Methyltransferase-independent function of enhancer of zeste homologue 2 maintains tumorigenicity induced by human oncogenic papillomavirus and polyomavirus.

Merkel cell polyomavirus (MCV) and high-risk human papillomavirus (HPV) are human tumor viruses that cause Merkel cell carcinoma (MCC) and oropharyngeal squamous cell carcinoma (OSCC), respectively. HPV E7 and MCV large T (LT) oncoproteins target the retinoblastoma tumor suppressor protein (pRb) through the conserved LxCxE motif. We identified enhancer of zeste homolog 2 (EZH2) as a common host oncoprotein activated by both viral oncoproteins through the pRb binding motif. EZH2 is a catalytic subunit of the polycomb 2 (PRC2) complex that trimethylates histone H3 at lysine 27 (H3K27me3). In MCC tissues EZH2 was highly expressed, irrespective of MCV status. Loss-of-function studies revealed that viral HPV E6/E7 and T antigen expression are required for Ezh2 mRNA expression and that EZH2 is essential for HPV(+)OSCC and MCV(+)MCC cell growth. Furthermore, EZH2 protein degraders reduced cell viability efficiently and rapidly in HPV(+)OSCC and MCV(+)MCC cells, whereas EZH2 histone methyltransferase inhibitors did not affect cell proliferation or viability within the same treatment period. These results suggest that a methyltransferase-independent function of EZH2 contributes to tumorigenesis downstream of two viral oncoproteins, and that direct targeting of EZH2 protein expression could be a promising strategy for the inhibition of tumor growth in HPV(+)OSCC and MCV(+)MCC patients.

Longer Poly(U) Stretches in the 3'UTR Are Essential for Replication of the Hepatitis C Virus Genotype 4a Clone in in vitro and in vivo.

The 3' untranslated region (UTR) of the hepatitis C virus (HCV) genome plays a significant role in replication including the poly(U) tract (You and Rice, 2008). Here we established an HCV clone that is infectious in vitro and in vivo, from an Egyptian patient with chronic HCV infection and hepatocellular carcinoma (HCC). First, we inoculated the patient plasma into a humanized chimeric mouse and passaged. We observed HCV genotype 4a propagation in the chimeric mouse sera at 1.7 × 107 copies/mL after 6 weeks. Next, we cloned the entire HCV sequence from the HCV-infected chimeric mouse sera using RT-PCR, and 5' and 3' RACE methodologies. We obtained first a shorter clone (HCV-G4 KM short, GenBank: AB795432.1), which contained 9,545 nucleotides with 341 nucleotides of the 5'UTR and 177 nucleotides of the 3'UTR, and this was frequently obtained for unknown reasons. We also obtained a longer clone by dividing the HCV genome into three fragments and the poly (U) sequences. We obtained a longer 3'UTR sequence than that of the HCV-G4 KM short clone, which contained 9,617 nucleotides. This longer clone possessed a 3'-UTR of 249 nucleotides (HCV-G4 KM long, GenBank: AB795432.2), because of a 71-nucleotide longer poly (U) stretch. The HCV-G4-KM long clone, but not the HCV-G4-KM short clone, could establish infection in human hepatoma HuH-7 cells. HCV RNAs carrying a nanoluciferase (NL) reporter were also constructed and higher replication activity was observed with G4-KM long-NL in vitro. Next, both short and long RNAs were intra-hepatically injected into humanized chimeric mice. Viral propagation was only observed for the chimeric mouse injected with the HCV-G4 KM long RNA in the sera after 21 days (1.64 × 106 copies/mL) and continued until 10 weeks post inoculation (wpi; 1.45-4.74 × 107 copies/mL). Moreover, sequencing of the HCV genome in mouse sera at 6 wpi revealed the sequence of the HCV-G4-KM long clone. Thus, the in vitro and in vivo results of this study indicate that the sequence of the HCV-G4-KM long RNA is that of an infectious clone.

