A recombinant human immunoglobulin with coherent avidity to hepatitis B virus surface antigens of various viral genotypes and clinical mutants.

The hepatitis B virus (HBV) envelope is composed of a lipid bilayer and three glycoproteins, referred to as the large (L), middle (M), and small (S) hepatitis B virus surface antigens (HBsAg). S protein constitutes the major portion of the viral envelope and an even greater proportion of subviral particles (SVP) that circulate in the blood. Recombinant S proteins are currently used as a preventive vaccine, while plasma fractions isolated from vaccinated people, referred to as hepatitis B immune globulin (HBIG), are used for short-term prophylaxis. Here, we characterized a recombinant human IgG1 type anti-S antibody named Lenvervimab regarding its binding property to a variety of cloned S antigens. Immunochemical data showed an overall consistent avidity of the antibody to S antigens of most viral genotypes distributed worldwide. Further, antibody binding was not affected by the mutations in the antigenic 'a' determinant found in many clinical variants, including the immune escape mutant G145R. In addition, mutations in the S gene sequence that confer drug resistance to the viral polymerase did not interfere with the antibody binding. These results support for a preventive use of the antibody against HBV infection.

Aurora kinase A promotes hepatitis B virus replication and expression.

Cellular protein kinases play critical roles in various steps of the hepatitis B virus life cycle. We found that viral replication in infected or transfected hepatoma cell was markedly inhibited by treatment with A-443654, a specific inhibitor of Akt. The antiviral mechanism of the drug mainly depended on the downregulation of Aurora A, a protein kinase that plays an essential role in mitosis but has not been implicated in the viral life cycle. Our data indicated that Aurora kinase A enhances viral replication and expression independently of its kinase activity required for mitotic function. Our findings suggest that mitotic kinases, considered to be an attractive target of antitumor agents, also provide a novel target for the development of antiviral therapy.

Inhibition of hepatitis B virus replication by a dNTPase-dependent function of the host restriction factor SAMHD1.

SAMHD1 is a cellular protein that possesses dNTPase activity and inhibits retroviruses and DNA viruses through the depletion of cellular dNTPs. However, recent evidence suggests the existence of alternative or additional mechanisms that involve novel nuclease activities. Hepatitis B virus is a DNA virus but resembles retroviruses in that its DNA genome is synthesized via reverse transcription of an RNA transcript. SAMHD1 was shown to inhibit the expression and replication of a transfected HBV DNA. We further investigated the antiviral mechanisms in a newly developed infection assay. Our data indicated that SAMHD1 exerts a profound antiviral effect. In addition, unlike previous findings, our results demonstrate the essential role of SAMHD1 dNTPase. SAMHD1 did not affect virion-derived cccDNA and gene expression but specifically inhibited viral DNA synthesis. These results indicate that SAMHD1 inhibits HBV replication at the reverse transcription step, most likely through the depletion of cellular dNTPs.

Development and pre-validation of an in vitro transactivation assay for detection of (anti)androgenic potential compounds using 22Rv1/MMTV cells.

The endocrine-disrupting effects of androgenic signaling play crucial roles in several androgen-related diseases. In attempting to develop an in vitro cell line to be used in androgen receptor (AR)-mediated reporter gene assays, we developed a stable 22Rv1/MMTV cell line, which is a human prostate cancer cell line that endogenously expresses functional AR, to evaluate AR-mediated transcriptional activation (TA). Using 22Rv1/MMTV cells, we established and optimized a test protocol for the AR-TA assay and validated the proposed assay using 20 compounds recommended by the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM). All the performance parameters for agonist and antagonist assays were 91-100% comparable between the 22Rv1/MMTV assay and the ICCVAM report. In conclusion, the AR-TA assay using 22Rv1/MMTV cells might be a quick and relatively inexpensive method for screening large numbers of chemicals for their potential to activate or inhibit AR-mediated gene transcription.

AAM-B Interacts with Nonstructural 4B and Regulates Hepatitis C Virus Propagation.

