HAND2 suppresses favipiravir efficacy in treatment of Borna disease virus infection.

Borna disease virus (BoDV-1) is a bornavirus prototype that infects the central nervous system of various animal species and can cause fatal encephalitis in various animals including humans. Among the reported anti-BoDV-1 treatments, favipiravir (T-705) is one of the best candidates since it has been shown to be effective in reducing various bornavirus titers in cell culture. However, T-705 effectiveness on BoDV-1 is cell type-dependent, and the molecular mechanisms that explain this cell type-dependent difference remain unknown. In this study, we noticed a fact that T-705 efficiently suppressed BoDV-1 in infected 293T cells, but not in infected SH-SY5Y cells, and sought to identify protein(s) responsible for this cell-type-dependent difference in T-705 efficacy. By comparing the transcriptomes of BoDV-1-infected 293T and SH-SY5Y cells, we identified heart- and neural crest derivatives-expressed protein 2 (HAND2) as a candidate involved in T-705 interference. HAND2 overexpression partly attenuated the inhibitory effect of T-705, whereas HAND2 knockdown enhanced this effect. We also demonstrated an interaction between T-705 and HAND2. Furthermore, T-705 impaired HAND2-mediated host gene expression. Because HAND2 is an essential transcriptional regulator of embryogenesis, T-705 may exhibit its adverse effects such as teratogenicity and embryotoxicity through the impairment of HAND2 function. This study provides novel insights into the molecular mechanisms underlying T-705 interference in some cell types and inspires the development of improved T-705 derivatives for the treatment of RNA viruses.

Roles of Human Endogenous Retroviruses and Endogenous Virus-Like Elements in Cancer Development and Innate Immunity.

Human endogenous retroviruses (HERVs) are remnants of ancient retroviral infections in the host genome. Although mutations and silencing mechanisms impair their original role in viral replication, HERVs are believed to play roles in various biological processes. Long interspersed nuclear elements (LINEs) are non-LTR retrotransposons that have a lifecycle resembling that of retroviruses. Although LINE expression is typically silenced in somatic cells, it also contributes to various biological processes. The aberrant expression of HERVs and LINEs is closely associated with the development of cancer and/or immunological diseases, suggesting that they are integrated into various pathways related to the diseases. HERVs/LINEs control gene expression depending on the context as promoter/enhancer elements. Some RNAs and proteins derived from HERVs/LINEs have oncogenic potential, whereas others stimulate innate immunity. Non-retroviral endogenous viral elements (nrEVEs) are a novel type of virus-like element in the genome. nrEVEs may also be involved in host immunity. This article provides a current understanding of how these elements impact cellular physiology in cancer development and innate immunity, and provides perspectives for future studies.

Nectin-2 Acts as a Viral Entry Mediated Molecule That Binds to Human Herpesvirus 6B Glycoprotein B.

Human herpesvirus 6B (HHV-6B) is a T-lymphotropic virus and the etiological agent of exanthem subitum. HHV-6B is present in a latent or persistent form after primary infection and is produced in the salivary glands or transmitted to this organ. Infected individuals continue to secrete the virus in their saliva, which is thus considered a source for virus transmission. HHV-6B primarily propagates in T cells because its entry receptor, CD134, is mainly expressed by activated T cells. The virus then spreads to the host's organs, including the salivary glands, nervous system, and liver. However, CD134 expression is not detected in these organs. Therefore, HHV-6B may be entering cells via a currently unidentified cell surface molecule, but the mechanisms for this have not yet been investigated. In this study, we investigated a CD134-independent virus entry mechanism in the parotid-derived cell line HSY. First, we confirmed viral infection in CD134-membrane unanchored HSY cells. We then determined that nectin cell adhesion molecule 2 (nectin-2) mediated virus entry and that HHV-6B-insensitive T-cells transduced with nectin-2 were transformed into virus-permissive cells. We also found that virus entry was significantly reduced in nectin-2 knockout parotid-derived cells. Furthermore, we showed that HHV-6B glycoprotein B (gB) interacted with the nectin-2 V-set domain. The results suggest that nectin-2 acts as an HHV-6B entry-mediated protein.

A Human Endogenous Bornavirus-Like Nucleoprotein Encodes a Mitochondrial Protein Associated with Cell Viability.

