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Publications 10

Hepatitis E virus (HEV) is a causative agent of acute hepatitis and jaundice. The number of human infections is approximated to be over 20 million cases per year. The transmission is mainly via the fecal-oral route and contaminated water and food are considered to be a major source of infection. As a mouse model is not available, a recent development of a cell culture-adapted HEV strain (47832c) is considered as a very important tools for molecular analysis of HEV pathogenesis in cells. Previously, we demonstrated that HEV-encoded methyltransferase (MeT) encoded by the 47832c strain inhibits MDA5- and RIG-I-mediated activation of interferon β (IFN-β) promoter. Here, we report that MeT impairs the phosphorylation and activation of interferon regulatory factor 3 and the p65 subunit of NF-κB in a dose-dependent manner. In addition, the MeT encoded by the 47832c, but not that of HEV clinical or field isolates (SAR-55, Mex-14, KC-1, and ZJ-1), displays the inhibitory effect. A deeper understanding of MeTmediated suppression of IFN-β expression would provide basis of the cell culture adaptation of HEV.

Middle East respiratory syndrome coronavirus (MERS-CoV) is a causative agent of severe-to-fatal pneumonia especially in patients with pre-existing conditions, such as smoking and chronic obstructive pulmonary disease (COPD). MERS-CoV transmission continues to be reported in the Saudi Arabian Peninsula since its discovery in 2012. However, it has rarely been epidemic outside the area except one large outbreak in South Korea in May 2015. The genome of the epidemic MERS-CoV isolated from a Korean patient revealed its homology to previously reported strains. MERS-CoV encodes 5 accessory proteins and generally, they do not participate in the genome transcription and replication but rather are involved in viral evasion of the host innate immune responses. Here we report that ORF8b, an accessory protein of MERS-CoV, strongly inhibits both MDA5- and RIG-I-mediated activation of interferon beta promoter activity while downstream signaling molecules were left largely unaffected. Of note, MDA5 protein levels were significantly down-regulated by ORF8b and co-expression of ORF4a and ORF4b. These novel findings will facilitate elucidation of mechanisms of virus-encoded evasion strategies, thus helping design rationale antiviral countermeasures against deadly MERS-CoV infection.

Abstract Kaposi’s sarcoma-associated herpesvirus (KSHV) is causally associated with several malignant tumors: Kaposi’s sarcoma (KS), multicentric Castleman’s disease (MCD), and primary effusion lymphoma (PEL). KS remains the most common AIDS-related malignancy since the AIDS epidemic and thus has been extensively studied. KS is characterized as an angioproliferative disease with massive immune cell infiltration at the early stage. High levels of proinflammatory cytokines and growth factors are found in KS lesions, and their involvement in the survival and growth of tumor cells has been well characterized. However, little is known about the role of the inflammatory microenvironment in the regulation of KSHV gene expression and/or viral replication. In the present study, we demonstrated that IFN-γ and TNF-α profoundly inhibited KSHV progeny production in primary human lymphatic endothelial cells (LECs) as well as induced KSHV-producer cells (iSLK.219) with doxycycline. Of note, IFN-γ inhibited overall KSHV gene expression, while the effects of TNF-α were confined to a selected set of genes, which were also downregulated by IFN-γ. The addition of IFN-γ up to 36 hr after induction of viral lytic replication was effective in terms of the inhibition of infectious virion production, suggesting that its inhibitory effect is exerted at the early stages of KSHV life cycle. We believe these data have potentially important implications for rationalizing a therapeutic agent to treat KSHV-induced tumors in which lytic replication plays a critical role in their pathogenesis: KS and MCD.

Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of Kaposi's sarcoma(1), a highly vascularized tumor originating from lymphatic endothelial cells, and of at least two different B cell malignancies(2,3). A dimeric complex formed by the envelope glycoproteins H and L (gH-gL) is required for entry of herpesviruses into host cells(4). We show that the ephrin receptor tyrosine kinase A2 (EphA2) is a cellular receptor for KSHV gH-gL. EphA2 co-precipitated with both gH-gL and KSHV virions. Infection of human epithelial cells with a GFP-expressing recombinant KSHV strain, as measured by FACS analysis, was increased upon overexpression of EphA2. Antibodies against EphA(2) and siRNAs directed against EphA2 inhibited infection of endothelial cells. Pretreatment of KSHV with soluble EphA2 resulted in inhibition of KSHV infection by up to 90%. This marked reduction of KSHV infection was seen with all the different epithelial and endothelial cells used in this study. Similarly, pretreating epithelial or endothelial cells with the soluble EphA2 ligand ephrinA4 impaired KSHV infection. Deletion of the gene encoding EphA2 essentially abolished KSHV infection of mouse endothelial cells. Binding of gH-gL to EphA2 triggered EphA2 phosphorylation and endocytosis, a major pathway of KSHV entry(5,6). Quantitative RT-PCR and in situ histochemistry revealed a close correlation between KSHV infection and EphA2 expression both in cultured cells derived from human Kaposi's sarcoma lesions or unaffected human lymphatic endothelium, and in situ in Kaposi's sarcoma specimens, respectively. Taken together, our results identify EphA2, a tyrosine kinase with known functions in neovascularization and oncogenesis, as an entry receptor for KSHV.

