Inhibition of Influenza Virus Infection by Lentinus edodes Mycelia Extract Through Its Direct Action and Immunopotentiating Activity.

Lentinula edodes mycelia (LEM) solid culture extracts contain many bioactive compounds with diverse pharmacological activities such as antitumor, antiviral, and immunopotentiating effects. In this study, we examined the anti-influenza virus activity of LEM in vitro and in vivo. LEM directly inhibited influenza virus growth in vitro at early phases of infection, possibly at the entry process of viral particles to host cells. We also found that the nasal administration of LEM increased the survival rate of infected mice, and this was likely due to the direct action of LEM on the viral growth. The oral administration of LEM showed prolonged median survival time of infected mice. Histological analysis revealed that the moderate bronchiolitis was observed in infected mice by the oral administration with LEM, and the extent of alveolitis was dramatically reduced. The orally LEM-administered mice showed a rapid activation of IFN-ß gene expression upon influenza virus infection. These results suggest that the immunopotentiation activity of LEM on type I IFN pathway represses the virus spread to distal alveolar regions from peribronchiolar regions which are primary infection sites in the mouse model. We propose that LEM has anti-influenza virus activities through the direct action on viral growth and stimulatory activity of innate immunity.

Actin-myosin network is required for proper assembly of influenza virus particles.

Actin filaments are known to play a central role in cellular dynamics. After polymerization of actin, various actin-crosslinking proteins including non-muscle myosin II facilitate the formation of spatially organized actin filament networks. The actin-myosin network is highly expanded beneath plasma membrane. The genome of influenza virus (vRNA) replicates in the cell nucleus. Then, newly synthesized vRNAs are nuclear-exported to the cytoplasm as ribonucleoprotein complexes (vRNPs), followed by transport to the beneath plasma membrane where virus particles assemble. Here, we found that, by inhibiting actin-myosin network formation, the virus titer tends to be reduced and HA viral spike protein is aggregated on the plasma membrane. These results indicate that the actin-myosin network plays an important role in the virus formation.

Reconstitution of human rRNA gene transcription in mouse cells by a complete SL1 complex.

An important characteristic of the transcription of a ribosomal RNA gene (rDNA) mediated by DNA-dependent RNA polymerase (Pol) I is its stringent species specificity. SL1/TIF-IB is a key complex for species specificity, but its functional complex has not been reconstituted. Here, we established a novel and highly sensitive monitoring system for Pol I transcription to reconstitute the SL1 activity in which a transcript harboring a reporter gene synthesized by Pol I is amplified and converted into translatable mRNA by the influenza virus RNA-dependent RNA polymerase. Using this monitoring system, we reconstituted Pol I transcription from the human rDNA promoter in mouse cells by expressing four human TATA-binding protein (TBP)-associated factors (TAFIs) in the SL1 complex. The reconstituted SL1 also re-activated human rDNA transcription in mouse A9 cells carrying an inactive human chromosome 21 that contains the rDNA cluster. Chimeric SL1 complexes containing human and mouse TAFIs could be formed, but these complexes were inactive for human rDNA transcription. We conclude that four human TAFIs are necessary and sufficient to overcome the barrier of species specificity for human rDNA transcription in mouse cells.

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.

Sorting of influenza A virus RNA genome segments after nuclear export.

The genome of the influenza A virus consists of eight different segments. These eight segments are thought to be sorted selectively in infected cells. However, the cellular compartment where segments are sorted is not known. We examined using temperature sensitive (ts) mutant viruses and cell fusion where segments are sorted in infected cells. Different cells were infected with different ts mutant viruses, and these cells were fused. In fused cells, genome segments are mixed only in the cytoplasm, because M1 prevents their re-import into the nucleus. We made a marker ts53 virus, which has silent mutations in given segments and determined the reassortment frequency on all segments using ts1 and marker ts53. In both co-infected and fused cells, all of marker ts53 segments and ts1 segments were incorporated into progeny virions in a random fashion. These results suggest that influenza virus genome segments are sorted after nuclear export.