Endogenous retroviruses (ERVs) are sequences in animal genomes that originated from ancient retrovirus infections; they provide genetic novelty in hosts by being coopted as functional genes or elements during evolution. Recently, we demonstrated that endogenous elements from not only from retroviruses but also nonretroviral RNA viruses are a possible source of functional genes in host animals. The remnants of ancient bornavirus infections, called endogenous bornavirus-like elements (EBLs), are present in the genomes of a wide variety of vertebrate species, and some express functional products in host cells. Previous studies have predicted that the human EBL locus derived from bornavirus nucleoprotein, termed hsEBLN-2, expresses mRNA encoding a protein, suggesting that hsEBLN-2 has acquired a cellular function during evolution. However, the detailed function of the hsEBLN-2-derived product remains to be elucidated. In this study, we show that the hsEBLN-2-derived protein E2 acts as a mitochondrial protein that interacts with mitochondrial host factors associated with apoptosis, such as HAX-1. We also demonstrate that knockdown of hsEBLN-2-derived RNA increased the levels of PARP and caspase-3 cleavage and markedly decreased cell viability. In contrast, overexpression of E2 enhanced cell viability, as well as the intracellular stability of HAX-1, under stress conditions. Our results suggest that hsEBLN-2 has been coopted as a host gene, the product of which is involved in cell viability by interacting with mitochondrial proteins. IMPORTANCE Our genomes contain molecular fossils of ancient viruses, called endogenous virus elements (EVEs). Mounting evidence suggests that EVEs derived from nonretroviral RNA viruses have acquired functions in host cells during evolution. Previous studies have revealed that a locus encoding a bornavirus-derived EVE, hsEBLN-2, which was generated approximately 43 million years ago in a human ancestor, may be linked to the development of some tumors. However, the function of hsEBLN-2 has not been determined. In this study, we found that the E2 protein, an expression product of hsEBLN-2, interacts with apoptosis-related host proteins as a mitochondrial protein and affects cell viability. This study suggests that nonretroviral RNA viral EVEs have been coopted by hosts with more diverse functions than previously thought, showing a pivotal role for RNA virus infection in evolution.

Characterization of an active LINE-1 in the naked mole-rat genome.

Naked mole-rats (NMRs, Heterocephalus glaber) are the longest-living rodent species. A reason for their long lifespan is pronounced cancer resistance. Therefore, researchers believe that NMRs have unknown secrets of cancer resistance and seek to find them. Here, to reveal the secrets, we noticed a retrotransposon, long interspersed nuclear element 1 (L1). L1s can amplify themselves and are considered endogenous oncogenic mutagens. Since the NMR genome contains fewer L1-derived sequences than other mammalian genomes, we reasoned that the retrotransposition activity of L1s in the NMR genome is lower than those in other mammalian genomes. In this study, we successfully cloned an intact L1 from the NMR genome and named it NMR-L1. An L1 retrotransposition assay using the NMR-L1 reporter revealed that NMR-L1 was active retrotransposon, but its activity was lower than that of human and mouse L1s. Despite lower retrotrasposition activity, NMR-L1 was still capable of inducing cell senescence, a tumor-protective system. NMR-L1 required the 3' untranslated region (UTR) for retrotransposition, suggesting that NMR-L1 is a stringent-type of L1. We also confirmed the 5' UTR promoter activity of NMR-L1. Finally, we identified the G-quadruplex structure of the 3' UTR, which modulated the retrotransposition activity of NMR-L1. Taken together, the data indicate that NMR-L1 retrotranspose less efficiently, which may contribute to the cancer resistance of NMRs.

Effects of activation of the LINE-1 antisense promoter on the growth of cultured cells.

Long interspersed element 1 (LINE-1, or L1) is a retrotransposon that constitutes ~ 17% of the human genome. Although ~ 6000 full-length L1s spread throughout the human genome, their biological significance remains undetermined. The L1 5' untranslated region has bidirectional promoter activity with a sense promoter driving L1 mRNA production and an antisense promoter (ASP) driving the production of L1-gene chimeric RNAs. Here, we stimulated L1 ASP activity using CRISPR-Cas9 technology to evaluate its biological impacts. Activation of the L1 ASP upregulated the expression of L1 ASP-driven ORF0 and enhanced cell growth. Furthermore, the exogenous expression of ORF0 also enhanced cell growth. These results indicate that activation of L1 ASP activity fuels cell growth at least through ORF0 expression. To our knowledge, this is the first report demonstrating the role of the L1 ASP in a biological context. Considering that L1 sequences are desilenced in various tumor cells, our results indicate that activation of the L1 ASP may be a cause of tumor growth; therefore, interfering with L1 ASP activity may be a potential strategy to suppress the growth.