Hepatitis C virus (HCV) usurps host cellular lipid metabolism for production of infectious virus particles. Recently, we have screened a siRNA library targeting host factors that control lipid metabolism and lipid droplet (LD) formation in cell culture grown HCV (HCVcc)-infected cells. Of 10 final candidates, we selected the gene encoding AAM-B for further characterization. We showed that siRNA-mediated knockdown of AAM-B impaired HCV propagation in Jc1-infected cells. More precisely, knockdown of AAM-B abrogated production of infectious HCV particles in both Jc1 RNA electroporated cells and Jc1-infected cells. It is worth noting that knockdown of AAM-B exerted no effect on lipid droplet formation. Moreover, AAM-B interacted with nonstructural 4B (NS4B) through the C-terminal region of NS4B. Protein interplay between AAM-B and NS4B was verified in the context of HCV replication. Using either transient or stable expression of AAM-B, we verified that AAM-B colocalized with NS4B in the cytoplasm. Immunofluorescence data further showed that AAM-B might be involved in recruitment of NS4B to sites in close proximity to LDs to facilitate HCV propagation. Collectively, this study provides new insight into how HCV utilizes cellular AAM-B to facilitate viral propagation.

Inhibition of hepatitis B virus replication by ligand-mediated activation of RNase L.

RNase L is a cellular endoribonuclease that is activated by 2',5'-linked oligoadenylates (2-5A), which are unique and specific ligands synthesized by a family of interferon-inducible, dsRNA-activated enzymes named oligoadenylate synthetases. In the typical antiviral pathway, activated RNase L degrades viral and cellular RNAs, thus limiting viral replication and spread. Although the antiviral activity of RNase L has been demonstrated for several RNA viruses, there is little evidence regarding its role against DNA viruses. In the present study, the potential antiviral activity of RNase L against hepatitis B virus (HBV) was explored utilizing the recently reported infection protocol based on human hepatoma HepG2 cells stably complemented with the virus entry factor NTCP. Viral replication and expression in this cell type was markedly inhibited by poly(I:C)- or 2-5A-mediated activation of RNase L; however, the inhibition was significantly reversed by RNase L knockdown. Further analysis in HBV1.2-transfected Huh-7 hepatoma cells indicated that the antiviral activity of RNase L depends on its ribonuclease function. We also provide evidence for the specific roles of OAS family members in this process. These results suggest that HBV replication can be regulated through interferon-mediated RNA decay pathways and that activation of these host antiviral factors may represent a novel therapeutic strategy for HBV infection.

Hantaan virus nucleocapsid protein stimulates MDM2-dependent p53 degradation.

Apoptosis has been shown to be induced and downregulated by the Hantaan virus (HTNV) nucleocapsid (N) protein. To address these conflicting data, expression of the p53 protein, one of the key molecules involved in apoptosis, was assessed in the presence of the N protein in A549 and HeLa cells. The amount of p53, increased by drug treatment, was reduced when cells were infected with HTNV or transfected with an expression vector of the HTNV N protein. When cells were treated with a proteasome inhibitor (MG132) or an MDM2 antagonist (Nutlin-3), p53 expression was not reduced in N protein-overexpressed cells. We concluded that the HTNV N protein ubiquitinates and degrades p53 MDM2-dependently. Here we report downregulation of p53 expression through a post-translational mechanism: MDM2-dependent ubiquitination and degradation by the HTNV N protein. These results indicate that N protein-dependent p53 degradation through the ubiquitin proteasome system is one of the anti-apoptotic mechanisms employed by HTNV.

Characterization of the carbohydrate binding and ADP-ribosyltransferase activities of chemically detoxified pertussis toxins.

Pertussis toxin (PTx) is an essential component of the acellular pertussis (aP) vaccine. However, because PTx in its native form is considered too toxic for human vaccine use, it must be inactivated into a stable, nontoxic form by treatment with chemical detoxifying agents or by genetic modification. Therefore, testing for the residual PTx in the aP vaccine is a major quality control step for vaccine manufacturers and regulatory authorities. The histamine sensitization test is currently the standard safety test method for all aP vaccines, regardless of the vaccine formula or the detoxification process, except for those with genetically modified PTx. However, test result variability and ethical concerns regarding animal use necessitate an alternative method. In vitro assays based on the biochemical properties of PTx have been considered as potential alternatives to the histamine sensitization test. In this study, the suitability of assays based on the ADP-ribosyltransferase and carbohydrate binding activities of PTx was assessed for PTx after treatment with formaldehyde, glutaraldehyde or both denaturants in sequence. The results indicated a distinctive pattern of the biochemical activities depending on the detoxification methods and storage conditions. These results suggest that although a more careful study is needed, these in vitro biochemical assays can be considered potential alternatives to the histamine sensitization test, as they might provide more specific safety information of aP vaccines.

