3 protease inhibitors.

CONTEXT: Natural strain variation and rapid resistance development makes development of broad spectrum hepatitis C virus (HCV) drugs very challenging and evaluation of inhibitor selectivity and resistance must account for differences in the catalytic properties of enzyme variants. OBJECTIVE: To understand how to study selectivity and relationships between efficacy and genotype or resistant mutants for NS3 protease inhibitors. MATERIALS AND METHODS: The catalytic properties of NS3 protease from genotypes 1a, 1b and 3a, and their sensitivities to four structurally and mechanistically different NS3 protease inhibitors have been analysed under different experimental conditions. RESULTS: The optimisation of buffer conditions for each protease variant enabled the comparison of their catalytic properties and sensitivities to the inhibitors. All inhibitors were most effective against genotype 1a protease, with VX-950 having the broadest selectivity. DISCUSSION AND CONCLUSION: A new strategy for evaluation of inhibitors relevant for the discovery of broad spectrum HCV drugs was established.

Prevalence of active hepatitis C virus infections among general public of Lahore, Pakistan.

BACKGROUND: To find out the prevalence of active hepatitis C virus (HCV) infections among general public in Lahore city, since data concerning the prevalence of active HCV in this city is currently unavailable. METHODS: Blood samples were collected randomly from individuals visiting different clinical laboratories in Lahore. Serum was separated and processed by nested PCR qualitative assay for the detection of HCV RNA. The samples were categorized into different age groups on the basis of pre-test questionnaires in order to record the age-wise differences regarding the prevalence of active HCV. Data were analyzed statistically using Chi-Square test. RESULTS: Out of the 4246 blood samples analyzed in this study, 210 were confirmed to be positive for active HCV infection. Gender-wise active HCV prevalence revealed no significant difference [OR = 1.10 CI = (0.83-1.46), p > 0.05]. However, among the age groups the highest prevalence was observed in the age groups 20-29 (7.7%) and 30-39 years (6.4%) with odds of prevalence of 14.8% (OR = 2.48, CI = (1.40-4.38), p < 0.05) and 10.3% (OR = 2.03, CI = (1.10-3.71), respectively. In age groups above 40 years (40-49, 50-59 and >59 years), a decrease in levels of active HCV prevalence was observed. CONCLUSIONS: Among tested samples, 4.9% of the subjects were confirmed to harbour active HCV infections and the "middle aged" population in Lahore was found to be at a higher risk of the HCV ailments compared to both their younger and older peers.

Over-expression and characterization of NS3 and NS5A of Hepatitis C virus genotype 3a.

BACKGROUND: Hepatitis C virus (HCV) is a common and leading cause for liver cirrhosis and hepatocellular carcinoma. Current therapies to treat HCV infection are shown to be partially effective and poorly tolerated. Therefore, ample efforts are underway to rationally design therapies targeting the HCV non-structural proteins. Most of the work carried out in this direction has been focusing mainly on HCV genotype 1. Two direct-acting antiviral agents (DAAs) Telaprevir and Boceprevir are being used against genotype 1a infection in combination therapy with interferon and ribavirin. Unfortunately these DAAs are not effective against genotype 3a. Considering the wide spread infection by HCV genotype 3a in developing countries especially South Asia, we have focused on the recombinant production of antiviral drug targets NS3 and NS5A from HCV genotype 3a. These protein targets are to be used for screening of inhibitors. RESULTS: High-level expression of NS3 and NS5A was achieved at 25°C, using ~1 and 0.5 mM Isopropyl ß-D-1-thiogalactopyranoside (IPTG), respectively. Yields of the purified NS3 and NS5A were 4 and 1 mg per liter culture volume, respectively. Although similar amounts of purified NS3 were obtained at 25 and 14°C, specificity constant (Kcat/Km) was somewhat higher at expression temperature of 25°C. Circular dichroism (CD) and Fourier-transform infrared (FT-IR) spectroscopy revealed that both NS3 and NS5A contain a mixture of alpha-helix and beta-sheet secondary structures. For NS3 protein, percentages of secondary structures were similar to the values predicted from homology modeling. CONCLUSIONS: NS3 and NS5A were over-expressed and using Nickel-affinity method both proteins were purified to ~ 95% purity. Yield of the purified NS3 obtained is four fold higher than previous reports. CD spectroscopy revealed that difference in activity of NS3 expressed at various temperatures is not related to changes in global structural features of the protein. Moreover, CD and FT-IR analysis showed that NS3 and NS5A contain both alpha-helical and beta-sheet structures and for NS5A, the proportion is almost equal. The production of NS3 and NS5A in milligram quantities will allow their characterization by biophysical and biochemical means that will help in designing new strategies to fight against HCV infection.

