Redirecting the JAK-STAT signal blocks the SARS-CoV-2 replication.

The distinct disease progression patterns of severe acute respiratory syndrome coronavirus clade 2 (SARS-CoV-2) indicate diverse host immune responses. SARS-CoV-2 severely impairs type I interferon (IFN) cell signaling, resulting in uncontrolled late-phase lung damage in patients. For better pharmacological properties, cytokine modifications may sometimes result in a loss of biological activity against the virus. Here, we employed the genetic code expansion and engineered IFN-ß, a phase II clinical cytokine with 3-amino tyrosine (IFN-ß-A) that reactivates STAT2 expression in virus-infected human cells through JAK/STAT cell signaling without affecting signal activation and serum half-life. This study identified that genetically encoded IFN-ß-A might stabilize the protein-receptor complex and trigger JAK-STAT cell signaling, which is a promising modality for controlling SARS-CoV-2 infection.

Equine immunoglobulin fragment F(ab')2 displays high neutralizing capability against multiple SARS-CoV-2 variants.

Neutralizing antibody-based passive immunotherapy could be an important therapeutic option against COVID-19. Herein, we demonstrate that equines hyper-immunized with chemically inactivated SARS-CoV-2 elicited high antibody titers with a strong virus-neutralizing potential, and F(ab')2 fragments purified from them displayed strong neutralization potential against five different SARS-CoV-2 variants. F(ab')2 fragments purified from the plasma of hyperimmunized horses showed high antigen-specific affinity. Experiments in rabbits suggested that the F(ab')2 displays a linear pharmacokinetics with approximate plasma half-life of 47 h. In vitro microneutralization assays using the purified F(ab')2 displayed high neutralization titers against five different variants of SARS-CoV-2 including the Delta variant, demonstrating its potential efficacy against the emerging viral variants. In conclusion, this study demonstrates that F(ab')2 generated against SARS-CoV-2 in equines have high neutralization titers and have broad target-range against the evolving variants, making passive immunotherapy a potential regimen against the existing and evolving SARS-CoV-2 variants in combating COVID-19.

Retinoic Acid-Inducible Gene I-Like Receptors Activate Snail To Limit RNA Viral Infections.

Retinoic acid-inducible gene I-like receptors (RLRs) are important cytosolic pattern recognition receptors (PRRs) that sense viral RNA before mounting a response leading to the activation of type I IFNs. Several viral infections induce epithelial-mesenchymal transition (EMT), even as its significance remains unclear. Here, we show that EMT or an EMT-like process is a general response to viral infections. Our studies identify a previously unknown mechanism of regulation of an important EMT-transcription factor (EMT-TF) Snail during RNA viral infections and describe its possible implication. RNA viral infections, poly(I·C) transfection, and ectopic expression of RLR components induced Snail levels, indicating that RLR pathway could regulate its expression. Detailed examination using mitochondrial antiviral signaling protein knockout (MAVS-KO) cells established that MAVS is essential in this regulation. We identified two interferon-stimulated response elements (ISREs) in the SNAI1 promoter region and demonstrated that they are important in its transcriptional activation by phosphorylated IRF3. Increasing the levels of Snail activated RLR pathway and dramatically limited replication of the RNA viruses dengue virus, Japanese encephalitis virus (JEV), and vesicular stomatitis virus, pointing to their antiviral functions. Knockdown of Snail resulted in a considerable increase in the JEV titer, validating its antiviral functions. Finally, transforming growth factor ß-mediated IFNB activation was dependent on Snail levels, confirming its important role in type I IFN activation. Thus, EMT-TF Snail is transcriptionally coregulated with type I IFN by RLRs and, in turn, promotes the RLR pathway, further strengthening the antiviral state in the cell. Our work identified an interesting mechanism of regulation of Snail that demonstrates potential coregulation of multiple innate antiviral pathways triggered by RLRs. Identification of antiviral functions of Snail also provides an opportunity to expand the sphere of RLR signaling. IMPORTANCE RLRs sense viral genomic RNA or the double-stranded RNA intermediates and trigger the activation of type I IFNs. Snail transcription factor, commonly associated with epithelial-mesenchymal transition (EMT), has been reported to facilitate EMT in several viral infections. Many of these reports are based on oncoviruses, leading to the speculation that EMT induced during infection is an important factor in the oncogenesis triggered by these infections. However, our studies reveal that EMT or EMT-like processes during viral infections have important functions in antiviral response. We have characterized a new mechanism of transcriptional regulation of Snail by IRF3 through interferon-stimulated response elements in their promoters, and this finding could have importance in nonviral contexts as well. We also identify that EMT-TF Snail promotes antiviral status of the infected cells through the RLR pathway. This study characterizes a new regulatory mechanism of activation of Snail and establishes its unidentified function in antiviral response.

mTORC1 restricts hepatitis C virus RNA replication through ULK1-mediated suppression of miR-122 and facilitates post-replication events.

The mechanistic target of rapamycin (mTOR), an important kinase that assimilates several upstream signals, associates into two functional complexes, mTORC1 and mTORC2. In this study, we demonstrate that HCV infection activates mTORC1 that functions in important antiviral response. Pharmacological inhibition of mTOR complexes augmented cellular HCV RNA levels, the observation confirmed further by Raptor depletion, indicating antiviral roles of mTORC1. ULK1 depletion phenocopied mTOR inhibition and thus suggested that mTORC1 restricts HCV replication through ULK1. We reveal that ULK1 depletion augmented the levels of miR-122, a critical host factor for HCV replication, thus possibly regulating HCV replication. The increase in HCV RNA levels, however, failed to augment intracellular infectious virion production, reflecting a lower rate of virion assembly. Higher intracellular HCV RNA levels, however, did not result in a corresponding increase in HCV RNA and infectious titres in mTOR inhibited supernatants, but in contrast showed a consistent drop, confirming defective viral assembly caused by the inhibition. Consistent with this, the mTOR activator caused a significant drop in HCV RNA levels both in infected cells and in the supernatant. Our results demonstrate that ULK1 depletion did not affect autophagy, suggesting that ULK1-mediated HCV regulation is autophagy independent. Together, our data demonstrate that mTORC1 functions to suppress HCV RNA replication, but facilitates the virion packaging and release. Our studies reveal that the activation of mTOR by HCV infection is an antiviral measure by the cells.