The ribonuclease L-dependent antiviral roles of human 2',5'-oligoadenylate synthetase family members against hepatitis C virus.

The latent ribonuclease RNase L and the interferon-inducible 2',5'-oligoadenylate synthetase (OAS) have been implicated in the antiviral response against hepatitis C virus (HCV). However, the specific roles of these enzymes against HCV have not been fully elucidated. In this study, a scarce endogenous expression and RNA degrading activity of RNase L in human hepatoma Huh7 cells enabled us to demonstrate the antiviral activity of RNase L against HCV replication through the transient expression of the enzyme. The antiviral potential of specific members of the OAS family was further examined through overexpression and RNA interference approaches. Our data suggested that among the members of the OAS family, OAS1 p46 and OAS3 p100 mediate the RNase L-dependent antiviral activity against HCV.

Improved protocols for histamine sensitization testing of acellular pertussis vaccines.

The histamine sensitization test is a widely used method for measuring the residual toxicity of pertussis toxin in acellular pertussis vaccines. Although it has been used as a routine assay for decades, the current protocols are difficult to standardize because the test results vary considerably and are based on several factors, including mouse strain, age and sex. In this study, we observed that mice of strains CD1, ddY and C57/BL6 were sufficiently sensitive to pertussis toxin among six mice strains tested and that aged male mice were more sensitive to pertussis toxin than younger or female mice. Using this animal model, we showed pertussis toxin dose-dependent responses in the two histamine sensitization test protocols based on either lethal end-point determination or mouse rectal temperature measurement. Sensitivity to pertussis toxin was further enhanced by the addition of lipopolysaccharide in both methods. With these improvements, pertussis toxin activity can be estimated more accurately and reproducibly using a reduced number of animals.

Modulation of the type I interferon pathways by culture-adaptive hepatitis C virus core mutants.

Hepatitis C virus (HCV) often establishes a persistent infection that leads to chronic liver diseases. The viral core protein modulates various cellular activities involved in this process. We found two mutations, K23E and V31A, in the core gene of the transfected HCV JFH-1 genome, which had been replicated for a prolonged period. The mutant viruses escaped immunochemical detection by a core-specific antibody and demonstrated enhanced RNA replication and protein expression, compared to the parental virus. The mutant core proteins bound less tightly than the parental type core to the DEAD-box RNA helicase DDX3 and attenuated the TBK1-mediated activation of interferon-related promoters. These results suggest a mechanism by which the viruses adapt to attenuate cellular antiviral activity and to establish persistent infection.

Retrospective analysis of the results of acellular pertussis vaccine toxicity tests performed in Korea.

Specific toxicity test is a major quality control test for acellular pertussis (aP) vaccines performed by manufacturers and regulatory authorities. The 'mouse body weight gain test (MWGT)', the 'leukocytosis-promoting test (LPT)' and the 'histamine sensitization test (HIST)' have been conducted to check the specific toxicity of all batches of aP vaccines used in Korea through the national quality control program, which requires a lot of animals, labor and time. In this study, test results obtained in the past 9 y from a total of 258 lots of aP vaccines were examined retrospectively to evaluate the three test methods. A pairwise comparison of the test results indicated a good correlation between LPT and HIST, whereas MWGT showed no correlation with either LPT or HIST. Moreover, the reversion to toxicity was higher than the residual toxicity in the majority of lots tested by HIST, which indicated that the histamine-sensitizing toxicity, although rated within a safe range, increased during the vaccine storage. Thus, the vaccine safety test results accumulated in the past might be useful for the improvement of test protocols.

Phylogenetic Analysis of the Rotavirus Genotypes Originated from Children < 5 Years of Age in 16 Cities in South Korea, between 2000 and 2004.

