The Endocytic Recycling Compartment Serves as a Viral Factory for Hepatitis E Virus

Abstract

Background & Aims: 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 modelling as many positive-strand RNA viruses. HEV genome encodes three proteins, 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 aimed to probe infectious particles and viral factories in HEV-producing cells, using antibodies directed against the different ORF2 isoforms. Methods: We generated monoclonal antibodies that specifically recognize the particle-associated ORF2i form, and antibodies that recognize the different ORF2 isoforms. We used them in confocal and electron microscopy approaches to probe viral factories in HEV-producing cells. We performed an extensive colocalization study of viral proteins with subcellular markers. We analyzed the impact of silencing Rab11, a central player of the endocytic recycling compartment (ERC). Results: One of the antibodies, named P1H1 and targeting the N-terminus of ORF2i, recognized delipidated HEV particles. Confocal and ultrastructural microscopy analyses of HEV-producing cells revealed an unprecedented HEV-induced membrane network containing tubular and vesicular structures. These subcellular structures were enriched in ORF2 and ORF3 proteins, and were dependent on the ORF3 expression and ORF2i capsid protein assembly. Colocalization and silencing analyses revealed that these structures are derived from the ERC. Conclusions: Our study reveals that 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

“…No colocalization was found between V5-tagged ORF1 and PACSIN2 (PCC = 0.22 ± 0.11, Figure 9F and Supplementary Figure 3D ). In addition, contrary to a previous report ( Szkolnicka et al, 2019 ; Bentaleb et al, 2021 ), no colocalization was found between V5-tagged ORF1 and CD63 or CD81, two markers of multivesicular bodies, while CD81 labeling appeared surrounding the V5-tagged ORF1 signal ( Figure 9F and Supplementary Figures 3E,F ).…”

“…Recently, the ORF2 and ORF3 proteins were reported to colocalize with cellular markers of the endocytic recycling compartment (ERC) in perinuclear substructures ( Bentaleb et al, 2021 ). To further delineate whether the V5-tagged ORF1 was also present in these substructures, we conducted co-labeling experiments of the V5-tagged-ORF1 with ERC markers such as CD71, Rab11, EHD1 and PACSIN2 in PLC3 cells electroporated with p6-V1 ( Figure 9 and Supplementary Figure 3D ).…”

“…In addition, we observed perinuclear aggregations of the ORF1 protein that often coincide with a deformation of the nucleus. Recently, ORF2 and ORF3 proteins as well as cellular markers of the ERC such as Rab11 and CD71 were reported to locate to this region in HEV-producing cells ( Bentaleb et al, 2021 ). Interestingly, we confirmed partial overlaps of the ORF1 protein with ORF2 and ORF3 viral proteins as well as several cellular markers of the ERC, especially in the nugget-like perinuclear region.…”

Characterization of the hepatitis E virus replicase

“…No colocalization was found between V5-tagged ORF1 and PACSIN2 (PCC = 0.22 ± 0.11, Figure 9F, Supplementary Figure 3D). In addition, contrary to a previous report (Szkolnicka et al, 2019;Bentaleb et al, 2021), no colocalization was found between V5-tagged ORF1 and CD63 or CD81, two markers of multivesicular bodies, while CD81 labeling appeared surrounding the V5-tagged ORF1 signal (Figure 9F, Supplementary Figures 3E-F).…”

“…Recently, the ORF2 and ORF3 proteins were reported to colocalize with cellular markers of the endocytic recycling compartment (ERC) in perinuclear substructures (Bentaleb et al, 2021). To further delineate whether the V5-tagged ORF1 was also present in these substructures, we conducted co-labeling experiments of the V5-tagged-ORF1 with ERC markers such as CD71, Rab11, EHD1 and PACSIN2 in PLC3 cells electroporated with p6-V1 (Figure 9, Supplementary Figure 3D).…”