Soluble ORF2 protein enhances HEV replication and induces long-lasting antibody response and protective immunity in vivo.

BACKGROUND AND AIMS: The HEV is a small positive-sense RNA virus that encodes a cytoplasmic form of the capsid protein (ORF2c), essential for virion structure, and a secreted glycosylated form (ORF2s) that accumulates at high titer in serum and can mask neutralizing epitopes. We explored the contribution of ORF2s to HEV replication and its role in generating antibodies against ORF2 in a nonhuman primate model. APPROACH AND RESULTS: We used a recombinant HEV genotype 3 variant that does not express ORF2s due to the introduction of stop codons (ORF2s mut ). Rhesus macaques (RMs) were given intrahepatic injections of infectious wildtype HEV (ORF2s wt ) RNA or a variant lacking ORF2s expression (ORF2s mut ). The replication of the ORF2s mut virus was delayed by ~2 weeks compared with ORF2s wt , and peak titers were nearly tenfold lower. Reversions of the 3 mutations that blocked ORF2s expression were not detected in the ORF2s mut genomes, indicating genetic stability. However, serum antibodies against ORF2 were transiently detected in RMs infected with ORF2s mut , whereas they were long-lasting in RMs infected with ORF2s wt . Moreover, RMs infected with ORF2s mut were more susceptible to reinfection, as evidenced by the viral RNA detected in fecal samples and the expansion of HEV-specific CD8 + T cells. CONCLUSIONS: These findings indicate that ORF2s may be dispensable for viral replication in vivo but is required for long-lived antibody-mediated responses that protect against HEV re-exposure.

The Viral ORF3 Protein Is Required for Hepatitis E Virus Apical Release and Efficient Growth in Polarized Hepatocytes and Humanized Mice.

Hepatitis E virus (HEV), an enterically transmitted RNA virus, is a major cause of acute hepatitis worldwide. Additionally, HEV genotype 3 (gt3) can frequently persist in immunocompromised individuals with an increased risk for developing severe liver disease. Currently, no HEV-specific treatment is available. The viral open reading frame 3 (ORF3) protein facilitates HEV egress in vitro and is essential for establishing productive infection in macaques. Thus, ORF3, which is unique to HEV, has the potential to be explored as a target for antiviral therapy. However, significant gaps exist in our understanding of the critical functions of ORF3 in HEV infection in vivo. Here, we utilized a polarized hepatocyte culture model and a human liver chimeric mouse model to dissect the roles of ORF3 in gt3 HEV release and persistent infection. We show that ORF3's absence substantially decreased HEV replication and virion release from the apical surface but not the basolateral surface of polarized hepatocytes. While wild-type HEV established a persistent infection in humanized mice, mutant HEV lacking ORF3 (ORF3null) failed to sustain the infection despite transient replication in the liver and was ultimately cleared. Strikingly, mice inoculated with the ORF3null virus displayed no fecal shedding throughout the 6-week experiment. Overall, our results demonstrate that ORF3 is required for HEV fecal shedding and persistent infection, providing a rationale for targeting ORF3 as a treatment strategy for HEV infection. IMPORTANCE HEV infections are associated with significant morbidity and mortality. HEV gt3 additionally can cause persistent infection, which can rapidly progress to liver cirrhosis. Currently, no HEV-specific treatments are available. The poorly understood HEV life cycle hampers the development of antivirals for HEV. Here, we investigated the role of the viral ORF3 protein in HEV infection in polarized hepatocyte cultures and human liver chimeric mice. We found that two major aspects of the HEV life cycle require ORF3: fecal virus shedding and persistent infection. These results provide a rationale for targeting ORF3 to treat HEV infection.

An Optimized High-Throughput Neutralization Assay for Hepatitis E Virus (HEV) Involving Detection of Secreted Porf2.

Hepatitis E virus (HEV) is a common cause of acute hepatitis worldwide. Current methods for evaluating the neutralizing activity of HEV-specific antibodies include immunofluorescence focus assays (IFAs) and real-time PCR, which are insensitive and operationally complicated. Here, we developed a high-throughput neutralization assay by measuring secreted pORF2 levels using an HEV antigen enzyme-linked immunosorbent assay (ELISA) kit based on the highly replicating HEV genotype (gt) 3 strain Kernow. We evaluated the neutralizing activity of HEV-specific antibodies and the sera of vaccinated individuals (n = 15) by traditional IFA and the novel assay simultaneously. A linear regression analysis shows that there is a high degree of correlation between the two assays. Furthermore, the anti-HEV IgG levels exhibited moderate correlation with the neutralizing titers of the sera of vaccinated individuals, indicating that immunization with gt 1 can protect against gt 3 Kernow infection. We then determined specificity of the novel assay and the potential threshold of neutralizing capacity using anti-HEV IgG positive sera (n = 27) and anti-HEV IgG negative sera (n = 23). The neutralizing capacity of anti-HEV IgG positive sera was significantly stronger than that of anti-HEV IgG negative. In addition, ROC curve analysis shows that the potential threshold of neutralizing capacity of sera was 8.07, and the sensitivity and specificity of the novel assay was 88.6% and 100%, respectively. Our results suggest that the neutralization assay using the antigen ELISA kit could be a useful tool for HEV clinical research.

