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.

Hepatitis C Virus Envelope Glycoprotein E1 Forms Trimers at the Surface of the Virion.

UNLABELLED: In hepatitis C virus (HCV)-infected cells, the envelope glycoproteins E1 and E2 assemble as a heterodimer. To investigate potential changes in the oligomerization of virion-associated envelope proteins, we performed SDS-PAGE under reducing conditions but without thermal denaturation. This revealed the presence of SDS-resistant trimers of E1 in the context of cell-cultured HCV (HCVcc) as well as in the context of HCV pseudoparticles (HCVpp). The formation of E1 trimers was found to depend on the coexpression of E2. To further understand the origin of E1 trimer formation, we coexpressed in bacteria the transmembrane (TM) domains of E1 (TME1) and E2 (TME2) fused to reporter proteins and analyzed the fusion proteins by SDS-PAGE and Western blotting. As expected for strongly interacting TM domains, TME1-TME2 heterodimers resistant to SDS were observed. These analyses also revealed homodimers and homotrimers of TME1, indicating that such complexes are stable species. The N-terminal segment of TME1 exhibits a highly conserved GxxxG sequence, a motif that is well documented to be involved in intramembrane protein-protein interactions. Single or double mutations of the glycine residues (Gly354 and Gly358) in this motif markedly decreased or abrogated the formation of TME1 homotrimers in bacteria, as well as homotrimers of E1 in both HCVpp and HCVcc systems. A concomitant loss of infectivity was observed, indicating that the trimeric form of E1 is essential for virus infectivity. Taken together, these results indicate that E1E2 heterodimers form trimers on HCV particles, and they support the hypothesis that E1 could be a fusion protein. IMPORTANCE: HCV glycoproteins E1 and E2 play an essential role in virus entry into liver cells as well as in virion morphogenesis. In infected cells, these two proteins form a complex in which E2 interacts with cellular receptors, whereas the function of E1 remains poorly understood. However, recent structural data suggest that E1 could be the protein responsible for the process of fusion between viral and cellular membranes. Here we investigated the oligomeric state of HCV envelope glycoproteins. We demonstrate that E1 forms functional trimers after virion assembly and that in addition to the requirement for E2, a determinant for this oligomerization is present in a conserved GxxxG motif located within the E1 transmembrane domain. Taken together, these results indicate that a rearrangement of E1E2 heterodimer complexes likely occurs during the assembly of HCV particles to yield a trimeric form of the E1E2 heterodimer. Gaining structural information on this trimer will be helpful for the design of an anti-HCV vaccine.

Interacting regions of CD81 and two of its partners, EWI-2 and EWI-2wint, and their effect on hepatitis C virus infection.

CD81 is a tetraspanin protein that is involved in several essential cellular functions, as well as in the hepatitis C virus (HCV) infection. CD81 interacts with a high stoichiometry with its partner proteins EWI-2, EWI-2wint, and EWI-F. These latter proteins modify the functions of CD81 and can thereby potentially inhibit infection or modulate cell migration. Here, we characterized the cleavage of EWI-2 leading to the production of EWI-2wint, which has been shown to inhibit HCV infection. We determined the regions of EWI-2/EWI-2wint and CD81 that are important for their interaction and their functionality. More precisely, we identified a glycine zipper motif in the transmembrane domain of EWI-2/EWI-2wint that is essential for the interaction with CD81. In addition, we found that palmitoylation on two juxtamembranous cysteines in the cytosolic tail of EWI-2/EWI-2wint is required for their interaction with CD81 as well as with CD9, another tetraspanin. Thus, we have shown that palmitoylation of a tetraspanin partner protein can influence the interaction with a tetraspanin. We therefore propose that palmitoylation not only of tetraspanins, but also of their partner proteins is important in regulating the composition of complexes in tetraspanin networks. Finally, we identified the regions in CD81 that are necessary for its functionality in HCV entry and we demonstrated that EWI-2wint needs to interact with CD81 to exert its inhibitory effect on HCV infection.

The CD81 partner EWI-2wint inhibits hepatitis C virus entry.

Two to three percent of the world's population is chronically infected with hepatitis C virus (HCV) and thus at risk of developing liver cancer. Although precise mechanisms regulating HCV entry into hepatic cells are still unknown, several cell surface proteins have been identified as entry factors for this virus. Among these molecules, the tetraspanin CD81 is essential for HCV entry. Here, we have identified a partner of CD81, EWI-2wint, which is expressed in several cell lines but not in hepatocytes. Ectopic expression of EWI-2wint in a hepatoma cell line susceptible to HCV infection blocked viral entry by inhibiting the interaction between the HCV envelope glycoproteins and CD81. This finding suggests that, in addition to the presence of specific entry factors in the hepatocytes, the lack of a specific inhibitor can contribute to the hepatotropism of HCV. This is the first example of a pathogen gaining entry into host cells that lack a specific inhibitory factor.