OBJECTIVES: The purpose of this study was to examine the diversity of the G and P types of human rotavirus strains isolated in South Korea during 2000 to 2004. METHODS: We selected 38 Group A rotavirus isolates among 652 fecal samples, which were collected from infants and children < 5 years of age with acute gastroenteritis or diarrhea admitted in 8 hospitals representative of five provinces of South Korea between 2000 and 2004. Rotavirus P- and G-genotypes were determined by nucleotide sequencing and phylogenetic analysis was performed. RESULTS: One G1P[4] consisted G1-Id-P[4]-V; one G1P[6] consisted G1-Id-P[6]-Ia; nine G1P[8] consisted G1-Ib-P[8]-Ia (n=3), G1-Ic-P[8]-Ia (n=1), and G1-Id-P[8]-Ia (n=5); 13 G2P[4] consisted G2-V-P[4]-V; two G3P[4] consisted G3-IIId-P[4]-V; five G3P[8] consisted G3-IIId-P[8]-Ia; four G4P[6] consisted G4-Ie-P[6]-Ia; two G4P[8] consisted G4-Ie-P[8]-II; one G9P[6] consisted G9-III-P[6]-Ia. CONCLUSIONS: A considerable amount of rotavirus genotypic diversity was detected in South Korea from 2000 to 2004. These findings are important to develop the effective vaccines and to undertake epidemiologic studies.

c-Fos regulates hepatitis C virus propagation.

Hepatitis C virus (HCV) RNA replication requires cellular factors as well as viral non-structural proteins (NS protein). Using small interfering RNA (siRNA) library screening, we previously identified c-Fos as a host factor involved in HCV propagation. In the present study, we demonstrated that silencing of c-Fos expression resulted in decrease of HCV propagation in cell culture grown HCV (HCVcc)-infected cells; whereas overexpression of c-Fos significantly increased HCV propagation. We further confirmed the positive role of c-Fos in HCV propagation by both HCV-luciferase reporter assay and immunofluorescence analysis. We showed that c-Fos level was upregulated by HCV infection. Furthermore, phorbol 12-myristate 13-acetate (PMA)-induced c-Fos level was synergistically increased by HCV infection. These data suggest that c-Fos acts as a positive regulator of HCV propagation and may contribute to HCV-associated pathogenesis.

PKR-dependent mechanisms of interferon-α for inhibiting hepatitis B virus replication.

Interferon-α (IFN-α) inhibits the replication of hepatitis B virus (HBV) in vivo and in vitro, but the molecular mechanism of this inhibition has been elusive. We found that while HBV replication in transfected human hepatoma Huh-7 cell was severely inhibited by IFN-α treatment as reported previously, this inhibition was markedly impaired in the cell in which the expression of IFN-inducible, double-stranded RNA-dependent protein kinase (PKR) was stably and specifically suppressed through RNA-interference. Intracellular level of viral capsids was down-regulated likewise in a PKR-dependent manner, whereas that of HBV transcripts including the viral RNA pregenome was not affected by IFN-α treatment. Ectopic expression of PKR also resulted in the reduction of viral capsids with concomitant increase of phosphorylated eIF2α. These results suggested that PKR functions as a key mediator of IFN-α in opposing HBV replication, most likely through the inhibition of protein synthesis.

Cell-surface receptor for complement component C1q (gC1qR) is a key regulator for lamellipodia formation and cancer metastasis.

We previously demonstrated that the receptor for the complement component C1q (gC1qR) is a lipid raft protein that is indispensable for adipogenesis and insulin signaling. Here, we provide the first report that gC1qR is an essential component of lamellipodia in human lung carcinoma A549 cells. Cell-surface gC1qR was concentrated in the lamellipodia along with CD44, monosialoganglioside, actin, and phosphorylated focal adhesion kinase in cells stimulated with insulin, IGF-1, EGF, or serum. The growth factor-induced lamellipodia formation and cell migration were significantly decreased in gC1qR-depleted cells, with a concomitant blunt activation of the focal adhesion kinase and the respective receptor tyrosine kinases. Moreover, the gC1qR-depleted cells exhibited a reduced proliferation rate in culture as well as diminished tumorigenic and metastatic activities in grafted mice. We therefore conclude that cell-surface gC1qR regulates lamellipodia formation and metastasis via receptor tyrosine kinase activation.

HCV-induced PKR activation is stimulated by the mitogen- and stress-activated protein kinase MSK2.

