RNA Replication Research Articles

The Japanese encephalitis virus NS1 protein concentrates ER membranes in a cytoskeleton-independent manner to facilitate viral replication.

Orthoflaviviruses remodel the endoplasmic reticulum (ER) network to construct replication organelles (ROs) for RNA replication. In this study, we demonstrate that the Japanese encephalitis virus (JEV) NS1 protein concentrates ER membranes in the perinuclear region, which provides a substantial membrane source for viral replication. Subsequently, the virus forms main replication organelles within this membrane-concentrated area to facilitate efficient replication. This process relies on the ER localization signal, glycosylation, dimerization, and membrane-binding sites of the NS1 protein. In conclusion, our study highlights the role of the NS1 protein in the formation of the ROs by JEV, providing new insights into orthoflavivirus replication.IMPORTANCEOrthoflaviviruses use the endoplasmic reticulum (ER) membranes for replication by forming invaginations to assemble the replication organelles. Here, we found that Japanese encephalitis virus (JEV) utilizes the NS1 protein to concentrate a significant number of ER membranes in the perinuclear area, thereby providing a membrane source for viral replication and facilitating the formation of main replication organelles (MROs). This process depends on the ER localization signals of NS1, as well as its glycosylation, dimerization, and membrane-binding sites, but not on the cytoskeleton. In summary, our study highlights how NS1 remodels ER membranes to facilitate the formation of MROs for JEV, thereby accelerating viral replication.

Imaging of viral replication in live cells by using split fluorescent protein-tagged reporter flaviviruses.

The knowledge on the life cycle of flaviviruses is still incomplete, and no direct-acting antivirals against their infections are clinically available. Herein, by screening via a Zika virus (ZIKV) replicon assay, we found that the N-terminus of NS2A exhibited great tolerance to the insertions of different split fluorescent proteins (split-FPs). Furthermore, both ZIKV and dengue virus encoding a split-FP-tagged NS2A propagated efficiently, and the split-FP-tagged ZIKVs had good genetic stability. Robust green fluorescence was observed in the reporter cell lines infected with these viruses and the fluorescence responded to anti-flavivirus chemicals with high specificity and sensitivity. Moreover, the sites of viral RNA replication were illuminated in live cells. Interestingly, by blocking viral RNA synthesis with an NS5 inhibitor, we found a correlation between the morphological characteristics of potential replication organelles and RNA amplification, highlighting that the NS2A-tagged viruses are of great value for the in-depth understanding of flavivirus replication mechanisms.

Comparison of transcriptome responses in blood cells of Atlantic salmon infected by three genotypes of Piscine orthoreovirus.

Piscine orthoreovirus (PRV) infection is common in aquaculture of salmonids. The three known PRV genotypes (PRV-1-3) have host species specificity and cause different diseases, but all infect and replicate in red blood cells (RBCs) in early infection phase. PRV-1 is the causative agent of heart and skeletal muscle inflammation (HSMI) in farmed Atlantic salmon (Salmo salar), PRV-2 causes erythrocytic inclusion body syndrome (EIBS) in coho salmon (Oncorhynchus kisutch), while PRV-3 induces HSMI-like disease in farmed rainbow trout (Oncorhynchus mykiss). PRV-3 can also infect A. salmon without causing clinical disease and has been shown to cross-protect against PRV-1 infection and HSMI, while PRV-2 or inactivated adjuvanted PRV-1 vaccine only partially reduced HSMI pathologic changes. In the present work, we studied the transcriptional responses in blood cells of A. salmon two- and five-weeks post infection with PRV-1, PRV-2, PRV-3, or post injection with inactivated PRV-1 vaccine. PRV-1 and PRV-3 replicated well in A. salmon blood cells, and both induced the typical innate antiviral responses triggered by dsRNA viruses. Two weeks post infection, PRV-3 triggered stronger antiviral responses than PRV-1, despite their similar viral RNA replication levels, but after five weeks the induced responses were close to equal. PRV-2 and the InPRV-1 vaccine did not trigger the same typical antiviral responses as the replicating PRV-1 and PRV-3 genotypes, but induced genes involved in membrane trafficking and signaling pathways that may regulate physiological functions. These findings propose that the protection mediated by PRV-3 against a secondary infection by PRV-1 occur due to a potent and early activation of the same type of innate immune responses. The difference in the timing of antiviral responses may give PRV-1 an evolutionary edge, facilitating its dissemination to A. salmon heart, a critical step for HSMI development.

