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Pest resistance molecules, α-amylase/trypsin inhibitors from wheat, activate innate immune cells through engagement of TLR4 to elicit inflammatory responses in the intestine.

The coronavirus disease 2019 ) pandemic now has >2,000,000 confirmed cases worldwide. COVID-19 is currently diagnosed using quantitative RT-PCR methods, but the capacity of quantitative RT-PCR methods is limited by their requirement of high-level facilities and instruments. We developed and evaluated reverse transcription loop-mediated isothermal amplification (RT-LAMP) assays to detect genomic RNA of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative virus of COVID-19. RT-LAMP assays reported in this study can detect as low as 100 copies of SARS-CoV-2 RNA. Cross-reactivity of RT-LAMP assays to other human coronaviruses was not observed. A colorimetric detection method was adapted for this RT-LAMP assay to enable higher throughput.

Human hepatitis B virus (HBV) causes chronic hepatitis and is associated with the development of hepatocellular carcinoma. HBV infection alters mitochondrial metabolism. The selective removal of damaged mitochondria is essential for the maintenance of mitochondrial and cellular homeostasis. Here, we report that HBV shifts the balance of mitochondrial dynamics toward fission and mitophagy to attenuate the virus-induced apoptosis. HBV induced perinuclear clustering of mitochondria and triggered mitochondrial translocation of the dynamin-related protein (Drp1) by stimulating its phosphorylation at Ser616, leading to mitochondrial fission. HBV also stimulated the gene expression of Parkin, PINK1, and LC3B and induced Parkin recruitment to the mitochondria. Upon translocation to mitochondria, Parkin, an E3 ubiquitin ligase, underwent self-ubiquitination and facilitated the ubiquitination and degradation of its substrate Mitofusin 2 (Mfn2), a mediator of mitochondrial fusion. In addition to conventional immunofluorescence, a sensitive dual fluorescence reporter expressing mito-mRFP-EGFP fused in-frame to a mitochondrial targeting sequence was employed to observe the completion of the mitophagic process by delivery of the engulfed mitochondria to lysosomes for degradation. Furthermore, we demonstrate that viral HBx protein plays a central role in promoting aberrant mitochondrial dynamics either when expressed alone or in the context of viral genome. Perturbing mitophagy by silencing Parkin led to enhanced apoptotic signaling, suggesting that HBV-induced mitochondrial fission and mitophagy promote cell survival and possibly viral persistence. Altered mitochondrial dynamics associated with HBV infection may contribute to mitochondrial injury and liver disease pathogenesis.

Mitochondrial dynamics is crucial for the regulation of cell homeostasis. Our recent findings suggest that hepatitis C virus (HCV) promotes Parkin-mediated elimination of damaged mitochondria (mitophagy). Here we show that HCV perturbs mitochondrial dynamics by promoting mitochondrial fission followed by mitophagy, which attenuates HCV-induced apoptosis. HCV infection stimulated expression of dynamin-related protein 1 (Drp1) and its mitochondrial receptor, mitochondrial fission factor. HCV further induced the phosphorylation of Drp1 (Ser616) and caused its subsequent translocation to the mitochondria, followed by mitophagy. Interference of HCV-induced mitochondrial fission and mitophagy by Drp1 silencing suppressed HCV secretion, with a concomitant decrease in cellular glycolysis and ATP levels, as well as enhanced innate immune signaling. More importantly, silencing Drp1 or Parkin caused significant increase in apoptotic signaling, evidenced by increased cytochrome C release from mitochondria, caspase 3 activity, and cleavage of poly(ADP-ribose) polymerase. These results suggest that HCV-induced mitochondrial fission and mitophagy serve to attenuate apoptosis and may contribute to persistent HCV infection.HCV persistence | innate immunity | autophagy H epatitis C virus (HCV) infection often leads to chronic hepatitis that can progress to fibrosis, cirrhosis, and hepatocellular carcinoma (1). HCV is a hepatotropic, noncytopathic (2, 3), single-stranded, positive-sense RNA virus that replicates its RNA genome on the endoplasmic reticulum (ER)-derived membranous structures (4, 5). HCV stimulates lipogenesis, leading to the accumulation of lipid droplets that facilitate virion assembly and maturation (5-8). HCV infection also induces mitochondrial dysfunction via ER and oxidative stress that results in mitochondrial Ca 2+ overload, collapse of mitochondrial transmembrane potential (ΔΨm), elevated levels of reactive oxygen species, and disruption of mitochondrial respiration (9-15). Liver tissues of patients with chronic hepatitis C frequently exhibit traits of mitochondrial injury such as swollen, ruptured, and empty mitochondria (16).Mitochondria are dynamic organelles that constantly undergo fission, fusion, and mitophagy to facilitate mitochondrial quality control, which is crucial for maintaining cell viability and bioenergetics (17). Aberrant mitochondrial dynamics are associated with the pathogenesis of several genetic and neurological disorders, cardiac dysfunctions, cancer, and metabolic diseases such as diabetes and obesity (18). Depending on their physiological and cellular context, the balance between mitochondrial fission and fusion processes modulates the mitochondrial morphology (17). Mitochondrial fission/fragmentation is mediated by recruitment of cytosolic Drp1 to the mitochondria, forming spirals that constrict both the inner and outer mitochondrial membranes (19). The mitochondrial fission is modulated by mitochondrial outer membrane proteins, which include mitochondrial fission 1 (Fis1), mitocho...

