Assembly and infection efficacy of hepatitis B virus surface protein exchanges in 8 hepatitis D virus genotype isolates.

BACKGROUND & AIMS: Chronic HDV infections cause the most severe form of viral hepatitis. HDV requires HBV envelope proteins for hepatocyte entry, particle assembly and release. Eight HDV and 8 HBV genotypes have been identified. However, there are limited data on the replication competence of different genotypes and the effect that different HBV envelopes have on virion assembly and infectivity. METHODS: We subcloned complementary DNAs (cDNAs) of all HDV and HBV genotypes and systematically studied HDV replication, assembly and infectivity using northern blot, western blot, reverse-transcription quantitative PCR, and in-cell ELISA. RESULTS: The 8 HDV cDNA clones initiated HDV replication with noticeable differences regarding replication efficacy. The 8 HBV-HBsAg-encoding constructs all supported secretion of subviral particles, however variations in envelope protein stoichiometry and secretion efficacy were observed. Co-transfection of all HDV/HBV combinations supported particle assembly, however, the respective pseudo-typed HDVs differed with respect to assembly kinetics. The most productive combinations did not correlate with the natural geographic distribution, arguing against an evolutionary adaptation of HDV ribonucleoprotein complexes to HBV envelopes. All HDVs elicited robust and comparable innate immune responses. HBV envelope-dependent differences in the activity of the EMA-approved entry inhibitor bulevirtide were observed, however efficient inhibition could be achieved at therapeutically applied doses. Lonafarnib also showed pan-genotypic activity. CONCLUSIONS: HDVs from different genotypes replicate with variable efficacies. Variations in HDV genomes and HBV envelope proteins are both major determinants of HDV egress and entry efficacy, and consequently assembly inhibition by lonafarnib or entry inhibition by bulevirtide. These differences possibly influence HDV pathogenicity, immune responses and the efficacy of novel drug regimens. LAY SUMMARY: HDV requires the envelope protein of HBV for assembly and to infect human cells. We investigated the ability of different HDV genotypes to infect cells and replicate. We also assessed the effect that envelope proteins from different HBV genotypes had on HDV infectivity and replication. Herein, we confirmed that genotypic differences in HDV and HBV envelope proteins are major determinants of HDV assembly, de novo cell entry and consequently the efficacy of novel antivirals.

Nutrient Supply and Simulated Herbivory Differentially Alter the Metabolite Pools and the Efficacy of the Glucosinolate-Based Defense System in Brassica Species.

Environmental stress hinders growth of plants and commonly results in the accumulation of carbon-based defense compounds. However, the dynamics of nitrogen (N)-containing defense compounds are less predictable under environmental stress. The impact of nutrient deficiency on plant defenses that require the metabolic conversion of a less toxic compound to a more potent toxin is even more poorly understood. We evaluated the effects of nitrogen (N) and potassium (K) deficiency and simulated herbivory on the concentration of metabolites including glucosinolates (GSLs), on the conversion of GSLs to more toxic isothiocyanates (ITCs), and on the activity of myrosinase (MYR) in leaves of Brassica juncea and Brassica nigra. Both species contained GSLs, predominantly sinigrin, but also derivatives of glucobrassicin. Compared to the control, N deficiency increased the sinigrin concentration in both species. Methyl jasmonate (MeJA) application increased sinigrin production in B. junceae, whereas in B. nigra MeJA increased sinigrin only under K-deficiency. Compared to the aliphatic-glucosinolates, MeJA application produced a greater compositional change in the profiles of indolic-glucosinolates. In both species the increase in sinigrin content of the tissue was associated with a decrease in its overall nutritive value as assessed by the content of sugars and amino acids. In B. juncea, application of MeJA decreased the conversion of sinigrin to allyl isothiocyanate (AITC) under both N and K deficiency. The potential activity of MYR decreased in both species under N deficiency. The reduced conversion of sinigrin to AITC and the lower activity of MYR suggest that the GSL-ITC defense system might have a limited efficiency in deterring generalist herbivores under environmental stress.

Stem cell-derived polarized hepatocytes.

Human stem cell-derived hepatocyte-like cells (HLCs) offer an attractive platform to study liver biology. Despite their numerous advantages, HLCs lack critical in vivo characteristics, including cell polarity. Here, we report a stem cell differentiation protocol that uses transwell filters to generate columnar polarized HLCs with clearly defined basolateral and apical membranes separated by tight junctions. We show that polarized HLCs secrete cargo directionally: Albumin, urea, and lipoproteins are secreted basolaterally, whereas bile acids are secreted apically. Further, we show that enterically transmitted hepatitis E virus (HEV) progeny particles are secreted basolaterally as quasi-enveloped particles and apically as naked virions, recapitulating essential steps of the natural infectious cycle in vivo. We also provide proof-of-concept that polarized HLCs can be used for pharmacokinetic and drug-drug interaction studies. This novel system provides a powerful tool to study hepatocyte biology, disease mechanisms, genetic variation, and drug metabolism in a more physiologically relevant setting.

Cell Culture Models for Hepatitis E Virus.

Despite a growing awareness, hepatitis E virus (HEV) remains understudied and investigations have been historically hampered by the absence of efficient cell culture systems. As a result, the pathogenesis of HEV infection and basic steps of the HEV life cycle are poorly understood. Major efforts have recently been made through the development of HEV infectious clones and cellular systems that significantly advanced HEV research. Here, we summarize these systems, discussing their advantages and disadvantages for HEV studies. We further capitalize on the need for HEV-permissive polarized cell models to better recapitulate the entire HEV life cycle and transmission.

In vitro wound healing of tumor cells: inhibition of cell migration by selected cytotoxic alkaloids.

BACKGROUND: Cell migration is involved in several pathological processes such as tumor invasion, neoangiogenesis and metastasis. Microtubules are needed in directional migration. METHODS: To investigate the effects of microtubule-binding agents (paclitaxel, vinblastine, colchicine, podophyllotoxin), benzophenanthridine alkaloids (sanguinarine, chelerythrine, chelidonine) and other anti-tumor drugs (homoharringtonine, doxorubicin) on cell migration, we performed the in vitro wound healing assay. The interactions between selected alkaloids and microtubules were studied via U2OS cells expressing microtubule-GFP markers. RESULTS: The microtubule-binding natural products paclitaxel, vinblastine, colchicine and podophyllotoxin significantly altered microtubule dynamics in living cells and inhibited cell migration at concentrations below apparent cytotoxicity. The benzophenanthridine alkaloid sanguinarine, chelerythrine and chelidonine which affected microtubules in living cells, did not inhibit cell migration. Homoharringtonine (protein biosynthesis inhibitor) and doxorubicin significantly inhibited cell migration, however, they did not exert obvious effects on microtubules. CONCLUSION: In this study, we demonstrated that microtubule-binding agents are effective anti-migrating agents; moreover, homoharringtonine and doxorubicin can be referred as anti-migrating agents, but direct microtubule dynamics are not involved in their mode of action. Our study provides evidence that some alkaloids and other microtubule-binding natural products may be interesting candidates for the development of novel agents against metastasis.