Domperidone inhibits dengue virus infection by targeting the viral envelope protein and nonstructural protein 1

Dengue is a mosquito-borne disease caused by dengue virus (DENV) infection, which remains a major public health concern worldwide owing to the lack of specific treatments or antiviral drugs available. This study investigated the potential repurposing of domperidone, an antiemetic and gastrokinetic agent, to control DENV infection. Domperidone was identified by pharmacophore-based virtual screening as a small molecule that can bind to both the viral envelope (E) and the nonstructural protein 1 (NS1) of DENV. Molecular dynamics (MD) simulations and surface plasmon resonance (SPR) analysis were subsequently performed to determine specific interactions of domperidone with the DENV E and NS1 proteins and their binding affinity. Treatment of immortalized human hepatocyte-like cells (imHC) with domperidone could inhibit DENV production and NS1 secretion in a dose-dependent manner following infection with DENV serotype 2. These inhibitory effects were mediated by reduction in viral RNA replication and viral E and NS1 protein expression, but not by interference with virus entry into cells or NS1 oligomerization. The suppression of DENV production and NS1 secretion by domperidone was observed across all four DENV serotypes to varying degrees between different virus strains. The findings from our study suggest viral target-based repurposing of domperidone for modulating DENV.

P-2025. Resistance Analyses from the Remdesivir Phase 2/3 CARAVAN Study in Pediatric and Neonatal Participants Hospitalized with COVID-19

Abstract Background Remdesivir (RDV) is a nucleotide analog prodrug approved for treatment of COVID-19. Here we present comprehensive SARS-CoV-2 resistance analyses from all cohorts of the Phase 2/3 CARAVAN study, which demonstrated the safety of RDV for treatment of COVID-19 in pediatric participants from birth to < 18 years of age. Methods CARAVAN was a single-arm, open-label study wherein pediatric participants received IV RDV for ≤10 days. Full-genome deep sequencing of SARS-CoV-2 was performed on respiratory samples collected on Days 1 (baseline), 3, 5, 7, and/or 10. In vitro antiviral activity of RDV against substitutions observed in the SARS-CoV-2 RNA replication complex was evaluated using a subgenomic replicon system. Substitutions were phenotyped if they met any of the criteria: 1) baseline Nsp12 substitutions detected in ≥3 participants; 2) baseline replication complex substitutions detected in ≥2 participants with viral RNA increase postbaseline; 3) treatment-emergent postbaseline replication complex substitutions. Results Of 58 participants enrolled and treated, baseline sequencing data were obtained from 37 participants. Six baseline amino acid polymorphisms met criteria for phenotyping, none of which impacted RDV susceptibility (EC50 fold change ≤1.55). Baseline and postbaseline sequencing data were obtained from 28 participants. Five treatment-emergent substitutions in Nsp12 were identified in 3 participants as mixtures with wildtype. Of these, only Nsp12 V792I alone and in combination with V166L showed low-level reduced susceptibility to RDV (Table 1). The participant in whom V166L and V792I were observed achieved clinical recovery and was released from the hospital on Day 13. Structural modeling suggests V166L and V792I are located on or adjacent to motif D of the viral RNA-dependent RNA polymerase and may alter the dynamics of nucleoside triphosphate incorporation. Six treatment-emergent substitutions in Nsp9, Nsp10, and Nsp13 were identified in 3 participants; none impacted RDV susceptibility (fold change ≤0.74). Conclusion The results of resistance analyses from the CARAVAN study support a high barrier to clinically meaningful resistance to RDV in pediatric patients with COVID-19. Disclosures Jasmine Moshiri, PhD, Gilead Sciences, Inc.: Employee|Gilead Sciences, Inc.: Stocks/Bonds (Public Company) Jiani Li, PhD, Gilead Sciences, Inc.: Employee|Gilead Sciences, Inc.: Stocks/Bonds (Public Company) Lauren Rodriguez, PhD, Gilead Sciences, Inc.: Employee|Gilead Sciences, Inc.: Stocks/Bonds (Public Company) Dong Han, MS, Gilead Sciences, Inc.: Employee|Gilead Sciences, Inc.: Stocks/Bonds (Public Company) Simin Xu, MS, Gilead Sciences, Inc.: Employee|Gilead Sciences, Inc.: Stocks/Bonds (Public Company) Pui Yan Ho, PhD, Gilead Sciences, Inc.: Employee|Gilead Sciences, Inc.: Stocks/Bonds (Public Company) Nadine Peinovich, MPH, Gilead Sciences, Inc.: Employee|Gilead Sciences, Inc.: Stocks/Bonds (Public Company) Clarissa Martinez, MPH, Gilead Sciences, Inc.: Employee|Gilead Sciences, Inc.: Stocks/Bonds (Public Company) Silvia Chang, Masters, Gilead Sciences, Inc.: Employee|Gilead Sciences, Inc.: Stocks/Bonds (Public Company) Kathryn Kersey, MS, Gilead Sciences, Inc.: Employee|Gilead Sciences, Inc.: Stocks/Bonds (Public Company) Jason K. Perry, PhD, Gilead Sciences, Inc.: Employee|Gilead Sciences, Inc.: Stocks/Bonds (Public Company) Danielle P. Porter, PhD, Gilead Sciences, Inc.: Employee|Gilead Sciences, Inc.: Stocks/Bonds (Public Company) Charlotte Hedskog, PhD, Gilead Sciences, Inc.: Employee|Gilead Sciences, Inc.: Stocks/Bonds (Public Company)

