Chieko Kai

Long Noncoding RNA NEAT1-Dependent SFPQ Relocation from Promoter Region to Paraspeckle Mediates IL8 Expression upon Immune Stimuli

Although thousands of long noncoding RNAs (lncRNAs) are localized in the nucleus, only a few dozen have been functionally characterized. Here we show that nuclear enriched abundant transcript 1 (NEAT1), an essential lncRNA for the formation of nuclear body paraspeckles, is induced by influenza virus and herpes simplex virus infection as well as by Toll-like receptor3-p38 pathway-triggered poly I:C stimulation, resulting in excess formation of paraspeckles. We found that NEAT1 facilitates the expression of antiviral genes including cytokines such as interleukin-8 (IL8). We found that splicing factor proline/glutamine-rich (SFPQ), a NEAT1-binding paraspeckle protein, is a repressor of IL8 transcription, and that NEAT1 induction relocates SFPQ from the IL8 promoter to the paraspeckles, leading to transcriptional activation of IL8. Together, our data show that NEAT1 plays an important role in the innate immune response through the transcriptional regulation of antiviral genes by the stimulus-responsive cooperative action of NEAT1 and SFPQ.

GENETIC DIVERSITY OF ARGENTINE ISOLATES OF FELINE IMMUNODEFICIENCY VIRUS

We report the nucleotide sequence and genetic diversity of part of the envelope (env) gene of four strains of feline immunodeficiency virus (FIV) isolated from Argentine domestic cats. The DNA encoding the V3 to V5 regions of the env gene of the FIV isolates were amplified by PCR, cloned and sequenced. Phylogenetic analysis revealed that the Argentine isolates did not cluster into a single group; one isolate clustered with subtype B FIV isolated in the USA and Japan, whereas the others formed a new cluster of FIV which might represent a prototype sequence for subtype E.

Molecular and phylogenetic analyses of the haemagglutinin (H) proteins of field isolates of canine distemper virus from naturally infected dogs.

We isolated three strains of canine distemper virus (CDV)--the Ueno, Hamamatsu, and Yanaka strains--from dogs in Japan and analysed the molecular properties of their haemagglutinin (H) proteins. Immunoprecipitation of all three strains with a monoclonal antibody revealed H proteins with molecular masses of 84 kDa, which differs from the molecular mass (78 kDa) of the H protein of the Onderstepoort vaccine strain. However, after tunicamycin treatment immunoprecipitation identified H proteins of identical molecular mass (68 kDa) for all three field isolates and the vaccine strain. Sequence analysis showed nine potential sites for asparagine-linked glycosylation in the H proteins of the new isolates, in contrast to four in the H protein of the Onderstepoort strain. Thus, variation in glycosylation of the H proteins of the isolates and the vaccine strain may cause differences in antigenicity of the viruses. Sequences of the H genes showed that the new Japanese isolates have 99% identity with each other, 95% with other European and American isolates (from seals, a German dog, a ferret and large felids) and 90% with the vaccine strain. Phylogenetically, the new Japanese isolates form one cluster which is separate from recent European or American isolates, all of which are distinct from vaccine strains.

Establishment of a Nipah virus rescue system

Nipah virus (NiV), a paramyxovirus, was first discovered in Malaysia in 1998 in an outbreak of infection in pigs and humans and incurred a high fatality rate in humans. Fruit bats, living in vast areas extending from India to the western Pacific, were identified as the natural reservoir of the virus. However, the mechanisms that resulted in severe pathogenicity in humans (up to 70% mortality) and that enabled crossing the species barrier were not known. In this study, we established a system that enabled the rescue of replicating NiVs from a cloned DNA by cotransfection of a constructed full-length cDNA clone and supporting plasmids coding virus nucleoprotein, phosphoprotein, and polymerase with the infection of the recombinant vaccinia virus, MVAGKT7, expressing T7 RNA polymerase. The rescued NiV (rNiV), by using the newly developed reverse genetics system, showed properties in vitro that were similar to the parent virus and retained the severe pathogenicity in a previously established animal model by experimental infection. A recombinant NiV was also developed, expressing enhanced green fluorescent protein (rNiV-EGFP). Using the virus, permissibility of NiV was compared with the presence of a known cellular receptor, ephrin B2, in a number of cell lines of different origins. Interestingly, two cell lines expressing ephrin B2 were not susceptible for rNiV-EGFP, indicating that additional factors are clearly required for full NiV replication. The reverse genetics for NiV will provide a powerful tool for the analysis of the molecular mechanisms of pathogenicity and cross-species infection.