Human norovirus (hNoV) infections cause acute gastroenteritis, accounting for millions of disease cases and more than 200,000 deaths annually. However, the lack of in vitro infection models and robust small-animal models has posed barriers to the development of virus-specific therapies and preventive vaccines. Promising recent progress in the development of a norovirus infection model is reviewed in this article, as well as attempts and efforts made since the discovery of hNoV more than 40 years ago. Because suitable experimental animal models for human norovirus are lacking, attractive alternatives are also discussed.


Enterococcus faecalis, the type strain of the genus Enterococcus, is not only a commensal bacterium in the gastrointestinal tract in vertebrates and invertebrates, but also causes serious disease as an opportunistic pathogen. To date, genome sequences have been published for over four hundred E. faecalis strains; however, pathogenicity of these microbes remains complicated. To increase our knowledge of E. faecalis virulence factors, we isolated strain CBA7120 from the feces of an 81-year-old female from the Republic of Korea and performed a comparative genomic analysis.


The genome sequence of E. faecalis CBA7120 is 3,134,087 bp in length, with a G + C content of 37.35 mol%, and is comprised of four contigs with an N50 value of 2,922,046 bp. The genome showed high similarity with other strains of E. faecalis, including OG1RF, T13, 12107 and T20, based on OrthoANI values. Strain CBA7120 contains 374 pan-genome orthologous groups (POGs) as singletons, including “Phages, Prophages, Transposable elements, Plasmids,” “Carbohydrates,” “DNA metabolism,” and “Virulence, Disease and Defense” subsystems. Genes related to multidrug resistance efflux pumps were annotated in the genome.


The comparative genomic analysis of E. faecalis strains presented in this study was performed using a variety of analysis methods and will facilitate future identification of hypothetical proteins.


Chronic infection with Theiler’s murine encephalomyelitis virus (TMEV) in susceptible SJL/J mice induces an immune-mediated demyelinating disease and has extensively been used as a relevant infectious model for multiple sclerosis (MS). Infection of the host with many other viruses also leads to acute or chronic inflammatory diseases in the central nervous system (CNS). Levels of viral load in the host often play a critical role in the pathogenesis of virus-induced diseases. Thus, the inhibition of viral replication in the host against a broad spectrum of similar viruses is critically important for preventing the viral pathogenicity.


P2/P3-expressing transgenic (B6 X SJL)F1 founders were generated and bred onto the C57BL/6 and SJL/J backgrounds. Differences in the development of demyelinating disease were compared. Viral persistence, cytokine production, and immune responses in the CNS of infected control and P2/P3-Tg mice were analyzed after infection using quantitative PCR, ELISA, and flow cytometry. Various cell types from the control and P2/P3-Tg mice, as well as cells transfected in vitro with the P2 and/or P3 regions, were also analyzed for viral replication and innate cytokine production.


P2/P3-transgenic (P2/P3-Tg) mice carrying the viral non-structural protein genes displayed significantly reduced virus-specific T cell responses in the CNS against both the structural and non-structural proteins. Consequently, viral loads in the CNS were greater in the Tg mice during the chronic infection. However, P2/P3-Tg SJL mice exhibited reduced disease incidence and less severe clinical symptoms than did their non-transgenic littermates. Interestingly, P2/P3-Tg mice showed low viral loads in the CNS at a very early period after infection (1–3 days) with TMEV and related EMCV but not unrelated VSV. Cells from P2/P3-Tg mice and cells transfected with the P2 and/or P3 regions in vitro yielded also lower viral replication but higher IFN-α/β production.


This study demonstrates that the expression of viral non-structural genes in mice inhibits initial viral replication and suppresses sustaining pathogenic anti-viral immune responses to broad viral determinants. It appears that the elevation of innate immune cytokines produced in the cells expressing the non-structural viral genes upon viral infection is responsible for the inhibitions. The inhibition is partially virus-specific as it is more efficient for a related virus compared to an unrelated virus, suggesting a role for the similarity in the viral genome structures. Therefore, the expression of viral non-structural genes may serve as a useful new method to prevent a broadly virus-specific pathogenesis in the hosts.