Antiviral effects and mechanism of Qi pi pill against influenza viruses.

Qi pi pill (QPP), which contains Renshen, Baizhu, Fuling, Gancao, Chenpi, Shanyao, Lianzi, Shanzha, Liushenqu, Maiya, and Zexie, was recommended for preventing and treating COVID-19 in Shandong Province (China). However, the mechanism by which QPP treats infectious diseases remains unclear. This study aims to investigate the therapeutic effect of QPP invitro and on acute influenza infection in mice, exploring its mechanism of action against influenza A virus (IAV). The invitro activity of QPP was assessed using dose-response curve analysis and titer reduction assay, and its antiviral mechanism was identified invitro by real-time polymerase chain reaction (PCR), time-of-addition, and enzymatic assays. The antiviral efficacy of QPP was further evaluated invivo using BALB/c mice infected with IAV. At the same time, each single Chinese herbal medicine in QPP was evaluated to preliminarily identify those with antiviral effects. In vitro results showed that QPP exhibited a higher potency antiviral effect against both influenza A and B viruses, inhibiting viral RNA replication and release by targeting RNA-dependent RNA polymerase and neuraminidase. Additionally, QPP significantly decreased the expression of inflammatory cytokines in A549 cells. Invivo study revealed that QPP significantly reduced the lung index and viral load in lung tissue of mice infected with IAV. Renshen, Gancao, Zexie, and Lianzi were the Chinese herbal medicines from QPP that showed anti-IAV activity. The antiviral activity of QPP targets IAV replication and release, cytokine modulation in host cells, and provides protection in mice with acute influenza infection.

Influenza A virus NS2 protein acts on vRNA-resident polymerase to drive the transcription to replication switch.

The heterotrimeric RNA-dependent RNA polymerase (RdRp) of influenza A virus catalyzes viral RNA transcription (vRNA→mRNA) and replication (vRNA→cRNA→vRNA) by adopting different conformations. A switch from transcription to replication occurs at a relatively late stage of infection. We recently reported that the viral NS2 protein, expressed at later stages from a spliced transcript of the NS segment messenger RNA (mRNA), inhibits transcription, promotes replication and plays a key role in the transcription-to-replication switch. In this study, we performed comprehensive functional analyses to elucidate how NS2 promotes viral genome replication. Using a cell-based single-step RNP reconstitution assay, we found that NS2 specifically promotes the first-step vRNA-to-cRNA synthesis. Further investigation revealed that this promotion is tightly associated with the intrinsic properties of the 3'-vRNA promoter. Employing a highly sensitive complementation reporter assay, we demonstrated that NS2 associates more strongly with the vRNA-resident RdRp than the cRNA-resident RdRp. These findings were further validated through in vitro replication analyses. We, therefore, propose that influenza A virus NS2 protein targets vRNA-resident RdRp to drive the transcription-to-replication switch during infection.

Dengue virus is particularly sensitive to interference with long-chain fatty acid elongation and desaturation.

