Daisuke Fujikura

ZAPS is a potent stimulator of signaling mediated by the RNA helicase RIG-I during antiviral responses

The poly(ADP-ribose) polymerases (PARPs) participate in many biological and pathological processes. Here we report that the PARP-13 shorter isoform (ZAPS), rather than the full-length protein (ZAP), was selectively induced by 5′-triphosphate–modified RNA (3pRNA) and functioned as a potent stimulator of interferon responses in human cells mediated by the RNA helicase RIG-I. ZAPS associated with RIG-I to promote the oligomerization and ATPase activity of RIG-I, which led to robust activation of IRF3 and NF-κB transcription factors. Disruption of the gene encoding ZAPS resulted in impaired induction of interferon-α (IFN-α), IFN-β and other cytokines after viral infection. These results indicate that ZAPS is a key regulator of RIG-I signaling during the innate antiviral immune response, which suggests its possible use as a therapeutic target for viral control.

Induction of Src-suppressed C Kinase Substrate (SSeCKS) in Vascular Endothelial Cells by Bacterial Lipopolysaccharide

We isolated cDNA of the mouse homologue of the src-suppressed C kinase substrate (SSeCKS) and analyzed the effects of lipopolysaccharide (LPS) injection on the tissue expression pattern of this protein. Northern blotting analysis showed that SSeCKS mRNA was expressed abundantly in the testis but at undetectable levels in other tissues of untreated control mice. Intraperitoneal administration of LPS strongly induced SSeCKS mRNA expression in the lung, heart, liver, spleen, kidney, lymph node, adrenal gland, and pituitary gland, as well as in the brain. In lung and spleen, the SSeCKS mRNA levels increased almost 10-fold at 1 hr after LPS injection and persisted at high levels until 4 hr. Both in situ hybridization and immunohistochemical studies revealed that LPS administration conspicuously elevated expression of SSeCKS mRNA and protein in vascular endothelial cells of several organs. Ectopic expression of SSeCKS caused loss of cytoplasmic F-actin fibers in the mouse endothelial cell line LEII. These results indicate that SSeCKS is one of the major LPS-responsive proteins and may participate in alteration of cytoskeletal architecture in endothelial cells during inflammation.

Interaction between TIM-1 and NPC1 Is Important for Cellular Entry of Ebola Virus

ABSTRACT Multiple host molecules are known to be involved in the cellular entry of filoviruses, including Ebola virus (EBOV); T-cell immunoglobulin and mucin domain 1 (TIM-1) and Niemann-Pick C1 (NPC1) have been identified as attachment and fusion receptors, respectively. However, the molecular mechanisms underlying the entry process have not been fully understood. We found that TIM-1 and NPC1 colocalized and interacted in the intracellular vesicles where EBOV glycoprotein (GP)-mediated membrane fusion occurred. Interestingly, a TIM-1-specific monoclonal antibody (MAb), M224/1, prevented GP-mediated membrane fusion and also interfered with the binding of TIM-1 to NPC1, suggesting that the interaction between TIM-1 and NPC1 is important for filovirus membrane fusion. Moreover, MAb M224/1 efficiently inhibited the cellular entry of viruses from all known filovirus species. These data suggest a novel mechanism underlying filovirus membrane fusion and provide a potential cellular target for antiviral compounds that can be universally used against filovirus infections. IMPORTANCE Filoviruses, including Ebola and Marburg viruses, cause rapidly fatal diseases in humans and nonhuman primates. There are currently no approved vaccines or therapeutics for filovirus diseases. In general, the cellular entry step of viruses is one of the key mechanisms to develop antiviral strategies. However, the molecular mechanisms underlying the entry process of filoviruses have not been fully understood. In this study, we demonstrate that TIM-1 and NPC1, which serve as attachment and fusion receptors for filovirus entry, interact in the intracellular vesicles where Ebola virus GP-mediated membrane fusion occurs and that this interaction is important for filovirus infection. We found that filovirus infection and GP-mediated membrane fusion in cultured cells were remarkably suppressed by treatment with a TIM-1-specific monoclonal antibody that interfered with the interaction between TIM-1 and NPC1. Our data provide new insights for the development of antiviral compounds that can be universally used against filovirus infections.

