The use of human iPSC-derived alveolar organoids to explore SARS-CoV-2 variant infections and host responses.

Severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) primarily targets the respiratory system. Physiologically relevant human lung models are indispensable to investigate virus-induced host response and disease pathogenesis. In this study, we generated human induced pluripotent stem cell (iPSC)-derived alveolar organoids (AOs) using an established protocol that recapitulates the sequential steps of in vivo lung development. AOs express alveolar epithelial type II cell protein markers including pro-surfactant protein C and ATP binding cassette subfamily A member 3. Compared to primary human alveolar type II cells, AOs expressed higher mRNA levels of SARS-CoV-2 entry factors, angiotensin-converting enzyme 2 (ACE2), asialoglycoprotein receptor 1 (ASGR1) and basigin (CD147). Considering the localization of ACE2 on the apical side in AOs, we used three AO models, apical-in, sheared and apical-out for SARS-CoV-2 infection. All three models of AOs were robustly infected with the SARS-CoV-2 irrespective of ACE2 accessibility. Antibody blocking experiment revealed that ASGR1 was the main receptor for SARS-CoV2 entry from the basolateral in apical-in AOs. AOs supported the replication of SARS-CoV-2 variants WA1, Alpha, Beta, Delta, and Zeta and Omicron to a variable degree with WA1 being the highest and Omicron being the least. Transcriptomic profiling of infected AOs revealed the induction of inflammatory and interferon-related pathways with NF-κB signaling being the predominant host response. In summary, iPSC-derived AOs can serve as excellent human lung models to investigate infection of SARS-CoV-2 variants and host responses from both apical and basolateral sides.

SNHG15 aids SARS-CoV-2 entry via RABL2A.

Angiotensin-converting enzyme 2 (ACE2) and several proteins have been identified as entry factors for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, whether long noncoding RNAs are involved in SARS-CoV-2 entry remains unknown. In this study, we investigated the role of small nucleolar RNA host gene 15 (SNHG15) in SARS-CoV-2 entry using a SARS-CoV-2 spike pseudotyped lentivirus with a luciferase reporter. Overexpression of SNHG15 promoted but SNHG15 knockdown limited SARS-CoV-2 entry in a dose- and time-dependent manner. SNHG15 interacted with Rab-like protein 2A (RABL2A). Overexpression and knockdown of RABL2A produced similar effects on SARS-CoV-2 entry as those of SNHG15. Furthermore, RABL2A knockdown abolished the SNHG15-mediated increase in SARS-CoV-2 entry. In conclusion, SNHG15 is a critical regulatory factor that aids SARS-CoV-2 entry through RABL2A.

Axin1: A novel scaffold protein joins the antiviral network of interferon.

Acute respiratory infection by influenza virus is a persistent and pervasive public health problem. Antiviral innate immunity initiated by type I interferon (IFN) is the first responder to pathogen invasion and provides the first line of defense. We discovered that Axin1, a scaffold protein, was reduced during influenza virus infection. We also found that overexpression of Axin1 and the chemical stabilizer of Axin1, XAV939, reduced influenza virus replication in lung epithelial cells. This effect was also observed with respiratory syncytial virus and vesicular stomatitis virus. Axin1 boosted type I IFN response to influenza virus infection and activated JNK/c-Jun and Smad3 signaling. XAV939 protected mice from influenza virus infection. Thus, our studies provide new mechanistic insights into the regulation of the type I IFN response and present a new potential therapeutic of targeting Axin1 against influenza virus infection.

Regulation of influenza A virus infection by Lnc-PINK1-2:5.

Influenza virus causes approximately 291,000 to 646,000 human deaths worldwide annually. It is also a disease of zoonotic importance, affecting animals such as pigs, horses, and birds. Even though vaccination is being used to prevent influenza virus infection, there are limited options available to treat the disease. Long noncoding RNAs (lncRNAs) are RNA molecules with more than 200 nucleotides that do not translate into proteins. They play important roles in the physiological and pathological processes. In this study, we identified a novel transcript, Lnc-PINK1-2:5 that was upregulated by influenza virus. This lncRNA was predominantly located in the nucleus and was not affected by type I interferons. Overexpression of Lnc-PINK1-2:5 reduced the influenza viral mRNA and protein levels in cells as well as titres in culture media. Knockdown of Lnc-PINK1-2:5 using CRISPR interference enhanced the virus replication. Antiviral activity of Lnc-PINK1-2:5 was independent of influenza virus strains. RNA sequencing analysis revealed that Lnc-PINK1-2:5 upregulated thioredoxin interacting protein (TXNIP) during influenza virus infection. Overexpression of TXNIP reduced influenza virus infection, suggesting that TXNIP is an antiviral gene. Knockdown of TXNIP abolished the Lnc-PINK1-2:5-mediated increase in influenza virus infection. In conclusion, the newly identified Lnc-PINK1-2:5 isoform is an anti-influenza lncRNA acting through the upregulation of TXNIP gene expression.