A unique phosphorylation-dependent eIF4E assembly on 40S ribosomes co-ordinated by hepatitis C virus protein NS5A that activates internal ribosome entry site translation.

We previously reported that the HCV (hepatitis C virus) protein NS5A up-regulated mRNA cap binding eIF4F (eukaryotic initiation factor 4F) complex assembly through mTOR (mechanistic target of rapamycin)-4EBP1 (eIF4E-binding protein 1) pathway and that NS5A (non-structural protein 5A) physically interacted with translation apparatus. In the present study, we demonstrate that NS5A co-ordinates a unique assembly of the cap binding protein eIF4E and 40S ribosome to form a complex that we call ENR (eIF4E-NS5A-ribosome). Recruitment of NS5A and eIF4E to 40S ribosome was confirmed by polysome fractionation, subcellular fractionation and high-salt-wash immunoprecipitation. These observations were also confirmed in HCV-infected cells, validating its biological significance. eIF4E phosphorylation was critical for ENR assembly. 80S ribosome dissociation and RNase integrity assays revealed that, once associated, the ENR complex is stable and RNA interaction is dispensable. Both the N- and C-terminal regions of NS5A domain 1 were indispensable for this assembly and for the NS5A-induced HCV IRES (internal ribosome entry site) activation. The present study demonstrates that NS5A initially associates with phosphorylated eIF4E of eIF4F complex and subsequently recruits it to 40S ribosomes. This is the first time the interaction of viral protein with both eIF4E and ribosomes has been reported. We propose that this assembly would determine the outcome of HCV infection and pathogenesis through regulation of viral and host translation.

Easing diagnosis and pushing the detection limits of SARS-CoV-2.

Rigorous testing is the way forward to fight the coronavirus disease 2019 pandemic. Here we show that the currently used and most reliable reverse transcription-polymerase chain reaction-based severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) procedure can be further simplified to make it faster, safer, and economical by eliminating the RNA isolation step. The modified method is not only fast and convenient but also at par with the traditional method in terms of accuracy, and therefore can be used for mass screening. Our method takes about half the time and is cheaper by ∼40% compared to the currently used method. We also provide a variant of the new method that increases the efficiency of detection by ∼30% compared to the existing procedure. Taken together, we demonstrate a more effective and reliable method of SARS-CoV-2 detection.

Unconventional MAPK-GSK-3ß Pathway Behind Atypical Epithelial-Mesenchymal Transition In Hepatocellular Carcinoma.

We recently reported an atypical epithelial mesenchymal transition (EMT) in human hepatoma cell culture Huh7.5, which was non-responsive to the canonical EMT-transcription factors. Here we characterize major pathways regulating this atypical EMT through whole genome transcriptome profiling and molecular analysis, and identify a unique regulation of EMT by GSK-3ß. Our analysis reveals remarkable suppression of several key liver-specific markers in Huh7.5M cells indicating that EMT not only changes the epithelial properties, but alters the characteristics associated with hepatocytes as well. One key finding of this study is that GSK-3ß, a known antagonist to ß-Catenin signaling and a major pro-apoptotic regulator, is critical for the maintenance of EMT in Huh7.5M cells as its inhibition reversed EMT. Importantly, through these studies we identify that maintenance of EMT by GSK-3ß in Huh7.5M is regulated by p38MAPK and ERK1/2 that has not been reported elsewhere and is distinct from another metastatic non-hepatic cell line MDA-MB-231. These data showcase the existence of non-canonical mechanisms behind EMT. The atypicalness of this system underlines the existence of tremendous diversity in cancer-EMT and warrants the necessity to take a measured approach while dealing with metastasis and cancer drug resistance.

An Atypical System for Studying Epithelial-Mesenchymal Transition in Hepatocellular Carcinoma.

Intrahepatic and extrahepatic metastases are frequently detected in hepatocellular carcinoma (HCC). Epithelial-mesenchymal transition (EMT) is believed to drive metastasis. There are not many well-established model systems to study EMT in HCC. Here we identified an atypical EMT while characterizing a population of mesenchymal cells in Huh7.5 hepatoma cell cultures. Cells with distinct morphology appeared during geneticin treatment of Huh7.5 cultures. Molecular characterization of geneticin resistant Huh7.5M cells confirmed EMT. Huh7.5M cells expressed cancer stem cell markers. p38MAPK and ERK1/2 were substantially activated in Huh7.5M cells. Their Inhibition elevated E-Cadherin expression with concerted suppression of Vimentin and anchorage independent growth in Huh7.5M cells. TGFß could not induce EMT in Huh7.5 cultures, but enriched mesenchymal populations, similar to geneticin. Huh7.5M cells formed more aggressive solid tumors, primarily comprising cells with epithelial morphology, in nude mice. Canonical EMT-TFs did not participate in this atypical EMT, indicating that the established canonical EMT-TFs do not drive every EMT and there is a dire need to identify additional factors. The system that we characterized is a unique model to study EMT, MET and biphasic TGFß signaling in HCC and offers considerable potential to facilitate more insightful studies on deeper questions in tumor metastasis.