Development of an in vivo delivery system for CRISPR/Cas9-mediated targeting of hepatitis B virus cccDNA.

Chronic hepatitis B virus (HBV) infection constitutes a global health issue with limited current therapeutic efficacy owing to the persistence of viral episomal DNA (cccDNA). The CRISPR/Cas9 system, a newly developed, powerful tool for genome editing and potential gene therapy, requires efficient delivery of CRISPR components for successful therapeutic application. Here, we investigated the effects of lentiviral- or adeno-associated virus 2 (AAV2) vector-mediated delivery of 3 guide (g)RNAs/Cas9 selected from 16 gRNAs. These significantly suppressed HBV replication in cells, with WJ11/Cas9 exhibiting highest efficacy and chosen for in vivo study. AAV2/WJ11-Cas9 also significantly inhibited HBV replication and significantly reduced cccDNA in the tested cells. Moreover, AAV2/WJ11-Cas9 enhanced entecavir effects when used in combination, indicative of different modes of action. Notably, in humanized chimeric mice, AAV2/WJ11-Cas9 significantly suppressed HBcAg, HBsAg, and HBV DNA along with cccDNA in the liver tissues without significant cytotoxicity; accordingly, next generation sequencing data showed no significant genomic mutations. To our knowledge, this represents the first evaluation of the CRISPR/Cas9 system using an HBV natural infection mode. Therefore, WJ11/Cas9 delivered by comparatively safer AAV2 vectors may provide a new therapeutic strategy for eliminating HBV infection and serve as an effective platform for curing chronic HBV infection.

Conversion of Sox2-dependent Merkel cell carcinoma to a differentiated neuron-like phenotype by T antigen inhibition.

Viral cancers show oncogene addiction to viral oncoproteins, which are required for survival and proliferation of the dedifferentiated cancer cell. Human Merkel cell carcinomas (MCCs) that harbor a clonally integrated Merkel cell polyomavirus (MCV) genome have low mutation burden and require viral T antigen expression for tumor growth. Here, we showed that MCV+ MCC cells cocultured with keratinocytes undergo neuron-like differentiation with neurite outgrowth, secretory vesicle accumulation, and the generation of sodium-dependent action potentials, hallmarks of a neuronal cell lineage. Cocultured keratinocytes are essential for induction of the neuronal phenotype. Keratinocyte-conditioned medium was insufficient to induce this phenotype. Single-cell RNA sequencing revealed that T antigen knockdown inhibited cell cycle gene expression and reduced expression of key Merkel cell lineage/MCC marker genes, including HES6, SOX2, ATOH1, and KRT20 Of these, T antigen knockdown directly inhibited Sox2 and Atoh1 expression. MCV large T up-regulated Sox2 through its retinoblastoma protein-inhibition domain, which in turn activated Atoh1 expression. The knockdown of Sox2 in MCV+ MCCs mimicked T antigen knockdown by inducing MCC cell growth arrest and neuron-like differentiation. These results show Sox2-dependent conversion of an undifferentiated, aggressive cancer cell to a differentiated neuron-like phenotype and suggest that the ontology of MCC arises from a neuronal cell precursor.

Characterization of a Merkel Cell Polyomavirus-Positive Merkel Cell Carcinoma Cell Line CVG-1.