Hepatitis C virus hypervariable region 1 antibodies interrupt E2-SR-B1 interaction to suppress viral infection.

Whether hypervariable region 1 (HVR1)-targeting antibodies elicited during natural hepatitis C virus (HCV) infection contribute to virus clearance and what is the mechanism underlying remain unclear. Here, we demonstrated that treatment of HCV-infected hepatoma Huh7.5 cells with the IgGs purified from 2 of 28 (7.1%) chronic hepatitis C (CHC) patients efficiently controlled the infection, for which genotype 1b HVR1 (1bHVR1)-binding antibody was critical. Moreover, we found that 1bHVR1 peptide was superior to 2aHVR1 in rabbit immunization to elicit antibodies neutralizing genotypes 1a, 2a, 3a, and 4a. The neutralization effect of 1bHVR1 IgG could be augmented by HH-1, an antibody constructed from CHC memory B cells but without binding to HVR1 peptide. Mechanistic studies showed that 1bHVR1 antisera and IgGs disrupted the interaction of E2-SR-B1 receptor. This study highlights the neutralizing activity of HVR1 antibody elicited by CHC patients and generated by HVR1-immunization against the established infections of multiple HCV genotypes.

[Incidence and characteristics of benign liver space-occupying mass in 17 721 patients with chronic hepatitis B: a color Doppler ultrasound-based case-control study].

OBJECTIVE: To analyze the incidence and risk factors of benign liver space-occupying mass in patients with chronic hepatitis B (CHB) and the ultrasound features that differentiate these masses from small hepatocellular carcinoma. METHODS: We retrospectively analyzed the color Doppler and clinical data of 17 721 patients with CHB treated in the Hepatology Unit of Nanfang Hospital between January, 2016 and December, 2017. The data were compared with those of 21629 healthy control subjects undergoing routine physical examination in the Center of Heath Management of Nanfang Hospital during the same period. RESULTS: Compared with the control subjects, the patients with CHB had significantly higher incidences of hepatic cysts (11.8% vs 8.7%, P < 0.05), hepatic hemangioma (8.2% vs 1.6%, P < 0.05) and hepatic cirrhosis nodules (20.6% vs 2.4%, P < 0.05). The incidences of hepatic cysts and cirrhosis nodules increased with age and was significantly higher in male than in female patients (P < 0.001). The highest incidence of hepatic hemangioma was found in CHB patients aged 30-49 years without a gender difference (P>0.05). Sonographically, the benign liver masses commonly showed homogeneous echo within the lesion with clear boundaries and regular shape. Hepatic hemangioma was distinctively hyperechoic in 83.32% (1579/1895) of the patients, while small hepatocellular carcinoma presented with weaker peripheral and internal blood flow signals with a lower flow velocity in the arteries and a higher flow velocity in the portal vein. Liver cirrhosis nodules mostly showed a mixture of strong and weak echoes (79.60%; 7637/9595) without blood flow signal within or around the nodule; an increased volume of the nodule accompanied by heterogeneous echoes within the nodule indicated an increased probability of malignant lesion. Hepatic cysts often displayed no echo within the lesion, but the echo could be enhanced posteriorly. CONCLUSIONS: The patients with CHB are at a significantly higher risk of developing hepatic cysts, hepatic hemangiomas and hepatic cirrhosis nodules than the control population, and an older age and the male gender are associated with a higher incidence of hepatic cysts or cirrhosis. The differences in the sonographic and hemodynamic features can help to differentiate hepatic benign mass from malignant lesions, and kinetic changes in sonography can be used to monitor potential malignant transformation of the cirrhotic lesions.

Adaptive mutation F772S-enhanced p7-NS4A cooperation facilitates the assembly and release of hepatitis C virus and is associated with lipid droplet enlargement.

