Molecular bases for HOIPINs-mediated inhibition of LUBAC and innate immune responses.

The NF-κB and interferon antiviral signaling pathways play pivotal roles in inflammatory and innate immune responses. The LUBAC ubiquitin ligase complex, composed of the HOIP, HOIL-1L, and SHARPIN subunits, activates the canonical NF-κB pathway through Met1-linked linear ubiquitination. We identified small-molecule chemical inhibitors of LUBAC, HOIPIN-1 and HOIPIN-8. Here we show that HOIPINs down-regulate not only the proinflammatory cytokine-induced canonical NF-κB pathway, but also various pathogen-associated molecular pattern-induced antiviral pathways. Structural analyses indicated that HOIPINs inhibit the RING-HECT-hybrid reaction in HOIP by modifying the active Cys885, and residues in the C-terminal LDD domain, such as Arg935 and Asp936, facilitate the binding of HOIPINs to LUBAC. HOIPINs effectively induce cell death in activated B cell-like diffuse large B cell lymphoma cells, and alleviate imiquimod-induced psoriasis in model mice. These results reveal the molecular and cellular bases of LUBAC inhibition by HOIPINs, and demonstrate their potential therapeutic uses.

Eligibility of Feline Calicivirus for a Surrogate of Human Norovirus in Comparison with Murine Norovirus, Poliovirus and Coxsackievirus.

Feline calicivirus (FCV) is frequently used as a surrogate of human norovirus. We investigated eligibility of FCV for anti-viral assay by investigating the stability of infectivity and pH sensitivity in comparison with other viruses. We found that infectivities of FCV and murine norovirus (MNV) are relatively unstable in infected cells compared with those of coxsackievirus (CoV) and poliovirus (PoV) , suggesting that FCV and MNV have vulnerability. Western blotting indicated that inactivation of FCV was not due to viral protein degradation. We also demonstrated sensitivity of FCV to low pH, the 50% inhibitory pH value being ca. 3.9. Since human norovirus is thought to persist longer, in infectivity and to be a resistant virus, CoV, which is robust and not restrained in use as PoV, may be more appropriate as a test virus for disinfectants, rather than FCV and MNV.

Teneurin-4 is a novel regulator of oligodendrocyte differentiation and myelination of small-diameter axons in the CNS.

Myelination is essential for proper functioning of the CNS. In this study, we have identified a mouse mutation, designated furue, which causes tremors and hypomyelination in the CNS, particularly in the spinal cord, but not in the sciatic nerve of the PNS. In the spinal cord of the furue mice, myelination of small-diameter axons was dramatically reduced, and differentiation of oligodendrocytes, the myelin-forming cells in the CNS, was inhibited. We subsequently found that the furue mutation was associated with a transgene insertion into the teneurin-4 (Ten-4, Ten-m4/Odz4) gene, encoding a transmembrane protein of unknown function. Ten-4 was strongly expressed in the spinal cord of wild-type mice and was induced during normal oligodendrocyte differentiation. In contrast, in the furue mice, the expression of Ten-4 was absent. Differentiation and cellular process formation of oligodendrocytes were inhibited in primary cell culture from the furue mice. Cell differentiation and process formation were also inhibited in the oligodendrocyte progenitor cell line CG-4 after suppression of Ten-4 expression by shRNA. Furthermore, Ten-4 positively regulated focal adhesion kinase, an essential signaling molecule for oligodendrocyte process formation and myelination of small-diameter axons. These findings suggest that Ten-4 is a novel regulator of oligodendrocyte differentiation and that it plays a critical role in the myelination of small-diameter axons in the CNS.

Monitoring of S protein maturation in the endoplasmic reticulum by calnexin is important for the infectivity of severe acute respiratory syndrome coronavirus.