Heat Shock Protein 70 Modulates Influenza A Virus Polymerase Activity

Background: It has been shown that heat shock protein 70 (Hsp70) plays a role in influenza A virus replication. Results: A correlation between viral replication/transcription activities and nuclear/cytoplasmic shuttling of Hsp70 was observed. Conclusion: Hsp70 modulates the influenza A virus polymerase activity. Significance: This study, for the first time, suggests that Hsp70 may actually assist in influenza A virus replication. The role of heat shock protein 70 (Hsp70) in virus replication has been discussed for many viruses. The known suppressive role of Hsp70 in influenza virus replication is based on studies conducted in cells with various Hsp70 expression levels. In this study, we determined the role of Hsp70 in influenza virus replication in HeLa and HEK293T cells, which express Hsp70 constitutively. Co-immunoprecipitation and immunofluorescence studies revealed that Hsp70 interacted with PB2 or PB1 monomers and PB2/PB1 heterodimer but not with the PB1/PA heterodimer or PB2/PB1/PA heterotrimer and translocated into the nucleus with PB2 monomers or PB2/PB1 heterodimers. Knocking down Hsp70 resulted in reduced virus transcription and replication activities. Reporter gene assay, immunofluorescence assay, and Western blot analysis of nuclear and cytoplasmic fractions from infected cells demonstrated that the increase in viral polymerase activity during the heat shock phase was accompanied with an increase in Hsp70 and viral polymerases levels in the nuclei, where influenza virus replication takes place, whereas a reduction in viral polymerase activity was accompanied with an increase in cytoplasmic relocation of Hsp70 along with viral polymerases. Moreover, significantly higher levels of viral genomic RNA (vRNA) were observed during the heat shock phase than during the recovery phase. Overall, for the first time, these findings suggest that Hsp70 may act as a chaperone for influenza virus polymerase, and the modulatory effect of Hsp70 appears to be a sequel of shuttling of Hsp70 between nuclear and cytoplasmic compartments.

Putative endogenous filovirus VP35-like protein potentially functions as an IFN antagonist but not a polymerase cofactor

It has been proposed that some non-retroviral RNA virus genes are integrated into vertebrate genomes. Endogenous filovirus-like elements (EFLs) have been discovered in some mammalian genomes. However, their potential roles in ebolavirus infection are unclear. A filovirus VP35-like element (mlEFL35) is found in the little brown bat (Myotis lucifugus) genome. Putative mlEFL35-derived protein (mlEFL35p) contains nearly full-length amino acid sequences corresponding to ebolavirus VP35. Ebola virus VP35 has been shown to bind double-stranded RNA, leading to inhibition of type I interferon (IFN) production, and is also known as a viral polymerase cofactor that is essential for viral RNA transcription/replication. In this study, we transiently expressed mlEFL35p in human kidney cells and investigated its biological functions. We first found that mlEFL35p was coimmunoprecipitated with itself and ebolavirus VP35s but not with the viral nucleoprotein. Then the biological functions of mlEFL35p were analyzed by comparing it to ebolavirus VP35s. We found that the expression of mlEFL35p significantly inhibited human IFN-β promoter activity as well as VP35s. By contrast, expression of mlEFL35p did not support viral RNA transcription/replication and indeed slightly decrease the reporter gene expression in a minigenome assay. These results suggest that mlEFL35p potentially acts as an IFN antagonist but not a polymerase cofactor.

Genome Sequence of a Bacillus anthracis Outbreak Strain from Zambia, 2011

ABSTRACT In August 2011, an anthrax outbreak occurred among Hippopotamus amphibius hippopotamuses and humans in Zambia. Here, we report the draft genome sequence of the Bacillus anthracis outbreak strain CZC5, isolated from tissues of H. amphibius hippopotamuses that had died in the outbreak area.

Death receptor 6 contributes to autoimmunity in lupus-prone mice

Expansion of autoreactive follicular helper T (Tfh) cells is tightly restricted to prevent induction of autoantibody-dependent immunological diseases, such as systemic lupus erythematosus (SLE). Here we show expression of an orphan immune regulator, death receptor 6 (DR6/TNFRSF21), on a population of Tfh cells that are highly expanded in lupus-like disease progression in mice. Genome-wide screening reveals an interaction between syndecan-1 and DR6 resulting in immunosuppressive functions. Importantly, syndecan-1 is expressed specifically on autoreactive germinal centre (GC) B cells that are critical for maintenance of Tfh cells. Syndecan-1 expression level on GC B cells is associated with Tfh cell expansion and disease progression in lupus-prone mouse strains. In addition, Tfh cell suppression by DR6-specific monoclonal antibody delays disease progression in lupus-prone mice. These findings suggest that the DR6/syndecan-1 axis regulates aberrant GC reactions and could be a therapeutic target for autoimmune diseases such as SLE.