The alphavirus 6K protein activates endogenous ionic conductances when expressed in Xenopus oocytes.

The Alphavirus Sindbis 6K protein is involved in several functions. It contributes to the processing and membrane insertion of E1 and PE2 viral envelope glycoproteins and to virus budding. It also permeabilizes Escherichia coli and mammalian cells. These viroporin-like properties have been proposed to help virus budding by modifying membrane permeabilities. We expressed Sindbis virus 6K cRNA in Xenopus oocytes to further characterize the effect of 6K on membrane conductances and permeabilization. Although no intrinsic channel properties were seen, cell shrinkage was observed within 24 h. Voltage-clamp experiments showed that 6K upregulated endogenous currents: a hyperpolarization-activated inward current (I (in)) and a calcium-dependent chloride current (I (Cl)). 6K was located at both the plasma and the endoplasmic reticulum membranes. The plasma membrane current upregulation likely results from disruption of the calcium homeostasis of the cell at the endoplasmic reticulum level. Indeed, 6K cRNA expression induced reticular calcium store depletion and capacitative calcium entry activation. By experimental modifications of the incubation medium, we showed that downstream of these events cell shrinkage resulted from a 6K -induced KCl efflux (I (Cl) upregulation leads to chloride efflux, which itself electrically drives potassium efflux), which was responsible for an osmotic water efflux. Our data confirm that 6K specifically triggers a sequential cascade of events that leads to cytoplasmic calcium elevation and cell permeabilization, which likely play a role in the Sindbis virus life cycle.

Contribution of the charged residues of hepatitis C virus glycoprotein E2 transmembrane domain to the functions of the E1E2 heterodimer.

The envelope glycoproteins of Hepatitis C virus (HCV), E1 and E2, form a heterodimer that is retained in the endoplasmic reticulum (ER). The transmembrane (TM) domains play a major role in E1E2 heterodimerization and in ER retention. Two fully conserved charged residues in the middle of the TM domain of E2 (Asp and Arg) are crucial for these functions. Replacement of the Asp residue by a Leu impaired E1E2 heterodimerization, whereas the Arg-to-Leu mutation had a milder effect. Both Asp and Arg residues were shown to contribute to the ER retention function of E2. In addition, the entry function of HCV envelope glycoproteins was affected by these mutations. Together, these data indicate that the charged residues present in the TM domain of E2 play a major role in the biogenesis and the entry function of the E1E2 heterodimer. However, the Asp and Arg residues do not contribute equally to these functions.

Role of N-linked glycans in the functions of hepatitis C virus envelope glycoproteins.

Hepatitis C virus (HCV) encodes two viral envelope glycoproteins. E1 contains 4 or 5 N-linked glycosylation sites and E2 contains up to 11, with most of the sites being well conserved, suggesting that they play an essential role in some functions of these proteins. For this study, we used retroviral pseudotyped particles harboring mutated HCV envelope glycoproteins to study these glycans. The mutants were named with an N followed by a number related to the relative position of the potential glycosylation site in each glycoprotein (E1N1 to E1N4 for E1 mutants and E2N1 to E2N11 for E2 mutants). The characterization of these mutants allowed us to define three phenotypes. For the first group (E1N3, E2N3, E2N5, E2N6, E2N7, and E2N9), the infectivities of the mutants were close to that of the wild type. The second group (E1N1, E1N2, E1N4, E2N1, and E2N11) contained mutants that were still infectious but whose infectivities were reduced to <50% that of the wild type. The third group (E2N2, E2N4, E2N8, and E2N10) contained mutants that had almost totally lost infectivity. The absence of infectivity of the E2N8 and E2N10 mutants was due to the lack of incorporation of the E1E2 heterodimer into HCVpp, which was due to misfolding of the heterodimer, as shown by immunoprecipitation with conformation-sensitive antibodies and by a CD81 pull-down assay. The absence of infectivity of the E2N2 and E2N4 mutants indicated that these two glycans are involved in controlling HCV entry. Altogether, the data indicate that some glycans of HCV envelope glycoproteins play a major role in protein folding and others play a role in HCV entry.

Regulation of hepatitis C virus polyprotein processing by signal peptidase involves structural determinants at the p7 sequence junctions.