Severe acute respiratory syndrome coronavirus (SARS-CoV) is the etiological agent of SARS, a fatal pulmonary disorder with no effective treatment. We found that SARS-CoV spike glycoprotein (S protein), a key molecule for viral entry, binds to calnexin, a molecular chaperone in the endoplasmic reticulum (ER), but not to calreticulin, a homolog of calnexin. Calnexin bound to most truncated mutants of S protein, and S protein bound to all mutants of calnexin. Pseudotyped virus carrying S protein (S-pseudovirus) produced by human cells that were treated with small interfering RNA (siRNA) for calnexin expression (calnexin siRNA-treated cells) showed significantly lower infectivity than S-pseudoviruses produced by untreated and control siRNA-treated cells. S-pseudovirus produced by calnexin siRNA-treated cells contained S protein modified with N-glycan side chains differently from other two S proteins and consisted of two kinds of viral particles: those of normal density with little S protein and those of high density with abundant S protein. Treatment with peptide-N-glycosidase F (PNGase F), which removes all types of N-glycan side chains from glycoproteins, eliminated the infectivity of S-pseudovirus. S-pseudovirus and SARS-CoV produced in the presence of α-glucosidase inhibitors, which disrupt the interaction between calnexin and its substrates, showed significantly lower infectivity than each virus produced in the absence of those compounds. In S-pseudovirus, the incorporation of S protein into viral particles was obviously inhibited. In SARS-CoV, viral production was obviously inhibited. These findings demonstrated that calnexin strictly monitors the maturation of S protein by its direct binding, resulting in conferring infectivity on SARS-CoV.

Serial histopathological examination of the lungs of mice infected with influenza A virus PR8 strain.

Avian influenza H5N1 and pandemic (H1N1) 2009 viruses are known to induce viral pneumonia and subsequent acute respiratory distress syndrome (ARDS) with diffuse alveolar damage (DAD). The mortality rate of ARDS/DAD is extremely high, at approximately 60%, and no effective treatment for ARDS/DAD has been established. We examined serial pathological changes in the lungs of mice infected with influenza virus to determine the progress from viral pneumonia to ARDS/DAD. Mice were intranasally infected with influenza A/Puerto Rico/8/34 (PR8) virus, and their lungs were examined both macro- and micro-pathologically every 2 days. We also evaluated general condition, survival rate, body weight, viral loads in lung, and surfactant proteins in serum. As a result, all infected mice died within 9 days postinfection. At 2 days postinfection, inflammation in alveolar septa, i.e., interstitial pneumonia, was observed around bronchioles. From 4 to 6 days postinfection, interstitial pneumonia with alveolar collapse expanded throughout the lungs. From 6 to 9 days postinfection, DAD with severe alveolar collapse was observed in the lungs of all of dying and dead mice. In contrast, DAD was not observed in the live infected-mice from 2 to 6 days postinfection, despite their poor general condition. In addition, histopathological analysis was performed in mice infected with a dose of PR8 virus which was 50% of the lethal dose for mice in the 20-day observation period. DAD with alveolar collapse was observed in all dead mice. However, in the surviving mice, instead of DAD, glandular metaplasia was broadly observed in their lungs. The present study indicates that DAD with severe alveolar collapse is associated with death in this mouse infection model of influenza virus. Inhibition of the development of DAD with alveolar collapse may decrease the mortality rate in severe viral pneumonia caused by influenza virus infection.

Fully human monoclonal antibody directed to proteolytic cleavage site in severe acute respiratory syndrome (SARS) coronavirus S protein neutralizes the virus in a rhesus macaque SARS model.