A Traditional Chinese Medicine, Maoto, Suppresses Hepatitis B Virus Production.

Worldwide, millions of people suffer from hepatitis B virus (HBV) infection, putting them at a high risk of death from liver cirrhosis and cancer. Although effective anti-HBV drugs have been developed, current drugs have some limitations, as most of them have a risk of significant side effects. Therefore, the discovery of safe and effective anti-HBV drugs is still needed. Natural compounds are considered sources of novel, safe and effective therapeutics. In this study, we screened a library of Kampos, traditional herbal medicines, for suppression of HBV production. Among them, we found that maoto reduced extracellular HBV DNA but not extracellular HBsAg during HBV infection, suggesting that it suppressed HBV production by interfering with HBV nucleocapsid incorporation into viral particles. Furthermore, we revealed that maoto reduced the expression of a host gene, Tropomyosin ß chain (TPM2), whose downregulation also suppressed HBV production, similarly to maoto. Since the safety of maoto has been already confirmed, maoto can be considered a candidate anti-HBV agent if the effect is confirmed in vivo. In addition, our findings also suggest TPM2 as a novel molecular target for the development of anti-HBV agents.

Kaposi's sarcoma-associated herpesvirus is cell-intrinsically controlled in latency in microgravity.

In the next several decades, humans will explore deep space, including Mars. During long-term space flight, astronauts will be exposed to various physical stressors. Among these stressors, microgravity may compromise the immune system. Consistently, the reactivation of several latent herpesviruses has been reported in astronauts. Although herpesvirus infection status is determined by both cell-intrinsic and -extrinsic factors, it remains unclear which factors play major roles in the virus reactivation in microgravity. Here, using Kaposi's sarcoma-associated herpesvirus (KSHV)-infected cells, we found that KSHV is cell-intrinsically controlled in latency in microgravity. Innate immunity appeared to be unaffected in microgravity, while the expression of some restriction factors against KSHV, such as CTCF and AMPK, was upregulated. Collectively, the infected cells in microgravity can control KSHV in latency, possibly by unimpaired innate immunity and upregulated KSHV restriction factors. This is the first pilot study of the conflicts between cell-intrinsic defense systems and viruses in microgravity and provides fundamental information regarding host-virus interactions in microgravity.

Profiling of LINE-1-Related Genes in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is a prime public health concern that accounts for most of the primary liver malignancies in humans. The most common etiological factor of HCC is hepatitis B virus (HBV). Despite recent advances in treatment strategies, there has been little success in improving the survival of HCC patients. To develop a novel therapeutic approach, evaluation of a working hypothesis based on different viewpoints might be important. Long interspersed element 1 (L1) retrotransposons have been suggested to play a role in HCC. However, the molecular machineries that can modulate L1 biology in HBV-related HCC have not been well-evaluated. Here, we summarize the profiles of expression and/or activation status of L1-related genes in HBV-related HCC, and HBV- and HCC-related genes that may impact L1-mediated tumorigenesis. L1 restriction factors appear to be suppressed by HBV infection. Since some of the L1 restriction factors also limit HBV, these factors may be exhausted in HBV-infected cells, which causes de-suppression of L1. Several HBV- and HCC-related genes that interact with L1 can affect oncogenic processes. Thus, L1 may be a novel prime therapeutic target for HBV-related HCC. Studies in this area will provide insights into HCC and other types of cancers.

Latent infection with Kaposi's sarcoma-associated herpesvirus enhances retrotransposition of long interspersed element-1.