Antigenic differences in the H proteins of canine distemper viruses

Antigenic properties between new Japanese field isolates and vaccine strains of canine distemper virus (CDV) have been compared using four monoclonal antibodies (MAbs) (JD-5, JD-7, JD-11 and d-7) against the hemagglutinin (H) proteins of CDV. JD-5, JD-7 and JD-11 are newly established antibodies. Three MAbs, namely d-7, JD-5 and JD-11, reacted similarly to all the CDV strains examined. However, JD-7 reacted much more strongly with the vaccine strains and an old field isolate than the recent field isolates in immunofluorescence, radio immunoprecipitation and virus neutralization assays. These results indicate that an antigenic region in the H protein, concerned with neutralization and recognized by JD-7, has been altered in the recent field isolates.

CD147/EMMPRIN Acts as a Functional Entry Receptor for Measles Virus on Epithelial Cells

ABSTRACT Measles is a highly contagious human disease caused by measles virus (MeV) and remains the leading cause of death in children, particularly in developing countries. Wild-type MeV preferentially infects lymphocytes by using signaling lymphocytic activation molecule (SLAM), whose expression is restricted to hematopoietic cells, as a receptor. MeV also infects other epithelial and neuronal cells that do not express SLAM and causes pneumonia and diarrhea and, sometimes, serious symptoms such as measles encephalitis and subacute sclerosing panencephalitis. The discrepancy between the tissue tropism of MeV and the distribution of SLAM-positive cells suggests that there are unknown receptors other than SLAM for MeV. Here we identified CD147/EMMPRIN (extracellular matrix metalloproteinase inducer), a transmembrane glycoprotein, which acts as a receptor for MeV on epithelial cells. Furthermore, we found the incorporation of cyclophilin B (CypB), a cellular ligand for CD147, in MeV virions, and showed that inhibition of CypB incorporation significantly attenuated SLAM-independent infection on epithelial cells, while it had no effect on SLAM-dependent infection. To date, MeV infection was considered to be triggered by binding of its hemagglutinin (H) protein and cellular receptors. Our present study, however, indicates that MeV infection also occurs via CD147 and virion-associated CypB, independently of MeV H. Since CD147 is expressed in a variety of cells, including epithelial and neuronal cells, this molecule possibly functions as an entry receptor for MeV in SLAM-negative cells. This is the first report among members of the Mononegavirales that CD147 is used as a virus entry receptor via incorporated CypB in the virions.

Hepatitis C Virus Impairs p53 via Persistent Overexpression of 3β-Hydroxysterol Δ24-Reductase

Persistent infection with hepatitis C virus (HCV) induces tumorigenicity in hepatocytes. To gain insight into the mechanisms underlying this process, we generated monoclonal antibodies on a genome-wide scale against an HCV-expressing human hepatoblastoma-derived cell line, RzM6-LC, showing augmented tumorigenicity. We identified 3β-hydroxysterol Δ24-reductase (DHCR24) from this screen and showed that its expression reflected tumorigenicity. HCV induced the DHCR24 overexpression in human hepatocytes. Ectopic or HCV-induced DHCR24 overexpression resulted in resistance to oxidative stress-induced apoptosis and suppressed p53 activity. DHCR24 overexpression in these cells paralleled the increased interaction between p53 and MDM2 (also known as HDM2), a p53-specific E3 ubiquitin ligase, in the cytoplasm. Persistent DHCR24 overexpression did not alter the phosphorylation status of p53 but resulted in decreased acetylation of p53 at lysine residues 373 and 382 in the nucleus after treatment with hydrogen peroxide. Taken together, these results suggest that DHCR24 is elevated in response to HCV infection and inhibits the p53 stress response by stimulating the accumulation of the MDM2-p53 complex in the cytoplasm and by inhibiting the acetylation of p53 in the nucleus.

The nonstructural proteins of Nipah virus play a key role in pathogenicity in experimentally infected animals.