-methyladenosine (mA) RNA methylation is the most abundant epitranscriptomic modification of eukaryotic messenger RNAs (mRNAs). Previous reports have found mA on both cellular and viral transcripts and defined its role in regulating numerous biological processes, including viral infection. Here, we show that mA and its associated machinery regulate the life cycle of hepatitis B virus (HBV). HBV is a DNA virus that completes its life cycle via an RNA intermediate, termed pregenomic RNA (pgRNA). Silencing of enzymes that catalyze the addition of mA to RNA resulted in increased HBV protein expression, but overall reduced reverse transcription of the pgRNA. We mapped the mA site in the HBV RNA and found that a conserved mA consensus motif situated within the epsilon stem loop structure, is the site for mA modification. The epsilon stem loop is located in the 3' terminus of all HBV mRNAs and at both the 5' and 3' termini of the pgRNA. Mutational analysis of the identified mA site in the 5' epsilon stem loop of pgRNA revealed that mA at this site is required for efficient reverse transcription of pgRNA, while mA methylation of the 3' epsilon stem loop results in destabilization of all HBV transcripts, suggesting that mA has dual regulatory function for HBV RNA. Overall, this study reveals molecular insights into how mA regulates HBV gene expression and reverse transcription, leading to an increased level of understanding of the HBV life cycle.

Hepatitis C Virus (HCV) induces intracellular events that trigger mitochondrial dysfunction and promote host metabolic alterations. Here, we investigated selective autophagic degradation of mitochondria (mitophagy) in HCV-infected cells. HCV infection stimulated Parkin and PINK1 gene expression, induced perinuclear clustering of mitochondria, and promoted mitochondrial translocation of Parkin, an initial event in mitophagy. Liver tissues from chronic HCV patients also exhibited notable levels of Parkin induction. Using multiple strategies involving confocal and electron microscopy, we demonstrated that HCV-infected cells display greater number of mitophagosomes and mitophagolysosomes compared to uninfected cells. HCV-induced mitophagy was evidenced by the colocalization of LC3 puncta with Parkin-associated mitochondria and lysosomes. Ultrastructural analysis by electron microscopy and immunoelectron microscopy also displayed engulfment of damaged mitochondria in double membrane vesicles in HCV-infected cells. The HCV-induced mitophagy occurred irrespective of genotypic differences. Silencing Parkin and PINK1 hindered HCV replication suggesting the functional relevance of mitophagy in HCV propagation. HCV-mediated decline of mitochondrial complex I enzyme activity was rescued by chemical inhibition of mitophagy or by Parkin silencing. Overall our results suggest that HCV induces Parkin-dependent mitophagy, which may have significant contribution in mitochondrial liver injury associated with chronic hepatitis C.

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