Pooling of Nasopharyngeal Swab Samples To Overcome a Global Shortage of Real-Time Reverse Transcription-PCR COVID-19 Test Kits.

The global outbreak and rapid spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have created an urgent need for large-scale testing of populations. There is a demand for high-throughput testing protocols that can be used for efficient and rapid testing of clinical specimens. We evaluated a pooled PCR protocol for testing nasopharyngeal (NP) swabs using known positive/negative and untested clinical samples that were assigned to pools of 5 or 10. In total, 630 samples were used in this study. Individual positive samples with cycle threshold (CT ) values as high as 33 could be consistently detected when pooled with 4 negative samples (pool of 5), and individual positive samples with CT values up to 31 could be consistently detected when pooled with 9 negative samples (pool of 10). Pooling of up to 5 samples can be employed in laboratories for the diagnosis of COVID-19 for efficient utilization of resources, rapid screening of a greater number of people, and faster reporting of test results.

Interplay between host non-coding RNAs and influenza viruses.

Influenza virus infection through seasonal epidemics and occasional pandemics has been a major public health concern for decades. Incomplete protection from vaccination and increased antiviral resistance due to frequent mutations of influenza viruses have led to a continuous need for new therapeutic options. The functional significance of host protein and influenza virus interactions has been established, but relatively less is known about the interaction of host noncoding RNAs, including microRNAs and long noncoding RNAs, with influenza viruses. In this review, we summarize host noncoding RNA profiles during influenza virus infection and the regulation of influenza virus infection by host noncoding RNAs. Influenza viral non-coding RNAs are briefly discussed. Increased understanding of the molecular regulation of influenza viral replication will be beneficial in identifying potential therapeutic targets against the influenza virus.

miR-29a is a negative regulator of influenza virus infection through targeting of the frizzled 5 receptor.

Influenza A virus (IAV) infections result in a large number of deaths and substantial economic losses each year. MicroRNAs repress gene expression and are involved in virus-host interactions. miR-29a is known to have anti-tumor and anti-fibrotic effects. However, the role of miR-29a in IAV infection is unclear. In the present study, we investigated the effect of miR-29a on IAV infection and the mechanisms by which it functions. IAV infection was found to cause decreased miR-29a expression in lung epithelial A549 cells and mouse lungs. Overexpression of miR-29a reduced IAV mRNA and protein levels and progeny virus production in HEK293 and A549 cells. Inhibition of IAV infection by miR-29a was observed with different strains of IAV, including A/PR/8/34, A/WSN/1933, and clinical isolates A/OK/3052/09 and A/OK/309/06 H3N2. Knockout of miR-29a using CRISPR/Cas9 resulted in an increase in viral mRNA and protein levels, confirming that miR-29a suppresses IAV infection. A 3' untranslated region (3'-UTR) reporter assay showed that miR-29a had binding sites in the 3'-UTR of the Wnt-Ca2+ signaling receptor frizzled 5 gene, and overexpression of miR-29a reduced the level of the endogenous frizzled 5 protein. Wnt5a treatment of HEK293 and A549 cells enhanced IAV infection. Our results suggest that miR-29a inhibits IAV infection, probably via the frizzled 5 receptor.

Hypoxia and transforming growth factor ß1 regulation of long non-coding RNA transcriptomes in human pulmonary fibroblasts.