Hepatitis E virus persists in the presence of a type III interferon response.

The RIG-I-like RNA helicase (RLR)-mediated interferon (IFN) response plays a pivotal role in the hepatic antiviral immunity. The hepatitis A virus (HAV) and the hepatitis C virus (HCV) counter this response by encoding a viral protease that cleaves the mitochondria antiviral signaling protein (MAVS), a common signaling adaptor for RLRs. However, a third hepatotropic RNA virus, the hepatitis E virus (HEV), does not appear to encode a functional protease yet persists in infected cells. We investigated HEV-induced IFN responses in human hepatoma cells and primary human hepatocytes. HEV infection resulted in persistent virus replication despite poor spread. This was companied by a type III IFN response that upregulated multiple IFN-stimulated genes (ISGs), but type I IFNs were barely detected. Blocking type III IFN production or signaling resulted in reduced ISG expression and enhanced HEV replication. Unlike HAV and HCV, HEV did not cleave MAVS; MAVS protein size, mitochondrial localization, and function remained unaltered in HEV-replicating cells. Depletion of MAVS or MDA5, and to a less extent RIG-I, also diminished IFN production and increased HEV replication. Furthermore, persistent activation of the JAK/STAT signaling rendered infected cells refractory to exogenous IFN treatment, and depletion of MAVS or the receptor for type III IFNs restored the IFN responsiveness. Collectively, these results indicate that unlike other hepatotropic RNA viruses, HEV does not target MAVS and its persistence is associated with continuous production of type III IFNs.

Distinct Entry Mechanisms for Nonenveloped and Quasi-Enveloped Hepatitis E Viruses.

UNLABELLED: The hepatitis E virus (HEV) sheds into feces as nonenveloped virions but circulates in the blood in a membrane-associated, quasi-enveloped form (eHEV). Since the eHEV virions lack viral proteins on the surface, we investigated the entry mechanism for eHEV. We found that compared to nonenveloped HEV virions, eHEV attachment to the cell was much less efficient, requiring a longer inoculation time to reach its maximal infectivity. A survey of cellular internalization pathways identified clathrin-mediated endocytosis as the main route for eHEV entry. Unlike nonenveloped HEV virions, eHEV entry requires Rab5 and Rab7, small GTPases involved in endosomal trafficking, and blocking endosomal acidification abrogated eHEV infectivity. However, low pH alone was not sufficient for eHEV uncoating, suggesting that additional steps are required for entry. Supporting this concept, eHEV infectivity was substantially reduced in cells depleted of Niemann-Pick disease type C1, a lysosomal protein required for cholesterol extraction from lipid, or in cells treated with an inhibitor of lysosomal acid lipase. These data support a model in which the quasi-envelope is degraded within the lysosome prior to virus uncoating, a potentially novel mechanism for virus entry. IMPORTANCE: The recent discovery of quasi-enveloped viruses has shifted the paradigm of virus-host interactions. The impact of quasi-envelopment in the virus life cycle and pathogenesis is largely unknown. HEV is a highly relevant model to study these questions. HEV circulates as quasi-enveloped virions in the blood that are hidden from neutralizing antibodies. eHEV particles most likely are responsible for the cell-to-cell spread of the virus. Given the increasing concerns about persistent HEV infection and its potential for transmission via the blood supply, understanding how eHEV infects cells is important for understanding its pathogenesis and developing therapies. Our data provide evidence that eHEV uses a potentially novel mechanism for cellular entry. Several steps critical to eHEV entry were identified and may provide a basis for developing treatments for hepatitis E. Because quasi-enveloped viruses resemble exosomes, these data also may provide insights into the exosome-mediated intercellular communications.

HECT-E3 ligase ETC-1 regulates securin and cyclin B1 cytoplasmic abundance to promote timely anaphase during meiosis in C. elegans.

