TMPRSS2 gene polymorphism common in East Asians confers decreased COVID-19 susceptibility.

COVID-19 has a wide range of clinical presentations, and the susceptibility to SARS-CoV-2 infection and the mortality rate also vary by region and ethnicity. Here, we found that rs12329760 in the TMPRSS2 gene, a missense variant common in East Asian populations, contributes to protection against SARS-CoV-2 infection. TMPRSS2 is a protease responsible for SARS-CoV-2 entry and syncytium formation. rs12329760 (c.478G>A, p. V160M) was associated with a reduced risk of moderate symptoms. The enzymatic activity of Met160-TMPRSS2 was lower than that of Val160-TMPRSS2, and thus the viral entry and the syncytium formation of SARS-CoV-2 were impaired. Collectively, these results indicate that the genetic variation in TMPRSS2, which is common in East Asians, is one of the molecular determinants of COVID-19 susceptibility.

A case of restless legs syndrome after BNT162b2 mRNA COVID-19 vaccination.

Restless legs syndrome (RLS) can be secondary to several disorders. We present an 87-year-old woman who developed RLS 2 days after the first injection of BNT162b2 mRNA coronavirus disease 2019 vaccine. The symptoms of RLS tended to improve and eventually resolved with the administration of gabapentin.

ARHGAP1 Transported with Influenza Viral Genome Ensures Integrity of Viral Particle Surface through Efficient Budozone Formation.

Influenza viral particles are assembled at the plasma membrane concomitantly with Rab11a-mediated endocytic transport of viral ribonucleoprotein complexes (vRNPs). The mechanism of spatiotemporal regulation of viral budozone formation and its regulatory molecules on the endocytic vesicles remain unclear. Here, we performed a proximity-based proteomics approach for Rab11a and found that ARHGAP1, a Rho GTPase-activating protein, is transported through the Rab11a-mediated apical transport of vRNP. ARHGAP1 stabilized actin filaments in infected cells for the lateral clustering of hemagglutinin (HA) molecules, a viral surface membrane protein, to the budozone. Disruption of the HA clustering results in the production of virions with low HA content, and such virions were less resistant to protease and had enhanced antigenicity, presumably because reduced clustering of viral membrane proteins exposes hidden surfaces. Collectively, these results demonstrate that Rab11a-mediated endocytic transport of ARHGAP1 with vRNPs stimulates budozone formation to ensure the integrity of virion surface required for viral survival. IMPORTANCE The endocytic transport of the influenza viral genome triggers the clustering of viral membrane proteins at the plasma membrane to form the viral budozone. However, host factors that promote viral budozone formation in concert with viral genome transport have not been identified. Here, we found that ARHGAP1, a negative regulator of the Rho family protein, is transported with the viral genome and stabilizes actin filaments to promote budozone formation. We have shown that ARHGAP1-mediated efficient formation of viral budozone was crucial for the clustering of viral HA protein to the progeny viral particles. The clustering of HA proteins on the virions is responsible for the structural integrity of the viral particles, which promotes viral stability and viral immune evasion. This study highlights the molecular mechanism that works in concert with viral genome packaging to ensure the structural integrity of viral particles.

Arf6 exacerbates allergic asthma through cell-to-cell transmission of ASC inflammasomes.

Asthma is a chronic inflammatory disease of the airways associated with excess production of Th2 cytokines and lung eosinophil accumulation. This inflammatory response persists in spite of steroid administration that blocks autocrine/paracrine loops of inflammatory cytokines, and the detailed mechanisms underlying asthma exacerbation remain unclear. Here, we show that asthma exacerbation is triggered by airway macrophages through a prion-like cell-to-cell transmission of extracellular particulates, including ASC protein, that assemble inflammasomes and mediate IL-1ß production. OVA-induced allergic asthma and associated IL-1ß production were alleviated in mice with small GTPase Arf6-deficient macrophages. The extracellular ASC specks were slightly engulfed by Arf6-/- macrophages, and the IL-1ß production was reduced in Arf6-/- macrophages compared with that in WT macrophages. Furthermore, pharmacological inhibition of the Arf6 guanine nucleotide exchange factor suppressed asthma-like allergic inflammation in OVA-challenged WT mice. Collectively, the Arf6-dependent intercellular transmission of extracellular ASC specks contributes to the amplification of allergic inflammation and subsequent asthma exacerbation.

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.

Influenza A Virus NS1 Protein Suppresses JNK1-Dependent Autophagosome Formation Mediated by Rab11a Recycling Endosomes.