Orthoflaviviruses are emerging arthropod-borne pathogens whose replication cycle is tightly linked to host lipid metabolism. Previous lipidomic studies demonstrated that infection with the closely related hepatitis C virus (HCV) changes the fatty acid (FA) profile of several lipid classes. Lipids in HCV-infected cells had more very long-chain and desaturated FAs and viral replication relied on functional FA elongation and desaturation. Here, we systematically analyzed the role of FA elongases and desaturases in infection models of the most prevalent pathogenic orthoflaviviruses, dengue (DENV), Zika (ZIKV), West Nile (WNV), yellow fever (YFV), and tick-borne encephalitis virus (TBEV). Knockdown of desaturases and elongases in Huh7 cells only marginally affected ZIKV, WNV, YFV, and TBEV replication, while DENV titers were strongly reduced. This was most prominent for enzymes involved in very long-chain fatty acid synthesis. In detail, knockdown of the FA elongase ELOVL4, which catalyzes ultra long-chain FA synthesis, significantly reduced DENV titers, decreased the formation of replication intermediates, and lowered viral protein levels in DENV infected hepatoma cells, suggesting a function of ELOVL4 in DENV RNA replication. In contrast, the activity of FA desaturase FADS2, rate-limiting in poly-unsaturated FA biosynthesis, is not involved in viral RNA replication or translation, but is essentially required for formation of infectious DENV particles. Further, in immunocompetent immortalized microglial cells, FADS2 deletion additionally limits viral replication through increased expression of interferon-stimulated genes in response DENV infection. Taken together, enzymes involved in very long-chain FA synthesis are critical for different steps of DENV replication.

Structural and functional analysis of the Nipah virus polymerase complex.

Nipah virus (NiV) is a bat-borne, zoonotic RNA virus that is highly pathogenic in humans. The NiV polymerase, which mediates viral genome replication and mRNA transcription, is a promising drug target. We determined the cryoelectron microscopy (cryo-EM) structure of the NiV polymerase complex, comprising the large protein (L)and phosphoprotein (P), and performed structural, biophysical, and in-depth functional analyses of the NiV polymerase. The L protein assembles with a long P tetrameric coiled-coil that is capped by a bundle of ⍺-helices that we show are likely dynamic in solution. Docking studies with a known L inhibitor clarify mechanisms of antiviral drug resistance. In addition, we identified L protein features that are required for both transcription and RNA replication and mutations that have a greater impact on RNA replication than on transcription. Our findings have the potential to aid in the rational development of drugs to combat NiV infection.

Characterization of the First Marine Pestivirus, Phocoena Pestivirus (PhoPeV).

The first marine pestivirus, Phocoena pestivirus (PhoPeV), isolated from harbor porpoise, has been recently described. To further characterize this unique pestivirus, its host cell tropism and growth kinetics were determined in different cell lines. In addition, the interaction of PhoPeV with innate immunity in porcine epithelial cells and the role of selected cellular factors involved in the viral entry and RNA replication of PhoPeV were investigated in comparison to closely and distantly related pestiviruses. While Bungowannah pestivirus (BuPV), a unique porcine pestivirus closely related to PhoPeV, exhibits a broad cell tropism, PhoPeV only infects cells from pigs, cattle, sheep, and cats, as has been described for classical swine fever virus (CSFV). Viral titers correlate with the amount of intracellular PhoPeV-specific RNA detected in the tested cell lines. PhoPeV replicates most efficiently in the porcine kidney cell line SK6. Pestiviruses generally counteract the cellular innate immune response by degradation of interferon regulatory factor 3 (IRF3) mediated by the viral N-terminal protease (Npro). No degradation of IRF3 and an increased expression of the type 1 interferon-stimulated antiviral protein Mx1 was observed in porcine cells infected with PhoPeV whose genome lacks the Npro encoding region. Infection of a CD46-deficient porcine cell line suggested that CD46, which is implicated in the viral entry of several pestiviruses, is not a major factor for the viral entry of PhoPeV. Moreover, the results of this study confirmed that the cellular factor DNAJC14 plays a crucial role in viral RNA replication of non-cytopathic pestiviruses, including PhoPeV.

Single-molecule assay reveals the impact of composition, RNA duplex, and inhibitors on the binding dynamics of SARS-CoV-2 polymerase complex.