Merkel cell polyomavirus (MCV) plays a causal role in ∼80% of Merkel cell carcinomas (MCC). MCV is clonally integrated into the MCC tumor genome, which results in persistent expression of large T (LT) and small T (sT) antigen oncoproteins encoded by the early locus. In MCV-positive MCC tumors, LT is truncated by premature stop codons or deletions that lead to loss of the C-terminal origin binding (OBD) and helicase domains important for replication. The N-terminal Rb binding domain remains intact. MCV-positive cell lines derived from MCC explants have been valuable tools to study the molecular mechanism of MCV-induced Merkel cell carcinogenesis. Although all cell lines have integrated MCV and express truncated LT antigens, the molecular sizes of the LT proteins differ between cell lines. The copy number of integrated viral genome also varies across cell lines, leading to significantly different levels of viral protein expression. Nevertheless, these cell lines share phenotypic similarities in cell morphology, growth characteristics, and neuroendocrine marker expression. Several low-passage MCV-positive MCC cell lines have been established since the identification of MCV. We describe a new MCV-positive MCV cell line, CVG-1, with features distinct from previously reported cell lines. CVG-1 tumor cells grow in more discohesive clusters in loose round cell suspension, and individual cells show dramatic size heterogeneity. It is the first cell line to encode an MCV sT polymorphism resulting in a unique leucine (L) to proline (P) substitution mutation at amino acid 144. CVG-1 possesses a LT truncation pattern near identical to that of MKL-1 cells differing by the last two C-terminal amino acids and also shows an LT protein expression level similar to MKL-1. Viral T antigen knockdown reveals that, like other MCV-positive MCC cell lines, CVG-1 requires T antigen expression for cell proliferation.

Hepatitis C virus attenuates mitochondrial lipid ß-oxidation by downregulating mitochondrial trifunctional-protein expression.

UNLABELLED: The course of hepatitis C virus (HCV) infection and disease progression involves alterations in lipid metabolism, leading to symptoms such as hypocholesterolemia and steatosis. Steatosis can be induced by multiple mechanisms, including increases in lipid biosynthesis and uptake, impaired lipoprotein secretion, and/or attenuation of lipid ß-oxidation. However, little is known about the effects of HCV on lipid ß-oxidation. A previous proteomics study revealed that HCV interacted with both the α- and ß-subunits of the mitochondrial trifunctional protein (MTP), an enzyme complex which catalyzes the last 3 steps of mitochondrial lipid ß-oxidation for cellular energy production. Here we show that in HCV-infected Huh7.5 cells, lipid ß-oxidation was significantly attenuated. Consistently with this, MTP protein and mRNA levels were suppressed by HCV infection. A loss-of-function study showed that MTP depletion rendered cells less responsive to alpha interferon (IFN-α) treatment by impairing IFN-stimulated gene expression. These aspects of host-virus interaction explain how HCV alters host energy homeostasis and how it may also contribute to the establishment of persistent infection in the liver. IMPORTANCE: HCV infection triggers metabolic alterations, which lead to significant disease outcomes, such as fatty liver (steatosis). This study revealed that HCV impairs mitochondrial lipid ß-oxidation, which results in low lipid combustion. On the other hand, the HCV-induced defects in metabolic status played an important role in the control of the type I interferon system. Under the conditions of impaired lipid ß-oxidation, host cells were less responsive to the ability of exogenously added IFN-α to suppress HCV replication. This suggests that interference with lipid ß-oxidation may assist the virus in the establishment of a long-term, persistent infection. Further understanding of this aspect of virus-host interaction may lead to improvements in the current standard therapy.

Hepatitis C virus NS5A inhibits mixed lineage kinase 3 to block apoptosis.

Hepatitis C virus (HCV) infection results in the activation of numerous stress responses including oxidative stress, with the potential to induce an apoptotic state. Previously we have shown that HCV attenuates the stress-induced, p38MAPK-mediated up-regulation of the K(+) channel Kv2.1, to maintain the survival of infected cells in the face of cellular stress. We demonstrated that this effect was mediated by HCV non-structural 5A (NS5A) protein, which impaired p38MAPK activity through a polyproline motif-dependent interaction, resulting in reduction of phosphorylation activation of Kv2.1. In this study, we investigated the host cell proteins targeted by NS5A to mediate Kv2.1 inhibition. We screened a phage-display library expressing the entire complement of human SH3 domains for novel NS5A-host cell interactions. This analysis identified mixed lineage kinase 3 (MLK3) as a putative NS5A interacting partner. MLK3 is a serine/threonine protein kinase that is a member of the MAPK kinase kinase (MAP3K) family and activates p38MAPK. An NS5A-MLK3 interaction was confirmed by co-immunoprecipitation and Western blot analysis. We further demonstrate a novel role of MLK3 in the modulation of Kv2.1 activity, whereby MLK3 overexpression leads to the up-regulation of channel activity. Accordingly, coexpression of NS5A suppressed this stimulation. Additionally we demonstrate that overexpression of MLK3 induced apoptosis, which was also counteracted by NS5A. We conclude that NS5A targets MLK3 with multiple downstream consequences for both apoptosis and K(+) homeostasis.