Kaposi's sarcoma (KS)-associated herpesvirus (KSHV), a gamma-2 herpesvirus, is the causative agent of KS, primary effusion lymphoma (PEL), and a plasma cell variant of multicentric Castleman's disease. Although KSHV latency is detected in KS-related tumors, oncogenic pathways activated by KSHV latent infection are not fully understood. Here, we found that retrotransposition of long interspersed element-1 (L1), a retrotransposon in the human genome, was enhanced in PEL cells. Among the KSHV latent genes, viral FLICE-inhibitory protein (vFLIP) enhanced L1 retrotransposition in an NF-κB-dependent manner. Intracellular cell adhesion molecule-1 (ICAM-1), an NF-κB target, regulated the vFLIP-mediated enhancement of L1 retrotransposition. Furthermore, ICAM-1 downregulated the expression of Moloney leukemia virus 10 (MOV10), an L1 restriction factor. Knockdown of ICAM-1 or overexpression of MOV10 relieved the vFLIP-mediated enhancement of L1 retrotransposition. Collectively, during KSHV latency, vFLIP upregulates ICAM-1 in an NF-κB-dependent manner, which, in turn, downregulates MOV10 expression and thereby enhances L1 retrotransposition. Because active L1 retrotransposition can lead to genomic instability, which is commonly found in KS and PEL, activation of L1 retrotransposition during KSHV latency may accelerate oncogenic processes through enhancing genomic instability. Our results suggest that L1 retrotransposition may be a novel target for impeding tumor development in KSHV-infected patients.

Importance of Promyelocytic Leukema Protein (PML) for Kaposi's Sarcoma-Associated Herpesvirus Lytic Replication.

Many DNA virus replication-related proteins are associated with promyelocytic leukemia protein (PML), a component of nuclear domain 10 (ND10), which has been investigated for its potential involvement in viral replication. In the case of Kaposi's sarcoma-associated herpesvirus (KSHV) lytic gene products, K8 (K-bZIP), ORF59, and ORF75 have been shown to colocalize with PML, but its importance in KSHV lytic replication is still unclear. In this study, we analyzed the functional influence of PML on KSHV latency and lytic replication in KSHV-infected primary effusion lymphoma (PEL) cell lines. Stable PML-knockout (BC3-PMLKO) and PML-overexpressing BC3 cells (BC3PML) were successfully generated and the latency and reactivation status were analyzed. The results demonstrated that neither KSHV latency nor the episome copy number was affected in BC3-PMLKO cells. In the reactivation phase, the expression dynamics of KSHV immediate-early or early lytic proteins such as RTA, K9 (vIRF1), K5, K3, ORF59, and K8 (K-bZIP) were comparable between wild-type, control BC3, and BC3-PMLKO cells. Interestingly, KSHV lytic replication, virion production, and expression of late genes were downregulated in BC3-PMLKO cells and upregulated in BC3PML cells, compared to those in control or wild-type BC3 cells. Moreover, exogenous PML increased the size of the PML dots and recruited additional K8 (K-bZIP) to PML-NBs as dots. Therefore, PML would function as a positive regulator for KSHV lytic DNA replication by recruiting KSHV replication factors such as 8 (K-bZIP) or ORF59 to the PML-NBs.

Inhibition of LINE-1 Retrotransposition by Capsaicin.

Long interspersed nuclear element 1 (LINE-1 or L1) is a non-long terminal repeat (LTR) retrotransposon that constitutes approximately 17% of the human genome. Since approximately 100 copies are still competent for retrotransposition to other genomic loci, dysregulated retrotransposition of L1 is considered to be a major risk factor of endogenous mutagenesis in humans. Thus, it is important to find drugs to regulate this process. Although various chemicals are reportedly capable of affecting L1 retrotransposition, it is poorly understood whether phytochemicals modulate L1 retrotransposition. Here, we screened a library of compounds that were derived from phytochemicals for reverse transcriptase (RT) inhibition with an in vitro RT assay. We identified capsaicin as a novel RT inhibitor that also suppressed L1 retrotransposition. The inhibitory effect of capsaicin on L1 retrotransposition was mediated neither through its receptor, nor through its modulation of the L1 promoter and/or antisense promoter activity, excluding the possibility that capsaicin indirectly affected L1 retrotransposition. Collectively, capsaicin suppressed L1 retrotransposition most likely by inhibiting the RT activity of L1 ORF2p, which is the L1-encoded RT responsible for L1 retrotransposition. Given that L1-mediated mutagenesis can cause tumorigenesis, our findings suggest the potential of capsaicin for suppressing cancer development.

vFLIP upregulates IKKε, leading to spindle morphology formation through RelA activation.

