Immunofluorescence analysis of human eosinophils.

A prominent inflammatory cell type in allergic diseases is the eosinophil, a granulated white blood cell that releases pro-inflammatory cytokines. Eosinophil-derived cytokines, including interleukin-9 (IL-9) and interleukin-13 (IL-13), can skew the immune response towards an allergic phenotype. Unfortunately, it is challenging to immunolabel and collect quantifiable images of eosinophils given their innate autofluorescence and ability to nonspecifically bind to antibodies. Hence, it is important to optimize permeabilization, blocking, and imaging conditions for eosinophils. Here, we show enhanced protocols to ensure that measured immunofluorescence represents specific immunolabelling. To test this, eosinophils were purified from human blood, adhered to glass coverslips, stimulated with or without platelet-activating factor (PAF), fixed with paraformaldehyde, and then permeabilized with Triton X-100 or saponin. Cells were then blocked with goat serum or human serum and incubated with antibodies labelling cytokines (IL-9 and IL-13) and secretory organelles (CD63 for crystalloid granules and transferrin receptor [TfnRc] for recycling endosomes). Carefully selected isotype controls were used throughout, and cells were imaged using Deltavision super-resolution microscopy. Intensities of fluorescent probes were quantified using Volocity software. Our findings show that permeabilization with saponin, blockage with human serum, and using concentrations of antibodies up to 10 µg/ml allowed us to detect marked differences in fluorescence intensities between isotypes and test antibodies. With the achievement of sufficient qualitative and quantitative measures of increased test probe intensity compared to respective isotypes, these results indicate that our protocol allows for optimal immunolabelling of eosinophils. Using this protocol, future studies may provide further insights into trafficking mechanisms within this important inflammatory cell type.

The RNA Interference Effector Protein Argonaute 2 Functions as a Restriction Factor Against SARS-CoV-2.

Argonaute 2 (Ago2) is a key component of the RNA interference (RNAi) pathway, a gene-regulatory system that is present in most eukaryotes. Ago2 uses microRNAs (miRNAs) and small interfering RNAs (siRNAs) for targeting to homologous mRNAs which are then degraded or translationally suppressed. In plants and invertebrates, the RNAi pathway has well-described roles in antiviral defense, but its function in limiting viral infections in mammalian cells is less well understood. Here, we examined the role of Ago2 in replication of the betacoronavirus SARS-CoV-2, the etiologic agent of COVID-19. Microscopic analyses of infected cells revealed that a pool of Ago2 closely associates with viral replication sites and gene ablation studies showed that loss of Ago2 resulted in over 1,000-fold increase in peak viral titers. Replication of the alphacoronavirus 229E was also significantly increased in cells lacking Ago2. The antiviral activity of Ago2 was dependent on both its ability to bind small RNAs and its endonuclease function. Interestingly, in cells lacking Dicer, an upstream component of the RNAi pathway, viral replication was the same as in parental cells. This suggests that the antiviral activity of Ago2 is independent of Dicer processed miRNAs. Deep sequencing of infected cells by other groups identified several SARS-CoV-2-derived small RNAs that bind to Ago2. A mutant virus lacking the most abundant ORF7A-derived viral miRNA was found to be significantly less sensitive to Ago2-mediated restriction. This combined with our findings that endonuclease and small RNA-binding functions of Ago2 are required for its antiviral function, suggests that Ago2-small viral RNA complexes target nascent viral RNA produced at replication sites for cleavage. Further studies are required to elucidate the processing mechanism of the viral small RNAs that are used by Ago2 to limit coronavirus replication.

Identification of Human Host Substrates of the SARS-CoV-2 Mpro and PLpro Using Subtiligase N-Terminomics.

The recent emergence of SARS-CoV-2 in the human population has caused a global pandemic. The virus encodes two proteases, Mpro and PLpro, that are thought to play key roles in the suppression of host protein synthesis and immune response evasion during infection. To identify the specific host cell substrates of these proteases, active recombinant SARS-CoV-2 Mpro and PLpro were added to A549 and Jurkat human cell lysates, and subtiligase-mediated N-terminomics was used to capture and enrich protease substrate fragments. The precise location of each cleavage site was identified using mass spectrometry. Here, we report the identification of over 200 human host proteins that are potential substrates for SARS-CoV-2 Mpro and PLpro and provide a global mapping of proteolysis for these two viral proteases in vitro. Modulating proteolysis of these substrates will increase our understanding of SARS-CoV-2 pathobiology and COVID-19.

Mayaro Virus Non-Structural Protein 2 Circumvents the Induction of Interferon in Part by Depleting Host Transcription Initiation Factor IIE Subunit 2.