The C terminus of NS5A domain II is a key determinant of hepatitis C virus genome replication, but is not required for virion assembly and release.

The NS5A protein of hepatitis C virus (HCV) plays roles in both virus genome replication and the assembly of infectious virus particles. NS5A comprises three domains, separated by low-complexity sequences. Whilst the function of domain I appears to be predominantly involved with genome replication, the roles of domains II and III are less well defined. It has been reported previously that a deletion spanning the majority of domain II but retaining the C-terminal 35 residues had no effect on virus production; however, deletion of the entire domain II eliminated genome replication, pointing to a key role for the C terminus of this domain. Recent work has also highlighted this region as the potential binding site of the host factor cyclophilin A (CypA). To define this requirement for replication in more detail, and to investigate the involvement of CypA, we conducted a mutagenic study of the C-terminal 30 residues of domain II within the context of both the infectious JFH-1 virus and a JFH-1-derived subgenomic replicon. We showed that 12 of these residues were absolutely required for virus genome replication, whilst mutations of the remainder either had no phenotype or exhibited a partial reduction in genome replication. There was an absolute correlation between the datasets for virus and subgenomic replicon, indicating that this region is involved solely in the process of genome replication. Comparison of our data with a previously published analysis of the same region in genotype 1b revealed some important differences between the two genotypes of HCV.

Protein kinase D negatively regulates hepatitis C virus secretion through phosphorylation of oxysterol-binding protein and ceramide transfer protein.

Hepatitis C virus (HCV) RNA replicates its genome on specialized endoplasmic reticulum modified membranes termed membranous web and utilizes lipid droplets for initiating the viral nucleocapsid assembly. HCV maturation and/or the egress pathway requires host sphingolipid synthesis, which occur in the Golgi. Ceramide transfer protein (CERT) and oxysterol-binding protein (OSBP) play a crucial role in sphingolipid biosynthesis. Protein kinase D (PKD), a serine/threonine kinase, is recruited to the trans-Golgi network where it influences vesicular trafficking to the plasma membrane by regulation of several important mediators via phosphorylation. PKD attenuates the function of both CERT and OSBP by phosphorylation at their respective Ser(132) and Ser(240) residues (phosphorylation inhibition). Here, we investigated the functional role of PKD in HCV secretion. Our studies show that HCV gene expression down-regulated PKD activation. PKD depletion by shRNA or inhibition by pharmacological inhibitor Gö6976 enhanced HCV secretion. Overexpression of a constitutively active form of PKD suppressed HCV secretion. The suppression by PKD was subverted by the ectopic expression of nonphosphorylatable serine mutant CERT S132A or OSBP S240A. These observations imply that PKD negatively regulates HCV secretion/release by attenuating OSBP and CERT functions by phosphorylation inhibition. This study identifies the key role of the Golgi components in the HCV maturation process.

Hepatitis C virus hijacks host lipid metabolism.

Hepatitis C virus (HCV) enhances its replication by modulating host cell lipid metabolism. HCV circulates in the blood in association with lipoproteins. HCV infection is associated with enhanced lipogenesis, reduced secretion, and beta-oxidation of lipids. HCV-induced imbalance in lipid homeostasis leads to steatosis. Many lipids are crucial for the virus life cycle, and inhibitors of cholesterol/fatty acid biosynthetic pathways inhibit virus replication, maturation and secretion. HCV negatively modulates the synthesis and secretion of very low-density lipoproteins (VLDL). Components involved in VLDL assembly are also required for HCV morphogenesis/secretion, suggesting that HCV co-opts the VLDL secretory pathway for its own secretion. This review highlights HCV-altered lipid metabolic events that aid the virus life cycle and ultimately promote liver disease.