Kaposi's sarcoma (KS)-associated herpesvirus (KSHV) vFLIP, a latent gene of KSHV, was first identified as a FLICE-inhibitory protein (FLIP) protecting cells from apoptosis. The vFLIP protein has been shown to activate the NF-κB signaling involved in spindle morphology formation both in HUVECs infected with KSHV and Kaposi's sarcoma (KS) itself. In this study, we independently established stably vFLIP-expressing cells and showed that they exhibited upregulated NF-κB family protein expression independent of the ability of IKKs to bind vFLIP. Further, vFLIP induced upregulation of IKKε, phosphorylation of RelA at Ser468 (p-RelA S468) and nuclear localization of Re1A concomitant with spindle morphology formation, and these effects were reversed by knockdown of IKKε and treatment with Bay-11. Overexpression of IKKε alone also showed spindle morphology formation with p-RelA S468. In conclusion, the spindle cell morphology in KS should be induced by RelA activation (p-RelA S468) by IKKε upregulation in vFLIP-expressing EA hy926 cells.

A Single Amino Acid Substitution within the Paramyxovirus Sendai Virus Nucleoprotein Is a Critical Determinant for Production of Interferon-Beta-Inducing Copyback-Type Defective Interfering Genomes.

One of the first defenses against infecting pathogens is the innate immune system activated by cellular recognition of pathogen-associated molecular patterns (PAMPs). Although virus-derived RNA species, especially copyback (cb)-type defective interfering (DI) genomes, have been shown to serve as real PAMPs, which strongly induce interferon-beta (IFN-ß) during mononegavirus infection, the mechanisms underlying DI generation remain unclear. Here, for the first time, we identified a single amino acid substitution causing production of cbDI genomes by successful isolation of two distinct types of viral clones with cbDI-producing and cbDI-nonproducing phenotypes from the stock Sendai virus (SeV) strain Cantell, which has been widely used in a number of studies on antiviral innate immunity as a representative IFN-ß-inducing virus. IFN-ß induction was totally dependent on the presence of a significant amount of cbDI genome-containing viral particles (DI particles) in the viral stock, but not on deficiency of the IFN-antagonistic viral accessory proteins C and V. Comparison of the isolates indicated that a single amino acid substitution found within the N protein of the cbDI-producing clone was enough to cause the emergence of DI genomes. The mutated N protein of the cbDI-producing clone resulted in a lower density of nucleocapsids than that of the DI-nonproducing clone, probably causing both production of the DI genomes and their formation of a stem-loop structure, which serves as an ideal ligand for RIG-I. These results suggested that the integrity of mononegaviral nucleocapsids might be a critical factor in avoiding the undesirable recognition of infection by host cells.IMPORTANCE The type I interferon (IFN) system is a pivotal defense against infecting RNA viruses that is activated by sensing viral RNA species. RIG-I is a major sensor for infection with most mononegaviruses, and copyback (cb)-type defective interfering (DI) genomes have been shown to serve as strong RIG-I ligands in real infections. However, the mechanism underlying production of cbDI genomes remains unclear, although DI genomes emerge as the result of an error during viral replication with high doses of viruses. Sendai virus has been extensively studied and is unique in that its interaction with innate immunity reveals opposing characteristics, such as high-level IFN-ß induction and strong inhibition of type I IFN pathways. Our findings provide novel insights into the mechanism of production of mononegaviral cbDI genomes, as well as virus-host interactions during innate immunity.

Generation of a non-transmissive Borna disease virus vector lacking both matrix and glycoprotein genes.

Borna disease virus (BoDV), a prototype of mammalian bornavirus, is a non-segmented, negative strand RNA virus that often causes severe neurological disorders in infected animals, including horses and sheep. Unique among animal RNA viruses, BoDV transcribes and replicates non-cytopathically in the cell nucleus, leading to establishment of long-lasting persistent infection. This striking feature of BoDV indicates its potential as an RNA virus vector system. It has previously been demonstrated by our team that recombinant BoDV (rBoDV) lacking an envelope glycoprotein (G) gene develops persistent infections in transduced cells without loss of the viral genome. In this study, a novel non-transmissive rBoDV, rBoDV ΔMG, which lacks both matrix (M) and G genes in the genome, is reported. rBoDV-ΔMG expressing green fluorescence protein (GFP), rBoDV ΔMG-GFP, was efficiently generated in Vero/MG cells stably expressing both BoDV M and G proteins. Infection with rBoDV ΔMG-GFP was persistently maintained in the parent Vero cells without propagation within cell culture. The optimal ratio of M and G for efficient viral particle production by transient transfection of M and G expression plasmids into cells persistently infected with rBoDV ΔMG-GFP was also demonstrated. These findings indicate that the rBoDV ΔMG-based BoDV vector may provide an extremely safe virus vector system and could be a novel strategy for investigating the function of M and G proteins and the host range of bornaviruses.