The genome replication of SARS-CoV-2, the causative agent of COVID-19, involves a multi-subunit replication complex consisting of non-structural proteins (nsps) 12, 7 and 8. While the structure of this complex is known, the dynamic behavior of the subunits interacting with RNA is missing. Here we report a single-molecule protein-induced fluorescence enhancement (SM-PIFE) assay to monitor binding dynamics between the reconstituted or co-expressed replication complex and RNA. Increasing binding times were observed, in this order, with nsp7 (none) nsp8 and nsp12, in nsp8-nsp12 mixtures and in reconstituted mixtures bearing all three proteins. Unstable, transient, and stable binding modes were recorded in the latter case, indicating that complexation is dynamic, and the correct conformation must be achieved before stable RNA binding can occur. Notably, the co-expressed protein yields mostly stable binding even at low concentrations, while the reconstituted proteins exhibit unstable binding indicating inefficient complexation with reduced protein. The SM-PIFE assay distinguishes inhibitors that impact protein binding from those that prevent replication, as demonstrated with suramin and remdesivir, respectively. The data reveals a correlation between binding lifetime/affinity, and protein activity, and underscores differences between co-expressed vs reconstituted mixtures, suggesting the existence of trapped conformations that may not evolve to productive binding.

The Compound AT13148 Targeting AKT Suppresses Dengue Virus 2 Replication.

Background: Dengue virus (DENV) infection, caused by serotypes DENV 1-4, represents a significant global public health challenge, with no antiviral drugs currently available for treatment. The host Protein kinase B (AKT) signaling pathway is crucial for DENV infection, presenting a potential target for antiviral drug development. Objective: This study aimed to evaluate the antiviral activity of kinase inhibitors that target the AKT pathway, focusing on the compound AT13148. Methods: A mini-screening was conducted to identify kinase inhibitors with antiviral properties against DENV-2. The effects of AT13148 on viral RNA replication and translation were assessed in a dose- and time-dependent manner following DENV-2 entry. The mechanism of action was further investigated by evaluating the impact of AT13148 on AKT kinase activity and phosphorylation status. Results: AT13148 exhibited potent antiviral activity against DENV-2, significantly inhibiting viral RNA replication and translation post-entry. The compound was found to inhibit AKT kinase activity through hyperphosphorylation. Conclusion: The findings indicate that AT13148 effectively targets the AKT pathway, demonstrating potential as an antiviral therapeutic against DENV-2 by interfering with the virus's post-entry processes. Further in vivo studies are warranted to assess the efficacy of AT13148 in controlling DENV infection.

Molnupiravir clinical trial simulation suggests that polymerase chain reaction underestimates antiviral potency against SARS-CoV-2.

Molnupiravir is an antiviral medicine that induces lethal copying errors during SARS-CoV-2 RNA replication. Molnupiravir reduced hospitalization in one pivotal trial by 50% and had variable effects on reducing viral RNA levels in three separate trials. We used mathematical models to simulate these trials and closely recapitulated their virologic outcomes. Model simulations suggest lower antiviral potency against pre-omicron SARS-CoV-2 variants than against omicron. We estimate that in vitro assays underestimate in vivo potency 7-8 fold against omicron variants. Our model suggests that because polymerase chain reaction detects molnupiravir mutated variants, the true reduction in non-mutated viral RNA is underestimated by ∼0.5 log 10 in the two trials conducted while omicron variants dominated. Viral area under the curve estimates differ significantly between non-mutated and mutated viral RNA. Our results reinforce past work suggesting that in vitro assays are unreliable for estimating in vivo antiviral drug potency and suggest that virologic endpoints for respiratory virus clinical trials should be catered to the drug mechanism of action.

Orthogonal RNA replication enables directed evolution and Darwinian adaptation in mammalian cells.

Directed evolution in mammalian cells offers a powerful approach for advancing synthetic biology applications. However, existing mammalian-based directed evolution methods face substantial bottlenecks, including host genome interference, small library size and uncontrolled mutagenesis. Here we engineered an orthogonal alphaviral RNA replication system to evolve RNA-based devices, enabling RNA replicase-assisted continuous evolution (REPLACE) in proliferating mammalian cells. This system generates a large, continuously diversified library of replicative RNAs through replicase-limited mode of replication and inducible mutagenesis. Using REPLACE, we engineered fluorescent proteins and transcription factors. Notably, cells equipped with REPLACE can undergo Darwinian adaptation, allowing them to evolve in response to both cell-extrinsic and cell-intrinsic challenges. Collectively, this work establishes a powerful platform for advancing mammalian synthetic biology and cell engineering applications through directed evolution.