The replication of viral nucleic acids triggers cellular antiviral responses. The double-stranded RNA (dsRNA)-activated protein kinase (PKR) plays a key role in this antiviral response. We have recently reported that JFH-1 HCV replication in Huh-7 cells triggers PKR activation. Here we show that the HCV-induced PKR activation is further stimulated by the mitogen- and stress-activated protein kinase 2 (MSK2), a member of the 90kDa ribosomal S6 kinase (RSK) family that has emerged as an important downstream effector of ERK and p38 MAPK signaling pathways. We show that MSK2 binds PKR and stimulates PKR phosphorylation, whereas the closely related MSK1 and RSK2 have no effect. Our data further indicate that MSK2 functions as an adaptor in mediating PKR activation, apparently independent of its catalytic activity. These results suggest that, in addition to viral dsRNA, stress signaling contributes to the regulation of cellular antiviral response.

Proximity between the cap and 5' epsilon stem-loop structure is critical for the suppression of pgRNA translation by the hepatitis B viral polymerase.

The pregenomic RNA (pgRNA) of hepatitis B virus (HBV) serves as an mRNA as well as an RNA template for viral reverse transcription. We previously reported that HBV Pol (polymerase) suppresses translation of the pgRNA through a mechanism involving the 5 epsilon sequence [Virology 373:112-123(2008)]. Here, we found that the recognition of the 5 epsilon stem-loop structure by HBV Pol is essential for the translation suppression. Intriguingly, the translation suppression was observed only when the 5 epsilon sequence was positioned within approximately 60 nucleotides from the 5' end, which is striking reminiscent of the pgRNA encapsidation. This finding implicates that the translation suppression is mechanistically linked to encapsidation of the pgRNA. However, unexpectedly, the HBV Pol-eIF4E interaction, which we reported recently [J. Virol. 84:52-58(2010)], is not required for the translation suppression. Instead, the data suggested that the cap proximity of 5 epsilon sequence is necessary and sufficient for the translation suppression.

Nonstructural 5A protein activates beta-catenin signaling cascades: implication of hepatitis C virus-induced liver pathogenesis.

BACKGROUND/AIMS: The nonstructural 5A (NS5A) protein of hepatitis C virus (HCV) has been implicated in HCV-induced liver pathogenesis. Wnt/beta-catenin signaling has also been involved in tumorigenesis. To elucidate the molecular mechanism of HCV pathogenesis, we examined the potential effects of HCV NS5A protein on Wnt/beta-catenin signal transduction cascades. METHODS: The effects of NS5A protein on beta-catenin signaling cascades in hepatic cells were investigated by luciferase reporter gene assay, confocal microscopy, immunoprecipitation assay, and immunoblot analysis. RESULTS: beta-Catenin-mediated transcriptional activity is elevated by NS5A protein, in the context of HCV replication, and by infection of cell culture-produced HCV. NS5A protein directly interacts with endogenous beta-catenin and colocalizes with beta-catenin in the cytoplasm. NS5A protein inactivates glycogen synthase kinase 3beta and increases subsequent accumulation of beta-catenin in HepG2 cells. beta-Catenin was also accumulated in HCV patients' liver tissues. In addition, the accumulation of beta-catenin in HCV replicon cells requires both activation of phosphatidylinositol 3-kinase and inactivation of GSK3beta. CONCLUSIONS: NS5A activates beta-catenin signaling cascades through increasing the stability of beta-catenin. This modulation is accomplished by the protein interplay between viral and cellular signaling transducer. These data suggest that NS5A protein may directly be involved in Wnt/beta-catenin-mediated liver pathogenesis.

Proteome analysis of adipocyte lipid rafts reveals that gC1qR plays essential roles in adipogenesis and insulin signal transduction.

Since insulin receptors and their downstream signaling molecules are organized in lipid rafts, proteomic analysis of adipocyte lipid rafts may provide new insights into the function of lipid rafts in adipogenesis and insulin signaling. To search for proteins involved in adipocyte differentiation and insulin signaling, we analyzed detergent-resistant lipid raft proteins from 3T3-L1 preadipocytes and adipocytes by 2-DE. Eleven raft proteins were identified from adipocytes. One of the adipocyte-specific proteins was globular C1q receptor (gC1qR), an acidic 32 kDa protein known as the receptor for the globular domain of complement C1q. The targeting of gC1qR into lipid rafts was significantly increased during adipogenesis, as determined by immunoblotting and immunofluorescence. Since the silencing of gC1qR by small RNA interference abolished adipogenesis and blocked insulin-induced activation of insulin receptor, insulin receptor substrate-1 (IRS-1), Akt, and Erk1/2, we can conclude that gC1qR is an essential molecule involved in adipogenesis and insulin signaling.