One of the key characteristics of idiopathic pulmonary fibrosis (IPF) is accumulation of excess fibrous tissue in the lung, which leads to hypoxic conditions. Transforming growth factor (TGF) ß is a major mediator that promotes the differentiation of fibroblasts to myofibroblasts. However, how hypoxia and TGFß together contribute the pathogenesis of IPF is poorly understood. Long non-coding RNAs (lncRNAs) have regulatory effects on certain genes and are involved in many diseases. In this study, we determined the effects of hypoxia and/or TGFß on mRNA and lncRNA transcriptomes in pulmonary fibroblasts. Hypoxia and TGFß1 synergistically increased myofibroblast marker expression. RNA sequencing revealed that hypoxia and TGFß1 treatment resulted in significant changes in 669 lncRNAs and 2,676 mRNAs compared to 150 lncRNAs and 858 mRNAs in TGFß1 alone group and 222 lncRNAs and 785 mRNAs in hypoxia alone group. TGFß1 induced the protein expression of HIF-1α, but not HIF-2α. On the other hand, hypoxia enhanced the TGFß1-induced phosphorylation of Smad3, suggesting a cross-talk between these two signaling pathways. In all, 10 selected lncRNAs (five-up and five-down) in RNA sequencing data were validated using real-time PCR. Two lncRNAs were primarily located in cytoplasm, three in nuclei and five in both nuclei and cytoplasm. The silencing of HIF-1α and Smad3, but not Smad2 and HIF-2α rescued the downregulation of FENDRR by hypoxia and TGFß1. In conclusion, hypoxia and TGFß1 synergistically regulate mRNAs and lncRNAs involved in several cellular processes, which may contribute to the pathogenesis of IPF.

Long non-coding RNA PSMB8-AS1 regulates influenza virus replication.

Long non-coding RNAs (lncRNAs) are a new arm of gene regulatory mechanism as discovered by sequencing techniques and follow-up functional studies. There are only few studies on lncRNAs as related to gene expression regulation and anti-viral activity during influenza virus infection. We sought to identify and characterize lncRNAs involved in influenza virus replication. Using RNA sequencing analysis, we found that 1,912 lncRNAs were significantly changed in human lung epithelial A549 cells infected with influenza A/Puerto Rico/8/34. Gene ontology analysis on neighboring genes of these lncRNAs revealed that the genes involved in type I interferon signaling and cellular response were highly enriched. Seven selected up-regulated lncRNAs (AC015849.2, RP-1-7H24.1, PSMB8-AS1, CTD-2639E6.9, PSOR1C3, AC007283.5 and RP11-670E13.5) were verified by real-time PCR. These lncRNAs were also induced by other two influenza H1N1 virus strains (A/WSN/1933 and A/Oklahoma/3052/09) and interferon ß1. Repression of PSMB8 antisense RNA 1 (PSMB8-AS1) using CRISPR interference reduced viral mRNA and protein levels as well as the release of progeny influenza virus particles. Our study suggests that lncRNA PSMB8-AS1 could be a new host factor target for developing antiviral therapy against influenza virus infection.

A Critical Role for P2X7 Receptor-Induced VCAM-1 Shedding and Neutrophil Infiltration during Acute Lung Injury.

Pulmonary neutrophils are the initial inflammatory cells that are recruited during lung injury and are crucial for innate immunity. However, pathological recruitment of neutrophils results in lung injury. The objective of this study is to determine whether the novel neutrophil chemoattractant, soluble VCAM-1 (sVCAM-1), recruits pathological levels of neutrophils to injury sites and amplifies lung inflammation during acute lung injury. The mice with P2X7 receptor deficiency, or treated with a P2X7 receptor inhibitor or anti-VCAM-1 Abs, were subjected to a clinically relevant two-hit LPS and mechanical ventilation-induced acute lung injury. Neutrophil infiltration and lung inflammation were measured. Neutrophil chemotactic activities were determined by a chemotaxis assay. VCAM-1 shedding and signaling pathways were assessed in isolated lung epithelial cells. Ab neutralization of sVCAM-1 or deficiency or antagonism of P2X7R reduced neutrophil infiltration and proinflammatory cytokine levels. The ligands for sVCAM-1 were increased during acute lung injury. sVCAM-1 had neutrophil chemotactic activities and activated alveolar macrophages. VCAM-1 is released into the alveolar airspace from alveolar epithelial type I cells through P2X7 receptor-mediated activation of the metalloproteinase ADAM-17. In conclusion, sVCAM-1 is a novel chemoattractant for neutrophils and an activator for alveolar macrophages. Targeting sVCAM-1 provides a therapeutic intervention that could block pathological neutrophil recruitment, without interfering with the physiological recruitment of neutrophils, thus avoiding the impairment of host defenses.