Potential Links between Hepadnavirus and Bornavirus Sequences in the Host Genome and Cancer.

Various viruses leave their sequences in the host genomes during infection. Such events occur mainly in retrovirus infection but also sometimes in DNA and non-retroviral RNA virus infections. If viral sequences are integrated into the genomes of germ line cells, the sequences can become inherited as endogenous viral elements (EVEs). The integration events of viral sequences may have oncogenic potential. Because proviral integrations of some retroviruses and/or reactivation of endogenous retroviruses are closely linked to cancers, viral insertions related to non-retroviral viruses also possibly contribute to cancer development. This article focuses on genomic viral sequences derived from two non-retroviral viruses, whose endogenization is already reported, and discusses their possible contributions to cancer. Viral insertions of hepatitis B virus play roles in the development of hepatocellular carcinoma. Endogenous bornavirus-like elements, the only non-retroviral RNA virus-related EVEs found in the human genome, may also be involved in cancer formation. In addition, the possible contribution of the interactions between viruses and retrotransposons, which seem to be a major driving force for generating EVEs related to non-retroviral RNA viruses, to cancers will be discussed. Future studies regarding the possible links described here may open a new avenue for the development of novel therapeutics for tumor virus-related cancers and/or provide novel insights into EVE functions.

Links between Human LINE-1 Retrotransposons and Hepatitis Virus-Related Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) accounts for approximately 80% of liver cancers, the third most frequent cause of cancer mortality. The most prevalent risk factors for HCC are infections by hepatitis B or hepatitis C virus. Findings suggest that hepatitis virus-related HCC might be a cancer in which LINE-1 retrotransposon, often termed L1, activity plays a potential role. Firstly, hepatitis viruses can suppress host defense factors that also control L1 mobilization. Secondly, many recent studies also have indicated that hypomethylation of L1 affects the prognosis of HCC patients. Thirdly, endogenous L1 retrotransposition was demonstrated to activate oncogenic pathways in HCC. Fourthly, several L1 chimeric transcripts with host or viral genes are found in hepatitis virus-related HCC. Such lines of evidence suggest a linkage between L1 retrotransposons and hepatitis virus-related HCC. Here, I briefly summarize current understandings of the association between hepatitis virus-related HCC and L1. Then, I discuss potential mechanisms of how hepatitis viruses drive the development of HCC via L1 retrotransposons. An increased understanding of the contribution of L1 to hepatitis virus-related HCC may provide unique insights related to the development of novel therapeutics for this disease.

Influenza A Virus-Induced Expression of a GalNAc Transferase, GALNT3, via MicroRNAs Is Required for Enhanced Viral Replication.

UNLABELLED: Influenza A virus (IAV) affects the upper and lower respiratory tracts and rapidly induces the expression of mucins, which are common O-glycosylated proteins, on the epithelial surfaces of the respiratory tract. Although mucin production is associated with the inhibition of virus transmission as well as characteristic clinical symptoms, little is known regarding how mucins are produced on the surfaces of respiratory epithelial cells and how they affect IAV replication. In this study, we found that two microRNAs (miRNAs), miR-17-3p and miR-221, which target GalNAc transferase 3 (GALNT3) mRNA, are rapidly downregulated in human alveolar basal epithelial cells during the early stage of IAV infection. We demonstrated that the expression of GALNT3 mRNA is upregulated in an IAV replication-dependent fashion and leads to mucin production in bronchial epithelial cells. A lectin microarray analysis revealed that the stable expression of GALNT3 by human alveolar basal epithelial cells induces mucin-type O-glycosylation modifications similar to those present in IAV-infected cells, suggesting that GALNT3 promotes mucin-type O-linked glycosylation in IAV-infected cells. Notably, analyses using short interfering RNAs and miRNA mimics showed that GALNT3 knockdown significantly reduces IAV replication. Furthermore, IAV replication was markedly decreased in embryonic fibroblast cells obtained from galnt3-knockout mice. Interestingly, IAV-infected galnt3-knockout mice exhibited high mortality and severe pathological alterations in the lungs compared to those of wild-type mice. Our results demonstrate not only the molecular mechanism underlying rapid mucin production during IAV infection but also the contribution of O-linked glycosylation to the replication and propagation of IAV in lung cells. IMPORTANCE: Viral infections that affect the upper or lower respiratory tracts, such as IAV, rapidly induce mucin production on the epithelial surfaces of respiratory cells. However, the details of how mucin-type O-linked glycosylation is initiated by IAV infection and how mucin production affects viral replication have not yet been elucidated. In this study, we show that levels of two miRNAs that target the UDP-GalNAc transferase GALNT3 are markedly decreased during the early stage of IAV infection, resulting in the upregulation of GALNT3 mRNA. We also demonstrate that the expression of GALNT3 initiates mucin production and affects IAV replication in infected cells. This is the first report demonstrating the mechanism underlying the miRNA-mediated initiation of mucin-type O-glycosylation in IAV-infected cells and its role in viral replication. Our results have broad implications for understanding IAV replication and suggest a strategy for the development of novel anti-influenza approaches.