Mayaro virus (MAYV) is an emerging mosquito-transmitted virus that belongs to the genus Alphavirus within the family Togaviridae. Humans infected with MAYV often develop chronic and debilitating arthralgia and myalgia. The virus is primarily maintained via a sylvatic cycle, but it has the potential to adapt to urban settings, which could lead to large outbreaks. The interferon (IFN) system is a critical antiviral response that limits replication and pathogenesis of many different RNA viruses, including alphaviruses. Here, we investigated how MAYV infection affects the induction phase of the IFN response. Production of type I and III IFNs was efficiently suppressed during MAYV infection, and mapping revealed that expression of the viral non-structural protein 2 (nsP2) was sufficient for this process. Interactome analysis showed that nsP2 interacts with DNA-directed RNA polymerase II subunit A (Rpb1) and transcription initiation factor IIE subunit 2 (TFIIE2), which are host proteins required for RNA polymerase II-mediated transcription. Levels of these host proteins were reduced by nsP2 expression and during infection by MAYV and related alphaviruses, suggesting that nsP2-mediated inhibition of host cell transcription is an important aspect of how some alphaviruses block IFN induction. The findings from this study may prove useful in design of vaccines and antivirals, which are currently not available for protection against MAYV and infection by other alphaviruses.

SARS-CoV-2 Nonstructural Protein 1 Inhibits the Interferon Response by Causing Depletion of Key Host Signaling Factors.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the ongoing coronavirus disease 2019 (COVID-19) pandemic. While previous studies have shown that several SARS-CoV-2 proteins can antagonize the interferon (IFN) response, some of the mechanisms by which they do so are not well understood. In this study, we describe two novel mechanisms by which SARS-CoV-2 blocks the IFN pathway. Type I IFNs and IFN-stimulated genes (ISGs) were poorly induced during SARS-CoV-2 infection, and once infection was established, cells were highly resistant to ectopic induction of IFNs and ISGs. Levels of two key IFN signaling pathway components, Tyk2 and STAT2, were significantly lower in SARS-CoV-2-infected cells. Expression of nonstructural protein 1 (NSP1) or nucleocapsid in the absence of other viral proteins was sufficient to block IFN induction, but only NSP1 was able to inhibit IFN signaling. Mapping studies suggest that NSP1 prevents IFN induction in part by blocking IRF3 phosphorylation. In addition, NSP1-induced depletion of Tyk2 and STAT2 dampened ISG induction. Together, our data provide new insights into how SARS-CoV-2 successfully evades the IFN system to establish infection. IMPORTANCE SARS-CoV-2 is the causative agent of COVID-19, a serious disease that can have a myriad of symptoms from loss of taste and smell to pneumonia and hypercoagulation. The rapid spread of SARS-CoV-2 can be attributed in part to asymptomatic transmission, where infected individuals shed large amounts of virus before the onset of disease. This is likely due to the ability of SARS-CoV-2 to effectively suppress the innate immune system, including the IFN response. Indeed, we show that the IFN response is efficiently blocked during SARS-CoV-2 infection, a process that is mediated in large part by nonstructural protein 1 and nucleocapsid. Our study provides new insights on how SARS-CoV-2 evades the IFN response to successfully establish infection. These findings should be considered for the development and administration of therapeutics against SARS-CoV-2.

Cytokine trafficking of IL-9 and IL-13 through TfnRc+ vesicles in activated human eosinophils.

Eosinophils are granulocytes that are elevated in lung mucosa in approximately half of patients with allergic asthma. These highly granulated cells can synthesize and secrete many cytokines, including IL-9 and IL-13. We hypothesized that IL-9 and IL-13 are found as preformed mediators in crystalloid granules and secreted using distinct trafficking pathways. Human eosinophils were purified from peripheral venous blood, adhered to coverslips, and stimulated with platelet activating factor (PAF). Cells were immunolabeled with antibodies to IL-9 or IL-13 and colocalized with markers for secretory organelles, using CD63 for crystalloid granules and transferrin receptor (TfnRc) for vesicles. Fixed cells were imaged using super-resolution microscopy and quantified by colocalization using Pearson's correlation coefficient. IL-9 immunofluorescence increased in a time-dependent manner to PAF, whereas colocalization of IL-9 and CD63 significantly increased from 0.52 to 0.67 after 5 min PAF. Colocalization of IL-9 with TfnRc significantly increased at 60 min of stimulation with PAF (0.54 at 0 min to 0.60 at 60 min). IL-13 showed lower colocalization with CD63 (0.55) than TfnRc (0.63) in unstimulated cells. Upon PAF stimulation, IL-13 intensity transiently decreased at 5 and 60 min, whereas colocalization of IL-13 with CD63 decreased throughout stimulation to 0.43. While colocalization of IL-13 with TfnRc transiently increased to 0.66 at 5 min PAF, it returned to near baseline levels (0.64) after 15 min PAF. Our results suggest that IL-9 and IL-13 are stored in crystalloid granules as well as endosomal structures, and that IL-9 is primarily trafficked to the cell surface via TfnRc+ endosome-like vesicles.