BACKGROUND: There is still no effective method to prevent or treat severe acute respiratory syndrome (SARS), which is caused by SARS coronavirus (CoV). In the present study, we evaluated the efficacy of a fully human monoclonal antibody capable of neutralizing SARS-CoV in vitro in a Rhesus macaque model of SARS. METHODS: The antibody 5H10 was obtained by vaccination of KM mice bearing human immunoglobulin genes with Escherichia coli-producing recombinant peptide containing the dominant epitope of the viral spike protein found in convalescent serum samples from patients with SARS. RESULTS: 5H10, which recognized the same epitope that is also a cleavage site critical for the entry of SARS-CoV into host cells, inhibited propagation of the virus and pathological changes found in Rhesus macaques infected with the virus through the nasal route. In addition, we analyzed the mode of action of 5H10, and the results suggested that 5H10 inhibited fusion between the virus envelope and host cell membrane. 5H10 has potential for use in prevention and treatment of SARS if it reemerges. CONCLUSIONS: This study represents a platform to produce fully human antibodies against emerging infectious diseases in a timely and safe manner.

A novel KRAB-Zinc finger protein interacts with latency-associated nuclear antigen of Kaposi's sarcoma-associated herpesvirus and activates transcription via terminal repeat sequences.

Kaposi's sarcoma-associated herpesvirus (KSHV) establishes latent infection in various cells in vitro as well as KSHV-associated tumor cells in vivo. The latency-associated nuclear antigen (LANA) of KSHV is one of a small number of genes expressed in the latent phase of KSHV infection. This antigen is crucial for establishment of the latent infection, such as replication of KSHV genomic DNA and maintenance of infection via direct interaction with terminal repeats (TRs) in the viral genome. Using a yeast two-hybrid screening method, we isolated a novel LANA-interacting protein (designated as KZLP; KRAB Zinc finger LANA interacting Protein) from a human peripheral leukocyte cDNA library. KZLP encodes a KRAB domain and 12 Kruppel-type zinc fingers. Reverse transcription polymerase chain reaction showed that KZLP was expressed ubiquitously in various cell lines including those infected with KSHV. A luciferase assay showed that KZLP could activate the KSHV open reading frame K1 promoter containing TRs in 293T cells, and that such activation required multiple TR sequences. In contrast, LANA repressed the activity of the K1 promoter through TRs, and again this repression required multiple TR units. Moreover, LANA almost completely abrogated the KZLP-mediated transcriptional activation. Our results suggest that KZLP and LANA regulate gene expression through TRs in the KSHV viral genome, including the K1 gene in latent KSHV-infected cells.

Visualization of a functional KSHV episome-maintenance protein LANA in living cells.

The latency-associated nuclear antigen (LANA) of Kaposi's saroma-associated herpesvirus (KSHV) can maintain a plasmid containing the KSHV origin of DNA replication (oriP) as episomes in dividing human cells. Hence, LANA is considered to play crucial roles in persistent KSHV infection in human cells. In this study, we characterized a LANA fusion protein of green fluorescent protein (GFP-LANA). Like the wild-type LANA, GFP-LANA interacted tightly with mitotic chromosomes, and maintained the plasmid selectively with the KSHV oriP for more than three weeks in a human B cell line. Moreover, equivalent amount of GFP-LANA protein was segregated into two daughter cells in living metaphase cells. Our results suggested that the activity of LANA serves the segregation of equivalent amounts of viral genomes tethered with LANA into two daughter progeny cells during cell division. Thus, GFP-LANA is a useful tool for the analyses of the functions and dynamics of LANA in living cells.

Human T-cell leukemia virus type 1 Tax oncoprotein induces and interacts with a multi-PDZ domain protein, MAGI-3.

Human T-cell leukemia virus type 1 (HTLV-1) is the causative agent of adult T-cell leukemia (ATL), whereas the closely related virus HTLV-2 has not been associated with such malignant conditions. HTLV-1 Tax1 oncoprotein transforms a rat fibroblast cell line (Rat-1) much more efficiently than does HTLV-2 Tax2. By using a differential display analysis, we isolated MAGI-3 as a Tax1-inducible gene in Rat-1 cells. Reverse transcription-polymerase chain reaction (RT-PCR) analysis confirmed that Tax1 induced MAGI-3 in Rat-1 cells. MAGI-3 has multiple PDZ domains and interacted with Tax1 but not Tax2 in 293T cells. The interaction of Tax1 with MAGI-3 was dependent on a PDZ domain-binding motif, which is missing in Tax2. The interaction of Tax1 with MAGI-3 altered their respective subcellular localization, and moreover, the interaction correlated well with the high transforming activities of Tax1 in Rat-1 cells relative to Tax2. MAGI-3 mRNA and the allied MAGI-1, but not MAGI-2, were expressed in HTLV-1-infected T-cell lines. Our results suggest that the interaction of Tax1 and MAGI-3 alters their respective biological activities, which may play a role in transformation by Tax1 as well as in the pathogenesis of HTLV-1-associated diseases.