Th2 cytokines efficiently stimulate periostin production in gingival fibroblasts but periostin does not induce an inflammatory response in gingival epithelial cells.

OBJECTIVES: This study aims to clarify whether gingival fibroblasts produce periostin in response to Th2 cytokines which are elevated in periodontitis lesion and, if so, whether periostin affects the inflammatory response and matrix-protein metabolism. DESIGN: Human gingival fibroblasts, periodontal ligament cells and the gingival epithelial cell line epi4 were stimulated with interleukin-4 (IL-4), IL-13, tumour necrosis factor-α (TNF-α) and Porphyromonas gingivalis lipopolysaccharide (LPS). Periostin expression was analysed by real-time polymerase chain-reaction (PCR) and Western blotting. The expression of the IL-4 receptor α-chain was evaluated by immunocytochemistry. The effect of periostin on the production of inflammatory cytokines and the expression of matrix protein-related genes was analysed by real-time PCR and enzyme-linked immunosorbent assay (ELISA). RESULTS: While IL-4 and IL-13 significantly induced periostin production in gingival fibroblasts and periodontal ligament cells, no effect was observed in epi4 cells. No stimulatory effect of TNF-α or P. gingivalis LPS on the production of periostin was observed. The effect of periostin on the production of inflammatory cytokines was weak in gingival fibroblasts; however, little or no effect was observed on periodontal ligament cells or epi4 cells. No significant effect of periostin on the expression of matrix protein-related genes was found. CONCLUSION: The results suggest that gingival fibroblasts may be a source of periostin in periodontitis lesions but periostin has only a limited role either in the inflammatory response or in matrix-protein metabolism. Thus, the role of periostin in the cellular interaction between epithelial and mesenchymal cells in gingiva may be distinct from that of skin.

Evolutionarily conserved interaction between the phosphoproteins and X proteins of bornaviruses from different vertebrate species.

Bornavirus, a non-segmented, negative-strand RNA viruses, is currently classified into several genetically distinct genotypes, such as Borna disease virus (BDV) and avian bornaviruses (ABVs). Recent studies revealed that bornavirus genotypes show unique sequence variability in the putative 5' untranslated region (5' UTR) of X/P mRNA, a bicistronic mRNA for the X protein and phosphoprotein (P). In this study, to understand the evolutionary relationship among the bornavirus genotypes, we investigated the functional interaction between the X and P proteins of four bornavirus genotypes, BDV, ABV genotype 4 and 5 and reptile bornavirus (RBV), the putative 5' UTRs of which exhibit variation in the length. Immunofluorescence and immunoprecipitation analyses using mammalian and avian cell lines revealed that the X proteins of bornaviruses conserve the ability to facilitate the export of P from the nucleus to the cytoplasm via interaction with P. Furthermore, we showed that inter-genotypic interactions may occur between X and P among the genotypes, except for X of RBV. In addition, a BDV minireplicon assay demonstrated that the X and P proteins of ABVs, but not RBV, can affect the polymerase activity of BDV. This study demonstrates that bornaviruses may have conserved the fundamental function of a regulatory protein during their evolution, whereas RBV has evolved distinctly from the other bornavirus genotypes.