The endocytic recycling compartment serves as a viral factory for hepatitis E virus.

Although hepatitis E virus (HEV) is the major leading cause of enterically transmitted viral hepatitis worldwide, many gaps remain in the understanding of the HEV lifecycle. Notably, viral factories induced by HEV have not been documented yet, and it is currently unknown whether HEV infection leads to cellular membrane modeling as many positive-strand RNA viruses. HEV genome encodes the ORF1 replicase, the ORF2 capsid protein and the ORF3 protein involved in virion egress. Previously, we demonstrated that HEV produces different ORF2 isoforms including the virion-associated ORF2i form. Here, we generated monoclonal antibodies that specifically recognize the ORF2i form and antibodies that recognize the different ORF2 isoforms. One antibody, named P1H1 and targeting the ORF2i N-terminus, recognized delipidated HEV particles from cell culture and patient sera. Importantly, AlphaFold2 modeling demonstrated that the P1H1 epitope is exposed on HEV particles. Next, antibodies were used to probe viral factories in HEV-producing/infected cells. By confocal microscopy, we identified subcellular nugget-like structures enriched in ORF1, ORF2 and ORF3 proteins and viral RNA. Electron microscopy analyses revealed an unprecedented HEV-induced membrane network containing tubular and vesicular structures. We showed that these structures are dependent on ORF2i capsid protein assembly and ORF3 expression. An extensive colocalization study of viral proteins with subcellular markers, and silencing experiments demonstrated that these structures are derived from the endocytic recycling compartment (ERC) for which Rab11 is a central player. Hence, HEV hijacks the ERC and forms a membrane network of vesicular and tubular structures that might be the hallmark of HEV infection.

Processing and Subcellular Localization of the Hepatitis E Virus Replicase: Identification of Candidate Viral Factories.

Hepatitis E virus (HEV) is the major cause of acute hepatitis worldwide. HEV is a positive-sense RNA virus expressing three open reading frames (ORFs). ORF1 encodes the ORF1 non-structural polyprotein, the viral replicase which transcribes the full-length genome and a subgenomic RNA that encodes the structural ORF2 and ORF3 proteins. The present study is focused on the replication step with the aim to determine whether the ORF1 polyprotein is processed during the HEV lifecycle and to identify where the replication takes place inside the host cell. As no commercial antibody recognizes ORF1 in HEV-replicating cells, we aimed at inserting epitope tags within the ORF1 protein without impacting the virus replication efficacy. Two insertion sites located in the hypervariable region were thus selected to tolerate the V5 epitope while preserving HEV replication efficacy. Once integrated into the infectious full-length Kernow C-1 p6 strain, the V5 epitopes did neither impact the replication of genomic nor the production of subgenomic RNA. Also, the V5-tagged viral particles remained as infectious as the wildtype particles to Huh-7.5 cells. Next, the expression pattern of the V5-tagged ORF1 was compared in heterologous expression and replicative HEV systems. A high molecular weight protein (180 kDa) that was expressed in all three systems and that likely corresponds to the unprocessed form of ORF1 was detected up to 25 days after electroporation in the p6 cell culture system. Additionally, less abundant products of lower molecular weights were detected in both in cytoplasmic and nuclear compartments. Concurrently, the V5-tagged ORF1 was localized by confocal microscopy inside the cell nucleus but also as compact perinuclear substructures in which ORF2 and ORF3 proteins were detected. Importantly, using in situ hybridization (RNAScope ®), positive and negative-strand HEV RNAs were localized in the perinuclear substructures of HEV-producing cells. Finally, by simultaneous detection of HEV genomic RNAs and viral proteins in these substructures, we identified candidate HEV factories.

In silico and in vitro screening of licensed antimalarial drugs for repurposing as inhibitors of hepatitis E virus.

ABSTRACT: Hepatitis E virus (HEV) infection is emerging in Cameroon and represents one of the most common causes of acute hepatitis and jaundice. Moreover, earlier reports showed evidence of falciparum malaria/HEVcoexistence. Although the Sofosbuvir/Ribavirin combination was recently proposed in the treatment of HEV-infected patients, no specific antiviral drug has been approved so far, thereby urging the search for new therapies. Fortunately, drug repurposing offers a good alternative to this end. In this study, we report the in silico and in vitro activities of 8 licensed antimalarial drugs and two anti-hepatitis C virus agents used as references (Sofosbuvir, and Ribavirin), for repurposing as antiviral inhibitors against HEV. Compounds were docked against five HEV-specific targets including the Zinc-binding non-structural protein (6NU9), RNA-dependent RNA polymerase (RdRp), cryoEM structure of HEV VLP, genotype 1 (6LAT), capsid protein ORF-2, genotype 3 (2ZTN), and the E2s domain of genotype 1 (3GGQ) using the iGEMDOCK software and their pharmacokinetic profiles and toxicities were predicted using ADMETlab2.0. Their in vitro effects were also assessed on a gt 3 p6Gluc replicon system using the luciferase reporter assay. The docking results showed that Sofosbuvir had the best binding affinities with 6NU9 (- 98.22 kcal/mol), RdRp (- 113.86 kcal/mol), 2ZTN (- 106.96 kcal/mol), while Ribavirin better collided with 6LAT (- 99.33 kcal/mol). Interestingly, Lumefantrine showed the best affinity with 3GGQ (-106.05 kcal/mol). N-desethylamodiaquine and Amodiaquine presented higher binding scores with 6NU9 (- 93.5 and - 89.9 kcal/mol respectively vs - 80.83 kcal/mol), while Lumefantrine had the greatest energies with RdRp (- 102 vs - 84.58), and Pyrimethamine and N-desethylamodiaquine had stronger affinities with 2ZTN compared to Ribavirin (- 105.17 and - 102.65 kcal/mol vs - 96.04 kcal/mol). The biological screening demonstrated a significant (P < 0.001) antiviral effect on replication with 1 µM N-desethylamodiaquine, the major metabolite of Amodiaquine. However, Lumefantrine showed no effect at the tested concentrations (1, 5, and 10 µM). The biocomputational analysis of the pharmacokinetic profile of both drugs revealed a low permeability of Lumefantrine and a specific inactivation by CYP3A2 which might partly contribute to the short half-time of this drug. In conclusion, Amodiaquine and Lumefantrine may be good antimalarial drug candidates for repurposing against HEV. Further in vitro and in vivo experiments are necessary to validate these predictions. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s40203-021-00093-y.