The hepatitis C virus genome encodes a polyprotein precursor that is co- and post-translationally processed by cellular and viral proteases to yield 10 mature protein products (C, E1, E2, p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B). Although most cleavages in hepatitis C virus polyprotein precursor proceed to completion during or immediately after translation, the cleavages mediated by a host cell signal peptidase are partial at the E2/p7 and p7/NS2 sites, leading to the production of an E2p7NS2 precursor. The sequences located immediately N-terminally of E2/p7 and p7/NS2 cleavage sites can function as signal peptides. When fused to a reporter protein, the signal peptides of p7 and NS2 were efficiently cleaved. However, when full-length p7 was fused to the reporter protein, partial cleavage was observed, indicating that a sequence located N-terminally of the signal peptide reduces the efficiency of p7/NS2 cleavage. Sequence analyses and mutagenesis studies have also identified structural determinants responsible for the partial cleavage at both the E2/p7 and p7/NS2 sites. Finally, the short distance between the cleavage site of E2/p7 or p7/NS2 and the predicted transmembrane alpha-helix within the P' region might impose additional structural constraints to the cleavage sites. The insertion of a linker polypeptide sequence between P-3' and P-4' of the cleavage site released these constraints and led to improved cleavage efficiency. Such constraints in the processing of a polyprotein precursor are likely essential for hepatitis C virus to post-translationally regulate the kinetics and/or the level of expression of p7 as well as NS2 and E2 mature proteins.

Characterization of the expression of the hepatitis C virus F protein.

Hepatitis C virus (HCV) is an important human pathogen that affects 170 million people worldwide. The HCV genome is approximately 9.6 kb in length and encodes a polyprotein that is proteolytically cleaved to generate at least 10 mature viral protein products. Recently, a new protein, named F, has been described to be expressed through a ribosomal frameshift within the capsid-encoding sequence, a mechanism unique among members of the family Flavidiridae: Here, expression of the F protein was investigated in an in vitro transcription/translation assay. Its expression in mammalian cells was confirmed using specific recombinant vaccinia viruses; under these conditions, protein expression is dependent on the HCV IRES. The F protein was tagged with firefly luciferase or the Myc epitope to facilitate its identification. Ribosomal frameshifting was dependent on the presence of mutations in the capsid-encoding sequence. No frameshifting was detected in the absence of any mutation. Furthermore, analysis of the F protein in time-course experiments revealed that the protein is very unstable and that its production can be stabilized by the proteasome inhibitor MG132. Finally, indirect immunofluorescence studies have localized the F protein in the cytoplasm, with notable perinuclear detection.

Overexpression of an ectopic H19 gene enhances the tumorigenic properties of breast cancer cells.

The maternally expressed H19 gene is transcribed as an untranslated RNA that serves as a riboregulator. We have previously reported that this transcript accumulates in epithelial cells in approximately 10% of breast cancers. To gain further insight on how the overexpression of the H19 gene affects the phenotype of human breast epithelial cells, we investigated the oncogenic potential of RNA that was abundantly expressed from MDA-MB-231 breast cancer cells stably transfected with the genomic sequence of the human H19 gene. The amount of H19 RNA did not affect cell proliferation capacity, timing of cell cycle phases or anchorage-dependent ability of H19-transfected clones in vitro. But in anchorage-independent growth assays the H19-recombined cells formed more and larger colonies in soft-agar versus control cells. To explore this phenotypic change, we analysed tumour development after subcutaneous injection of H19-recombined cells into scid mice. Results showed that H19 overexpression promotes tumour progression. These data support the hypothesis that an overload of H19 transcript is associated with cells exhibiting higher tumorigenic phenotypes and therefore we conclude that the H19 gene has oncogenic properties in breast epithelial cells.

Human monocytic cell lines transformed in vitro by Epstein-Barr virus display a type II latency and LMP-1-dependent proliferation.

Epstein-Barr virus (EBV) classically infects and transforms B lymphocytes in vitro, yielding lymphoblastoid cell lines (LCLs). In contrast to other herpesviruses, EBV is not described as an infectious agent for monocytes. However, recent papers described in vitro infection of monocytes leading to abortive or transient viral expression. In the present study, we report the characterization of E1, a monocytic cell line infected and transformed by EBV. This cell line was derived from an LCL by a drastic electroporation and selection of neomycin-resistant cells, unfavorable to B-cell outgrowth. E1 expressed surface molecules of monocytic lineage (CD14, major histocompatibility complex class II, and CD80) and the c-fms gene, a highly specific marker for the monocytic lineage. This cell line is able to phagocytose and secrete proinflammatory monokines tumor necrosis factor alpha, interleukin-6 (IL-6), and IL-8. E1 cells are tumorigenic after injection in nude mice, and a monocytic cell line obtained from one of these tumors (TE1) displayed immunophenotype and functional properties similar to those of E1. We detected the presence of the EBV genome in both cell lines, as well as expression of the EBNA-1 and LMP-1, but not EBNA-2, viral genes, characteristic of a type II latency. LMP-1 influences the phenotype of these monocytic cell lines, as demonstrated by down-regulation of cell proliferation and membrane intercellular adhesion molecule 1 expression due to an LMP-1 antisense strategy. This is the first description of a latently infected human monocytic cell line and the first direct demonstration of an instrumental role for LMP-1 in the proliferation of EBV-transformed cell lines expressing a type II latency.