Nuclear domain 10 (ND10) components are restriction factors that inhibit herpesviral replication. Effector proteins of different herpesviruses can antagonize this restriction by a variety of strategies, including degradation or relocalization of ND10 proteins. We investigated the interplay of Kaposi's Sarcoma-Associated Herpesvirus (KSHV) infection and cellular defense by nuclear domain 10 (ND10) components. Knock-down experiments in primary human cells show that KSHV-infection is restricted by the ND10 components PML and Sp100, but not by ATRX. After KSHV infection, ATRX is efficiently depleted and Daxx is dispersed from ND10, indicating that these two ND10 components can be antagonized by KSHV. We then identified the ORF75 tegument protein of KSHV as the viral factor that induces the disappearance of ATRX and relocalization of Daxx. ORF75 belongs to a viral protein family (viral FGARATs) that has homologous proteins in all gamma-herpesviruses. Isolated expression of ORF75 in primary cells induces a relocalization of PML and dispersal of Sp100, indicating that this viral effector protein is able to influence multiple ND10 components. Moreover, by constructing a KSHV mutant harboring a stop codon at the beginning of ORF75, we could demonstrate that ORF75 is absolutely essential for viral replication and the initiation of viral immediate-early gene expression. Using recombinant viruses either carrying Flag- or YFP-tagged variants of ORF75, we could further corroborate the role of ORF75 in the antagonization of ND10-mediated intrinsic immunity, and show that it is independent of the PML antagonist vIRF3. Members of the viral FGARAT family target different ND10 components, suggesting that the ND10 targets of viral FGARAT proteins have diversified during evolution. We assume that overcoming ND10 intrinsic defense constitutes a critical event in the replication of all herpesviruses; on the other hand, restriction of herpesviral replication by ND10 components may also promote latency as the default outcome of infection.

Kaposi sarcoma (KS), a human herpes virus 8 (HHV-8; also called KSHV)-induced endothelial tumor, develops only in a small fraction of individuals infected with HHV-8. We hypothesized that inborn errors of immunity to HHV-8 might underlie the exceedingly rare development of classic KS in childhood. We report here autosomal recessive OX40 deficiency in an otherwise healthy adult with childhood-onset classic KS. OX40 is a co-stimulatory receptor expressed on activated T cells. Its ligand, OX40L, is expressed on various cell types, including endothelial cells. We found OX40L was abundantly expressed in KS lesions. The mutant OX40 protein was poorly expressed on the cell surface and failed to bind OX40L, resulting in complete functional OX40 deficiency. The patient had a low proportion of effector memory CD4(+) T cells in the peripheral blood, consistent with impaired CD4(+) T cell responses to recall antigens in vitro. The proportion of effector memory CD8(+) T cells was less diminished. The proportion of circulating memory B cells was low, but the antibody response in vivo was intact, including the response to a vaccine boost. Together, these findings suggest that human OX40 is necessary for robust CD4(+) T cell memory and confers apparently selective protective immunity against HHV-8 infection in endothelial cells.

During the lytic phase of infection, the gamma herpesvirus Kaposi's Sarcoma-Associated Herpesvirus (KSHV) expresses a highly abundant, 1.1 kb nuclear noncoding RNA of unknown function. We observe that this polyadenylated nuclear (PAN) RNA avidly binds host poly(A)-binding protein C1 (PABPC1), which normally functions in the cytoplasm to bind the poly(A) tails of mRNAs, regulating mRNA stability and translation efficiency. During the lytic phase of KSHV infection, PABPC1 is re-localized to the nucleus as a consequence of expression of the viral shutoff exonuclease (SOX) protein; SOX also mediates the host shutoff effect in which host mRNAs are downregulated while viral mRNAs are selectively expressed. We show that whereas PAN RNA is not required for the host shutoff effect or for PABPC1 re-localization, SOX strongly upregulates the levels of PAN RNA in transient transfection experiments. This upregulation is destroyed by the same SOX mutation that ablates the host shutoff effect and PABPC1 nuclear re-localization or by removal of the poly(A) tail of PAN. In cells induced into the KSHV lytic phase, depletion of PAN RNA using RNase H-targeting antisense oligonucleotides reveals that it is necessary for the production of late viral proteins from mRNAs that are themselves polyadenylated. Our results add to the repertoire of functions ascribed to long noncoding RNAs and suggest a mechanism of action for nuclear noncoding RNAs in gamma herpesvirus infection.