Linking the function of cis-acting RNA elements to coronavirus replication using interactomes.

RNA structures that are functionally important are defined as cis-acting RNA elements because their functions cannot be compensated for in trans. The cis-acting RNA elements in the 3' UTR of coronaviruses are important for replication; however, the mechanism linking the cis-acting RNA elements to their replication function remains to be established. In the present study, a comparison of the biological processes of the interactome and the replication efficiency between the 3' UTR cis-acting RNA elements in coronaviruses, including severe acute respiratory syndrome coronavirus 2, suggests that (i) the biological processes, including translation, protein folding and protein stabilization, derived from the analysis of the cis-acting RNA element interactome and (ii) the architecture of the cis-acting RNA elements and their interactomes are highly correlated with coronavirus replication. In addition, alteration of the interactome using the compound 5-benzyloxygramine can cause reduced coronavirus replication, reinforcing the connection between cis-acting RNA elements and replication by interactome. Together, these results link cis-acting RNA elements to the coronavirus replication and establish a model to analyse the cis-acting RNA elements in the replication of RNA viruses by interactome.

Ubiquitin-like modifier-activating enzyme 1 interacts with Zika virus NS5 and promotes viral replication in the infected cell.

Translation errors, impaired folding or environmental stressors (e.g. infection) can all lead to an increase in the presence of misfolded proteins. These activate cellular responses to their removal, including intracellular protein degradation activities. Protein ubiquitylation is involved in two major degradation pathways, the ubiquitin-proteasome system and selective autophagy. In humans, the ubiquitin-like modifier-activating enzyme 1 (UBA1) is the primary E1 enzyme in the ubiquitin conjugation cascade. Viruses have evolved to exploit protein degradation pathways to complete their infection cycles. Zika virus (ZIKV) is an emerging orthoflavivirus causing serious neurologic disorders in neonates (congenital microcephaly) and adults (Guillain-Barré syndrome). Non-structural protein 5 (NS5), the largest and most conserved protein in the orthoflaviviruses, catalyses the synthesis and capping of new viral genomes. In addition to viral RNA replication in the cytoplasm, ZIKV NS5 is translocated into the nucleus to interfere with host antiviral responses. Here, we demonstrate that ZIKV NS5 co-immunoprecipitates with cellular UBA1. Immunofluorescence assays suggest that this interaction takes place primarily in the nucleus of an infected cell, although colocalization of both proteins is also detected in the cytosol. RNA interference-mediated depletion of UBA1 leads to reduced virus titres in the infected cells, while transient overexpression of UBA1 favours faster replication kinetics, with higher virus titres and protein levels detected. Moreover, UBA1-targeting drugs cause significant drops in virus infectivity. These results support a proviral role for UBA1 during ZIKV infection and encourage the potential use of inhibitors against this enzyme or its NS5-interacting epitopes as potential therapeutic targets.

Genome-wide analysis of alternative splicing differences in hepatic ischemia reperfusion injury

Alternative splicing (AS) contributes to transcript and protein diversity, affecting their structure and function. However, the specific transcriptional regulatory mechanisms underlying AS in the context of hepatic ischemia reperfusion (IR) injury in mice have not been extensively characterized. In this study, we investigated differentially alternatively spliced (DAS) genes and differentially expressed transcripts (DETs) in a mouse model of hepatic IR injury using the high throughput RNA sequencing (RNA-seq) analysis and replicate multivariate analysis of transcript splicing (rMATS) analysis. We further conducted Gene ontology (GO) term enrichment, the Kyoto Encyclopedia of Genes and Genomes (KEGG) database and the protein-protein interaction (PPI) network. A total of 898 DAS genes (p ≤ 0.05) were screened out in the hepatic IR group compared to the sham group, while functional enrichment analysis revealed that DETs and DAS genes were significantly associated with the ATP-dependent chromain, splicesome and metabolic pathways. The expression level of the DAS genes: Gabpb2, Smg1, Tnrc6c, Mettl17, Smpd4, Kcnt2, D16Ertd472e, Rab3gap2, Echdc2 and Ssx2ip were verified by RT-PCR and qRT-PCR. Our findings provide a comprehensive genome-wide view of AS events in hepatic IR injury in mice, enhancing our understanding of AS dynamics and the molecular mechanisms governing alternative pre-mRNA splicing.