The anaphase inhibitor securin plays a crucial role in regulating the timing of sister chromatid separation during mitosis. When sister chromatid pairs become bioriented, the E3 ligase anaphase promoting complex/cyclosome (APC/C) ubiquitylates securin for proteolysis, triggering sister chromatid separation. Securin is also implicated in regulating meiotic progression. Securin protein levels change sharply during cell cycle progression, enabling its timely action. To understand the mechanism underlying the tightly regulated dynamics of securin, we analyzed the subcellular localization of the securin IFY-1 during C. elegans development. IFY-1 was highly expressed in the cytoplasm of germ cells. The cytoplasmic level of IFY-1 declined immediately following meiosis I division and remained low during meiosis II and following mitoses. We identified a C. elegans homolog of another type of E3 ligase, UBE3C, designated ETC-1, as a regulator of the cytoplasmic IFY-1 level. RNAi-mediated depletion of ETC-1 stabilized IFY-1 and CYB-1 (cyclin B1) in post-meiosis I embryos. ETC-1 knockdown in a reduced APC function background caused an embryonic lethal phenotype. In vitro, ETC-1 ubiquitylates IFY-1 and CYB-1 in the presence of the E2 enzyme UBC-18, which functions in pharyngeal development. Genetic analysis revealed that UBC-18 plays a distinct role together with ETC-1 in regulating the cytoplasmic level of IFY-1 during meiosis. Our study reports a novel mechanism, mediated by ETC-1, that co-operates with APC/C to maintain the meiotic arrest required for proper cell cycle timing during reproduction.

Sprouty proteins inhibit receptor-mediated activation of phosphatidylinositol-specific phospholipase C.

Sprouty (Spry) proteins are negative regulators of receptor tyrosine kinase signaling; however, their exact mechanism of action remains incompletely understood. We identified phosphatidylinositol-specific phospholipase C (PLC)-γ as a partner of the Spry1 and Spry2 proteins. Spry-PLCγ interaction was dependent on the Src homology 2 domain of PLCγ and a conserved N-terminal tyrosine residue in Spry1 and Spry2. Overexpression of Spry1 and Spry2 was associated with decreased PLCγ phosphorylation and decreased PLCγ activity as measured by production of inositol (1,4,5)-triphosphate (IP(3)) and diacylglycerol, whereas cells deficient for Spry1 or Spry1, -2, and -4 showed increased production of IP(3) at baseline and further increased in response to growth factor signals. Overexpression of Spry 1 or Spry2 or small-interfering RNA-mediated knockdown of PLCγ1 or PLCγ2 abrogated the activity of a calcium-dependent reporter gene, suggesting that Spry inhibited calcium-mediated signaling downstream of PLCγ. Furthermore, Spry overexpression in T-cells, which are highly dependent on PLCγ activity and calcium signaling, blocked T-cell receptor-mediated calcium release. Accordingly, cultured T-cells from Spry1 gene knockout mice showed increased proliferation in response to T-cell receptor stimulation. These data highlight an important action of Spry, which may allow these proteins to influence signaling through multiple receptors.

Comprehensive genomic screens identify a role for PLZF-RARalpha as a positive regulator of cell proliferation via direct regulation of c-MYC.

The t(11;17)(q23;q21) translocation is associated with a retinoic acid (RA)-insensitive form of acute promyelocytic leukemia (APL), involving the production of reciprocal fusion proteins, promyelocytic leukemia zinc finger-retinoic acid receptor alpha (PLZF-RARalpha) and RARalpha-PLZF. Using a combination of chromatin immunoprecipitation promotor arrays (ChIP-chip) and gene expression profiling, we identify novel, direct target genes of PLZF-RARalpha that tend to be repressed in APL compared with other myeloid leukemias, supporting the role of PLZF-RARalpha as an aberrant repressor in APL. In primary murine hematopoietic progenitors, PLZF-RARalpha promotes cell growth, and represses Dusp6 and Cdkn2d, while inducing c-Myc expression, consistent with its role in leukemogenesis. PLZF-RARalpha binds to a region of the c-MYC promoter overlapping a functional PLZF site and antagonizes PLZF-mediated repression, suggesting that PLZF-RARalpha may act as a dominant-negative version of PLZF by affecting the regulation of shared targets. RA induced the differentiation of PLZF-RARalpha-transformed murine hematopoietic cells and reduced the frequency of clonogenic progenitors, concomitant with c-Myc down-regulation. Surviving RA-treated cells retained the ability to be replated and this was associated with sustained c-Myc expression and repression of Dusp6, suggesting a role for these genes in maintaining a self-renewal pathway triggered by PLZF-RARalpha.