Effect of water-soluble fraction from lysozyme-treated Enterococcus faecalis FK-23 on mortality caused by influenza A virus in mice.

To maintain homeostasis of the immune system is considered important for the prevention of influenza A virus infection. Aberrant systemic inflammation is frequently induced by influenza A virus infection, and the severity of the symptoms is associated with pathogenicity of the virus. Lactic acid bacteria are known to have a positive effect in maintaining the immune system. Furthermore, preparations of a lactic acid bacteria strain, Enterococcus faecalis FK-23 (FK-23), have been reported to exert preferable homeostatic effects on immune diseases such as allergic rhinitis and early asthmatic responses. In this study, we examined the efficacy of the water-soluble fraction of lysed and heat-treated FK-23 (SLFK) against a lethal influenza A virus challenge. Mice were orally administered SLFK from day -7 to day 20, and intranasally infected with influenza virus A/Puerto Rico/8/34 (H1N1) at 10(3) PFU on day 0. The survival rate of SLFK-administered mice after influenza A virus infection was significantly improved compared with that of control mice. In addition, the mRNA expression level of the anti-inflammatory cytokine interleukin-10 (IL-10) in lung tissues was enhanced by the oral administration of SLFK after influenza A virus infection. These observations suggest that the oral administration of SLFK exerts a protective effect against influenza virus infection through the activation of the anti-inflammatory response.

A novel multiplex PCR discriminates Bacillus anthracis and its genetically related strains from other Bacillus cereus group species.

Anthrax is an important zoonotic disease worldwide that is caused by Bacillus anthracis, a spore-forming pathogenic bacterium. A rapid and sensitive method to detect B. anthracis is important for anthrax risk management and control in animal cases to address public health issues. However, it has recently become difficult to identify B. anthracis by using previously reported molecular-based methods because of the emergence of B. cereus, which causes severe extra-intestinal infection, as well as the human pathogenic B. thuringiensis, both of which are genetically related to B. anthracis. The close genetic relation of chromosomal backgrounds has led to complexity of molecular-based diagnosis. In this study, we established a B. anthracis multiplex PCR that can screen for the presence of B. anthracis virulent plasmids and differentiate B. anthracis and its genetically related strains from other B. cereus group species. Six sets of primers targeting a chromosome of B. anthracis and B. anthracis-like strains, two virulent plasmids, pXO1 and pXO2, a bacterial gene, 16S rRNA gene, and a mammalian gene, actin-beta gene, were designed. The multiplex PCR detected approximately 3.0 CFU of B. anthracis DNA per PCR reaction and was sensitive to B. anthracis. The internal control primers also detected all bacterial and mammalian DNAs examined, indicating the practical applicability of this assay as it enables monitoring of appropriate amplification. The assay was also applied for detection of clinical strains genetically related to B. anthracis, which were B. cereus strains isolated from outbreaks of hospital infections in Japan, and field strains isolated in Zambia, and the assay differentiated B. anthracis and its genetically related strains from other B. cereus group strains. Taken together, the results indicate that the newly developed multiplex PCR is a sensitive and practical method for detecting B. anthracis.

Aureobasidium pullulans-cultured fluid induces IL-18 production, leading to Th1-polarization during influenza A virus infection

Several microbial molecules with pathogen-associated molecular patterns stimulate host innate immune responses. The innate immune system plays a crucial role in activating acquired immune response via cytokine production and antigen presentation. Previous studies have shown that Aureobasidium pullulans-cultured fluid (AP-CF), which contains β-glucan, exhibits adjuvant activity and renders mice resistance to influenza A virus infection; however, the underlying mechanism remains elusive. In this study, we investigated the innate immune response to AP-CF. We found that intraperitoneal administration of AP-CF increased the serum level of IL-18 and the number of splenic IFN-γ producing CD4+ cells during influenza A virus infection. The adjuvant effect of AP-CF was distinct from that of alum, which is known to have the ability to stimulate a Th2 immune response. In addition, AP-CF injection barely increased the number of peritoneal neutrophils and inflammatory macrophages, whereas alum injection markedly increased the number of neutrophils and inflammatory macrophages, suggesting that AP-CF is a weak inducer of inflammation compared to alum. AP-CF induced IL-18 production by DC2.4 cells, a dendritic cell line, and by peritoneal exudate cells that include peritoneal macrophages. Collectively, our findings indicate that AP-CF is an adjuvant that promotes the Th1 response during influenza A virus infection.