Annual SZ: An Alternative Immunotherapy for COVID-19 and Long COVID.

Since the outbreak of coronavirus disease 2019 (COVID-19) in late 2019 and early 2020, the identification of drugs to control severe acute respiratory syndrome coro-navirus 2 (SARS-CoV-2) infection and its symptoms has been a pressing focus of research. Cytokine storm and acute respiratory distress syndrome (ARDS) are the leading causes of mortality following infection. In this review, we discuss immune pathogenesis and four medications, including Remdesivir, Tocilizumab, Dexamethasone, and Annual SZ for COVID-19. A comparison of the effectiveness and therapeutic usage of drugs as reported in clinical trials and reports was made at different disease levels as well. Clinical studies indicate that Annual SZ with mild side effects was more affordable and might be more effective than other medications. Additionally, Annual SZ was capable of reducing the lev-els of pro-inflammatory cytokines as well as viral attachment and RNA replication.

Role of Rab13, Protein Kinase A, and Zonula Occludens-1 in Hepatitis E Virus Entry and Cell-to-Cell Spread: Comparative Analysis of Quasi-Enveloped and Non-Enveloped Forms.

Hepatitis E virus (HEV) exists in two distinct forms: a non-enveloped form (neHEV), which is present in feces and bile, and a quasi-enveloped form (eHEV), found in circulating blood and culture supernatants. This study aimed to elucidate the roles of Ras-associated binding 13 (Rab13) and protein kinase A (PKA) in the entry mechanisms of both eHEV and neHEV, utilizing small interfering RNA (siRNA) and chemical inhibitors. The results demonstrated that the entry of both viral forms is dependent on Rab13 and PKA. Further investigation into the involvement of tight junction (TJ) proteins revealed that the targeted knockdown of zonula occludens-1 (ZO-1) significantly impaired the entry of both eHEV and neHEV. In addition, in ZO-1 knockout (KO) cells inoculated with either viral form, HEV RNA levels in culture supernatants did not increase, even up to 16 days post-inoculation. Notably, the absence of ZO-1 did not affect the adsorption efficiency of eHEV or neHEV, nor did it influence HEV RNA replication. In cell-to-cell spread assays, ZO-1 KO cells inoculated with eHEV showed a lack of expression of HEV ORF2 and ORF3 proteins. In contrast, neHEV-infected ZO-1 KO cells showed markedly reduced ORF2 and ORF3 protein expression within virus-infected foci, compared to non-targeting knockout (NC KO) cells. These findings underscore the crucial role of ZO-1 in facilitating eHEV entry and mediating the cell-to-cell spread of neHEV in infected cells.

Small-molecule inhibition of SARS-CoV-2 NSP14 RNA cap methyltransferase.

Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)1. The rapid development of highly effective vaccines2,3 against SARS-CoV-2 has altered the trajectory of the pandemic, and antiviral therapeutics4 have further reduced the number of COVID-19 hospitalizations and deaths. Coronaviruses are enveloped, positive-sense, single-stranded RNA viruses that encode various structural and non-structural proteins, including those critical for viral RNA replication and evasion from innate immunity5. Here we report the discovery and development of a first-in-class non-covalent small-molecule inhibitor of the viral guanine-N7 methyltransferase (MTase) NSP14. High-throughput screening identified RU-0415529, which inhibited SARS-CoV-2 NSP14 by forming a unique ternary S-adenosylhomocysteine (SAH)-bound complex. Hit-to-lead optimization of RU-0415529 resulted in TDI-015051 with a dissociation constant (Kd) of 61 pM and a half-maximal effective concentration (EC50) of 11 nM, inhibiting virus infection in a cell-based system. TDI-015051 also inhibited viral replication in primary small airway epithelial cells and in a transgenic mouse model of SARS CoV-2 infection with an efficacy comparable with the FDA-approved reversible covalent protease inhibitor nirmatrelvir6. The inhibition of viral cap methylases as an antiviral strategy is also adaptable to other pandemic viruses.