Pathogenesis of hepatitis C virus infection in Tupaia belangeri.

The lack of a small-animal model has hampered the analysis of hepatitis C virus (HCV) pathogenesis. The tupaia (Tupaia belangeri), a tree shrew, has shown susceptibility to HCV infection and has been considered a possible candidate for a small experimental model of HCV infection. However, a longitudinal analysis of HCV-infected tupaias has yet to be described. Here, we provide an analysis of HCV pathogenesis during the course of infection in tupaias over a 3-year period. The animals were inoculated with hepatitis C patient serum HCR6 or viral particles reconstituted from full-length cDNA. In either case, inoculation caused mild hepatitis and intermittent viremia during the acute phase of infection. Histological analysis of infected livers revealed that HCV caused chronic hepatitis that worsened in a time-dependent manner. Liver steatosis, cirrhotic nodules, and accompanying tumorigenesis were also detected. To examine whether infectious virus particles were produced in tupaia livers, naive animals were inoculated with sera from HCV-infected tupaias, which had been confirmed positive for HCV RNA. As a result, the recipient animals also displayed mild hepatitis and intermittent viremia. Quasispecies were also observed in the NS5A region, signaling phylogenic lineage from the original inoculating sequence. Taken together, these data suggest that the tupaia is a practical animal model for experimental studies of HCV infection.

Role of oxysterol binding protein in hepatitis C virus infection.

Hepatitis C virus (HCV) RNA genome replicates within the ribonucleoprotein (RNP) complex in the modified membranous structures extended from endoplasmic reticulum. A proteomic analysis of HCV RNP complexes revealed the association of oxysterol binding protein (OSBP) as one of the components of these complexes. OSBP interacted with the N-terminal domain I of the HCV NS5A protein and colocalized to the Golgi compartment with NS5A. An OSBP-specific short hairpin RNA that partially downregulated OSBP expression resulted in a decrease of the HCV particle release in culture supernatant with little effect on viral RNA replication. The pleckstrin homology (PH) domain located in the N-terminal region of OSBP targeted this protein to the Golgi apparatus. OSBP deletion mutation in the PH (DeltaPH) domain failed to localize to the Golgi apparatus and inhibited the HCV particle release. These studies suggest a possible functional role of OSBP in the HCV maturation process.

Sialadenitis in patients with chronic hepatitis C is not directly related to hepatitis C virus.

Sjogren's syndrome has been suspected to be an extrahepatic manifestation of chronic hepatitis C virus (HCV) infection. To evaluate the association of sialadenitis with HCV infection, serum levels of salivary amylase (s-isoamylase) and antibodies to Ro (SS-A) and La (SS-B) were analyzed in 114 patients with chronic hepatitis C. Serum s-isoamylase levels were monitored before and after HCV was eradicated by interferon therapy. Immunohistochemistry and Western blotting using anti-HCV antibodies, and in situ hybridization of HCV-RNA were performed in the salivary gland. Serum s-isoamylase levels were elevated in patients with chronic hepatitis C (P<0.0001). The s-isoamylase remained high even after HCV was eradicated. The in situ hybridization did not show the presence of HCV-RNA in the salivary gland from patients with chronic hepatitis C. A protein reacting with anti-HCV-E2 antibodies was found in the cytosol fraction of normal salivary gland from HCV-negative cases. Latent sialadenitis is frequently observed in chronic hepatitis C, which is not directly related to HCV per se. The presence of a common epitope between antigenic protein in the salivary gland and the HCV-derived protein may be a possible pathogenetic mechanisms such as molecular mimicry.