Nipah virus (NiV) P gene encodes P protein and three accessory proteins (V, C and W). It has been reported that all four P gene products have IFN antagonist activity when the proteins were transiently expressed. However, the role of those accessory proteins in natural infection with NiV remains unknown. We generated recombinant NiVs lacking V, C or W protein, rNiV(V−), rNiV(C−), and rNiV(W−), respectively, to analyze the functions of these proteins in infected cells and the implications in in vivo pathogenicity. All the recombinants grew well in cell culture, although the maximum titers of rNiV(V−) and rNiV(C−) were lower than the other recombinants. The rNiV(V−), rNiV(C−) and rNiV(W−) suppressed the IFN response as well as the parental rNiV, thereby indicating that the lack of each accessory protein does not significantly affect the inhibition of IFN signaling in infected cells. In experimentally infected golden hamsters, rNiV(V−) and rNiV(C−) but not the rNiV(W−) virus showed a significant reduction in virulence. These results suggest that V and C proteins play key roles in NiV pathogenicity, and the roles are independent of their IFN-antagonist activity. This is the first report that identifies the molecular determinants of NiV in pathogenicity in vivo.

Feline immunodeficiency virus can infect a human cell line (MOLT-4) but establishes a state of latency in the cells.

Infectivity of feline immunodeficiency virus (FIV) in feline and human lymphoblastoid cell lines was examined using homogeneous populations of FIV derived from infectious molecular clones of strains TMZ and Petaluma, and two recombinant chimeric clones carrying gag, pol, vif and ORF-A from the heterologous virus. FIV from the clones with the env region of the Petaluma strain was shown to infect and establish provirus in a human lymphoid cell line (MOLT-4), although the FIV-infected cells did not produce any infectious viruses. By treatment of the infected MOLT-4 cells with a phorbol ester, infectious virus was rescued. To examine which stage of the life-cycle of FIV is blocked in these cells, we analysed transcription of FIV-14 in the cells by RT-PCR. FIV-specific RNA expression could not be detected. These results strongly suggest that latency of the virus in MOLT-4 cells is due to a failure in transcription.

Prior Immunization with Severe Acute Respiratory Syndrome (SARS)-Associated Coronavirus (SARS-CoV) Nucleocapsid Protein Causes Severe Pneumonia in Mice Infected with SARS-CoV

The details of the mechanism by which severe acute respiratory syndrome-associated coronavirus (SARS-CoV) causes severe pneumonia are unclear. We investigated the immune responses and pathologies of SARS-CoV-infected BALB/c mice that were immunized intradermally with recombinant vaccinia virus (VV) that expressed either the SARS-CoV spike (S) protein (LC16m8rVV-S) or simultaneously all the structural proteins, including the nucleocapsid (N), membrane (M), envelope (E), and S proteins (LC16m8rVV-NMES) 7–8 wk before intranasal SARS-CoV infection. The LC16m8rVV-NMES-immunized group exhibited as severe pneumonia as the control groups, although LC16m8rVV-NMES significantly decreased the pulmonary SARS-CoV titer to the same extent as LC16m8rVV-S. To identify the cause of the exacerbated pneumonia, BALB/c mice were immunized with recombinant VV that expressed the individual structural proteins of SARS-CoV (LC16mOrVV-N, -M, -E, -S) with or without LC16mOrVV-S (i.e., LC16mOrVV-N, LC16mOrVV-M, LC16mOrVV-E, or LC16mOrVV-S alone or LC16mOrVV-N + LC16mOrVV-S, LC16mOrVV-M + LC16mOrVV-S, or LC16mOrVV-E + LC16mOrVV-S), and infected with SARS-CoV more than 4 wk later. Both LC16mOrVV-N-immunized mice and LC16mOrVV-N + LC16mOrVV-S-immunized mice exhibited severe pneumonia. Furthermore, LC16mOrVV-N-immunized mice upon infection exhibited significant up-regulation of both Th1 (IFN-γ, IL-2) and Th2 (IL-4, IL-5) cytokines and down-regulation of anti-inflammatory cytokines (IL-10, TGF-β), resulting in robust infiltration of neutrophils, eosinophils, and lymphocytes into the lung, as well as thickening of the alveolar epithelium. These results suggest that an excessive host immune response against the nucleocapsid protein of SARS-CoV is involved in severe pneumonia caused by SARS-CoV infection. These findings increase our understanding of the pathogenesis of SARS.