Hepatitis C virus (HCV) infection is a major cause of chronic hepatitis and liver cancer worldwide. Adaptive mutations play important roles in the development of the HCV replicon and its infectious clones. We and others have previously identified the p7 mutation F772S and the co-presence of NS4A mutations in infectious HCV full-length clones and chimeric recombinants. However, the underlying mechanism of F772S function remains incompletely understood. Here, we investigated the functional role of F772S using an efficient JFH1-based reporter virus with Core-NS2 from genotype 2a strain J6, and we designated J6-p7/JFH1-4A according to the strain origin of the p7 and NS4A sequences. We found that replacing JFH1-4A with J6-4A (wild-type or mutated NS4A) or genotype 2b J8-4A severely attenuated the viability of J6-p7/JFH1-4A. However, passage-recovered viruses that contained J6-p7 all acquired F772S. Introduction of F772S efficiently rescued the viral spread and infectivity titers of J6-p7/J6-4A, which reached the levels of the original J6-p7/JFH1-4A and led to a concomitant increase in RNA replication, assembly and release of viruses with J6-specific p7 and NS4A. These data suggest that an isolate-specific cooperation existed between p7 and NS4A. NS4A exchange- or substitution-mediated viral attenuation was attributed to the RNA sequence, and no p7-NS4A protein interaction was detected. Moreover, we found that F772S-enhanced p7-NS4A cooperation was associated with the enlargement of intracellular lipid droplets. This study therefore provides new insights into the mechanisms of adaptive mutations and facilitates studies on the HCV life cycle and virus-host interaction.

Identification of weak points of hepatitis C virus NS3 protease inhibitors using surface plasmon resonance biosensor-based interaction kinetic analysis and genetic variants.

To aid the design of next generation hepatitis C virus (HCV) drugs, the kinetics of the interactions between NS3 protease inhibitors and enzyme from genotypes 1a, 1b, and 3a have been characterized. The linear mechanism-based inhibitors VX-950 (telaprevir) and SCH 503034 (boceprevir) benefited from covalent adduct formation. However, the apparent affinities were rather weak (VX-950, K(D)* of 340, 8.5, and 1000 nM for genotypes 1a, 1b and 3a, respectively; SCH 503034, K(D)* of 90 and 3.9 nM for 1b and 3a, respectively). The non-mechanism-based macrocyclic inhibitors BILN-2016 (ciluprevir) and ITMN-191 (danoprevir) had faster association and slower dissociation kinetics, indicating that rigidification is kinetically favorable. ITMN-191 had nanomolar affinities for all genotypes (K(D)* of 0.13, 1.6, and 0.52 nM), suggesting that a broad spectrum drug is conceivable. The data show that macrocyclic scaffolds and mechanism-based inhibition are advantageous but that there is considerable room for improvement of the kinetics of HCV protease targeted drugs.

Comparative molecular dynamics simulation of Hepatitis C Virus NS3/4A protease (Genotypes 1b, 3a and 4b) predicts conformational instability of the catalytic triad in drug resistant strains.

The protease domain of the Hepatitis C Virus (HCV) nonstructural protein 3 (NS3) has been targeted for inhibition by several direct-acting antiviral drugs. This approach has had marked success to treat infections caused by HCV genotype 1 predominant in the USA, Europe, and Japan. However, genotypes 3 and 4, dominant in developing countries, are resistant to a number of these drugs and little progress has been made towards understanding the structural basis of their drug resistivity. We have previously developed a 4D computational methodology, based on 3D structure modeling and molecular dynamics simulation, to analyze the active sites of the NS3 proteases of HCV-1b and 4a in relation to their catalytic activity and drug susceptibility. Here, we improved the methodology, extended the analysis to include genotype 3a (predominant in South Asia including Pakistan), and compared the results of the three genotypes (1b, 3a and 4a). The 4D analyses of the interactions between the catalytic triad residues (His57, Asp81, and Ser139) indicate conformational instability of the catalytic site in HCV-3a and 4a compared to that of HCV-1b NS3 protease. The divergence is gradual and genotype-dependent, with HCV-1b being the most stable, HCV-4a being the most unstable and HCV-3a representing an intermediate state. These results suggest that the structural dynamics behavior, more than the rigid structure, could be related to the altered catalytic activity and drug susceptibility seen in NS3 proteases of HCV-3a and 4a.