PDZ domain-binding motif of human T-cell leukemia virus type 1 Tax oncoprotein augments the transforming activity in a rat fibroblast cell line.

While human T-cell leukemia virus type 1 (HTLV-1) is associated with the development of adult T-cell leukemia (ATL), HTLV-2 has not been reported to be associated with such malignant leukemias. HTLV-1 Tax1 oncoprotein transforms a rat fibroblast cell line (Rat-1) to form multiple large colonies in soft agar, and this activity is much greater than that of HTLV-2 Tax2. We have demonstrated here that the increased number of transformed colonies induced by Tax1 relative to Tax2 was mediated by a PDZ domain-binding motif (PBM) in Tax1, which is absent in Tax2. Tax1 PBM mediated the interaction of Tax1 with the discs large (Dlg) tumor suppressor containing PDZ domains, and the interaction correlated well with the transforming activities of Tax1 and the mutants. Through this interaction, Tax1 altered the subcellular localization of Dlg from the detergent-soluble to the detergent-insoluble fraction in a fibroblast cell line as well as in HTLV-1-infected T-cell lines. These results suggest that the interaction of Tax1 with PDZ domain protein(s) is critically involved in the transforming activity of Tax1, the activity of which may be a crucial factor in malignant transformation of HTLV-1-infected cells in vivo.

Latency-associated nuclear antigen of Kaposi's sarcoma-associated herpesvirus interacts with human myeloid cell nuclear differentiation antigen induced by interferon alpha.

Kaposi's sarcoma-associated herpesvirus (KSHV)/human herpes virus type 8 (HHV-8) is tightly linked to the development of Kaposi's sarcoma, primary effusion lymphoma (PEL) and some cases of multicentric Castleman's disease. Latency-associated nuclear antigen (LANA) is one of a limited number of KSHV genes consistently expressed in these diseases as well as in KSHV-infected cell lines derived from PEL, and has been shown to play crucial role in persistence of KSHV genomes in the infected cells. In this study, we explored the cellular factors that interact with LANA using yeast two-hybrid screening, and isolated a part of gene encoding human myeloid cell nuclear differentiation antigen (MNDA). MNDA is a hematopoietic interferon-inducible nuclear proteins with a HIN-200 family member with conserved 200-amino acid repeats. Immunoprecipitation assay revealed that LANA interacted with MNDA in a mammalian embryonic kidney cell line. MNDA transcript was undetectable in three PEL cell lines by reverse-transcription polymerase chain reaction, but it was induced by interferon alpha (IFNalpha). Moreover, LANA and MNDA were co-localized in the nuclei of MNDA-expressing PEL cells. Our results suggest that LANA interacts with MNDA in KSHV-infected cells exposed to IFNalpha. Such interaction may modulate IFN-mediated host defense activities.

Homotypic cell-cell adhesion induced by human T cell leukemia virus type 1 tax protein in T cell lines.