Autophagy is an essential process for cellular metabolism and homeostasis, but also functions as one of innate immune responses against pathogen infection. However, in contrast to cellular metabolism and homeostasis pathways, less is known about how virus infection leads to autophagosome formation. Here, we showed that influenza A virus NS1 protein inhibits the formation of autophagosomes. The autophagosome formation was induced by infection with NS1 mutant virus lacking the dsRNA-binding activity for inhibition of innate immune responses (R38AK41A) or the activation of PI3K-Akt signaling pathway (Y89F). R38AK41A mutant infection induced phosphorylation of JNK1 and up-regulated the expression of autophagy-related genes which are downstream of JNK1 signaling pathway. We also found that the amount of phosphorylated TSC2, which activates mTOR, increased in wild type-infected cells but not in Y89F mutant-infected cells. These findings suggest that NS1 inhibits the autophagosome formation through both the inhibition of JNK1 and the activation of PI3K-Akt-mTOR pathway. Further, viral ribonucleoprotein (vRNP) complexes were selectively sequestered into autophagosomes, and knockdown of Rab11a, which is responsible for the apical transport of vRNP complexes, impaired not only engulfment of vRNP complexes by autophagosomes but also the formation of autophagosomes in R38AK41A mutant-infected cells. This indicates that Rab11a-positive recycling endosomes function as a donor membrane for the phagophore elongation and an autophagic receptor for the selective engulfment of viral RNP complexes. Based on these results, we propose that NS1 inhibits JNK1-mediated autophagy induction and the sequestration of vRNP complexes into autophagosomes.

RLR-mediated antiviral innate immunity requires oxidative phosphorylation activity.

Mitochondria act as a platform for antiviral innate immunity, and the immune system depends on activation of the retinoic acid-inducible gene I (RIG-I)-like receptors (RLR) signaling pathway via an adaptor molecule, mitochondrial antiviral signaling. We report that RLR-mediated antiviral innate immunity requires oxidative phosphorylation (OXPHOS) activity, a prominent physiologic function of mitochondria. Cells lacking mitochondrial DNA or mutant cells with respiratory defects exhibited severely impaired virus-induced induction of interferons and proinflammatory cytokines. Recovery of the OXPHOS activity in these mutants, however, re-established RLR-mediated signal transduction. Using in vivo approaches, we found that mice with OXPHOS defects were highly susceptible to viral infection and exhibited significant lung inflammation. Studies to elucidate the molecular mechanism of OXPHOS-coupled immune activity revealed that optic atrophy 1, a mediator of mitochondrial fusion, contributes to regulate the antiviral immune response. Our findings provide evidence for functional coordination between RLR-mediated antiviral innate immunity and the mitochondrial energy-generating system in mammals.

Probing the role of Asp-120(81) of metallo-beta-lactamase (IMP-1) by site-directed mutagenesis, kinetic studies, and X-ray crystallography.

Metallo-beta-lactamase IMP-1 is a di-Zn(II) metalloenzyme that efficiently hydrolyzes beta-lactam antibiotics. Wild-type (WT) IMP-1 has a conserved Asp-120(81) in the active site, which plays an important role in catalysis. To probe the catalytic role of Asp-120(81) in IMP-1, the IMP-1 mutants, D120(81)A and D120(81)E, were prepared by site-directed mutagenesis, and various kinetics studies were conducted. The IMP-1 mutants exhibited 10(2)-10(4)-fold drops in k(cat) values compared with WT despite the fact that they contained two Zn(II) ions in the active site. To evaluate the acid-base characteristics of Asp-120(81), the pH dependence for hydrolysis was examined by stopped-flow studies. No observable pK(a) values between pH 5 and 9 were found for WT and D120(81)A. The rapid mixing of equimolar amounts of nitrocefin and all enzymes failed to result in the detection of an anion intermediate of nitrocefin at 650 nm. These results suggest that Asp-120(81) of IMP-1 is not a factor in decreasing the pK(a) for the water bridging two Zn(II) ions and is not a proton donor to the anionic intermediate. In the case of D120(81)E, the nitrocefin hydrolysis product, which shows a maximum absorption at 460 nm, was bound to D120(81)E in the protonated form. The three-dimensional structures of D120(81)A and D120(81)E were also determined at 2.0 and 3.0 A resolutions, respectively. In the case of D120(81)E, the Zn-Zn distance was increased by 0.3 A compared with WT, due to the change in the coordination mode of Glu-120(81)OE1 and the positional shift in the conserved His-263(197) at the active site.