Cell-cell adhesion is involved in the processes of cell growth, activation and migration, and inflammation. T cells infected with human T cell leukemia virus type 1 (HTLV-1) exhibit a high degree of homotypic cell-cell adhesion in vitro. In this study, we investigated the involvement of the viral protein Tax in such process. Expression of Tax in an interleukin (IL)-2-dependent mouse T cell line (CTLL-2) increased homotypic cell-cell adhesion; however, less cell adhesion was induced by Tax than that observed in HTLV-1-infected T cell lines. Moreover, Tax induced cell-cell adhesion in a human T cell line, in which the expression of Tax is inducible. Microscopic examination also revealed Tax-induced morphologic changes, including rounding of CTLL-2 cells, increased cell volume, and increased nucleus size. Taken together, our results suggest that Tax induces cell-cell adhesion and morphologic changes in HTLV-1-infected cells. Tax may thus play a role in persistent HTLV-1 infection and the pathogenesis of associated disease.

Chromosome binding site of latency-associated nuclear antigen of Kaposi's sarcoma-associated herpesvirus is essential for persistent episome maintenance and is functionally replaced by histone H1.

Latency-associated nuclear antigen 1 (LANA1) of Kaposi's sarcoma-associated herpesvirus (KSHV; human herpesvirus 8) persistently maintains a plasmid containing the KSHV latent origin of replication (oriP) as a closed circular episome in dividing cells. In this study, we investigated the involvement of chromosome binding activity of LANA1 in persistent episome maintenance. Deletion of the N-terminal 22 amino acids of LANA1 (DeltaN-LANA) inhibited the interaction with mitotic chromosomes in a human cell line, and the mutant concomitantly lost activity for the long-term episome maintenance of a plasmid containing viral oriP in a human B-cell line. However, a chimera of DeltaN-LANA with histone H1, a cellular chromosome component protein, rescued the association with mitotic chromosomes as well as the long-term episome maintenance of the oriP-containing plasmid. Our results suggest that tethering of KSHV episomes to mitotic chromosomes by LANA1 is crucial in mediating the long-term maintenance of viral episomes in dividing cells.

Castleman's disease of the buccal mucosa: report of a case and review of the literature of head and neck cases.

A case of Castleman's disease occurring in the buccal mucosa is described. An 84-year-old woman noticed that a mass in the left buccal mucosa that had been present for half a year. Computed tomography revealed a well-demarcated submucosal tumor, measuring 4.0 x 3.0 x 2.0 cm. The patient received no treatment at this time, and continued growth of the mass was observed. After incisional biopsy, the lesion was surgically removed. Histologically, the tumor consisted of an enlarged lymph node with conspicuous lymph follicles, in which vascular channels and deposits of eosinophilic material were noted. Laboratory examination showed an increase of serum antibody level of cytomegalovirus but of no other viruses. The patient was followed up for 1(1/2) years, with no clinical evidence of recurrence. This is the first report of Castleman's disease presenting in an oral site.

Human T-cell leukemia virus type 2 (HTLV-2) Tax protein transforms a rat fibroblast cell line but less efficiently than HTLV-1 Tax.

Human T-cell leukemia virus type 1 (HTLV-1) and HTLV-2 are retroviruses with similar biological properties. Whereas HTLV-1 is the causative agent of an aggressive T-cell leukemia, HTLV-2 has been associated with only a few cases of lymphoproliferative disorders. Tax1 and Tax2 are the transcriptional activators of HTLV-1 and HTLV-2, respectively. Here we show that Tax2 transformed a Rat-1 fibroblast cell line to form colonies in soft agar, but the size and number of the colonies were lower than those of Tax1. Use of a chimeric Tax protein showed that the C-terminal amino acids 300 to 353 were responsible for the high transforming activity of Tax1. Activation of cellular genes by Tax1 through transcription factor NF-kappa B is reportedly essential for the transformation of Rat-1 cells. Tax2 also activated the transcription through NF-kappa B in Rat-1 cells, and such activity was equivalent to that induced by Tax1. Thus, the high transforming activity of Tax1 is mediated by mechanisms other than NF-kappa B activation. Our results showed that Tax2 has a lower transforming activity than Tax1 and suggest that the high transforming activity of Tax1 is involved in the leukemogenic property of HTLV-1.