Bichromatic UV detection system for atomically-resolved imaging of ions

We present a compact and bichromatic imaging system, located outside of the vacuum chamber of a trapped ion apparatus, that collects the fluorescence of 230.6 nm and 369.5 nm photons simultaneously on a shared EMCCD camera. The system contains two lens doublets, consisting of a sphere and an asphere. It provides a numerical aperture of 0.45 and 0.40 at 230.6 nm and 369.5 nm, respectively, and enables spatially resolved state detection with a large field of view of 300 $\mu$m for long $^{115}$In$^+$/$^{172}$Yb$^+$ Coulomb crystals. Instead of diffraction limited imaging for one wavelength, the focus in this system is on simultaneous single-ion resolved imaging of both species over a large field with special attention to the deep UV wavelength (230.6 nm) and the low scattering rate of In$^+$ ions. The introduced concept is applicable to other dual-species applications

Interferon-stimulated gene (ISG)-expression screening reveals the specific antibunyaviral activity of ISG20

Bunyaviruses pose a significant threat to human health, prosperity and food security. In response to viral infections, interferons (IFNs) upregulate the expression of hundreds of interferon stimulated genes (ISGs) whose cumulative action can potently inhibit the replication of bunyaviruses. We used a flow cytometry-based method to screen the ability of ∼500 unique ISGs from humans and rhesus macaques to inhibit the replication of Bunyamwera orthobunyavirus (BUNV), the prototype of both the Peribunyaviridae family and Bunyavirales order. Candidates possessing antibunyaviral activity were further examined using a panel of divergent bunyaviruses. Interestingly, one candidate, ISG20, exhibited potent antibunyaviral activity against most viruses examined from the Peribunyaviridae, Hantaviridae and Nairoviridae families, whereas phleboviruses (Phenuiviridae) largely escaped inhibition. Similar to other viruses known to be targeted by ISG20, the antibunyaviral activity of ISG20 is dependent upon its functional ribonuclease activity. Through use of an infectious VLP assay (based on the BUNV minigenome system), we confirmed that gene expression from all 3 viral segments is strongly inhibited by ISG20. Using in vitro evolution, we generated a substantially ISG20-resistant BUNV and mapped the determinants of ISG20 sensitivity/resistance. Taken together, we report that ISG20 is a broad and potent antibunyaviral factor yet some bunyaviruses are remarkably ISG20 resistant. Thus, ISG20 sensitivity/resistance could influence the pathogenesis of bunyaviruses, many of which are emerging viruses of clinical or veterinary significance

A plasmid DNA-launched SARS-CoV-2 reverse genetics system and coronavirus toolkit for COVID-19 research

The recent emergence of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the underlying cause of Coronavirus Disease 2019 (COVID-19), has led to a worldwide pandemic causing substantial morbidity, mortality, and economic devastation. In response, many laboratories have redirected attention to SARS-CoV-2, meaning there is an urgent need for tools that can be used in laboratories unaccustomed to working with coronaviruses. Here we report a range of tools for SARS-CoV-2 research. First, we describe a facile single plasmid SARS-CoV-2 reverse genetics system that is simple to genetically manipulate and can be used to rescue infectious virus through transient transfection (without in vitro transcription or additional expression plasmids). The rescue system is accompanied by our panel of SARS-CoV-2 antibodies (against nearly every viral protein), SARS-CoV-2 clinical isolates, and SARS-CoV-2 permissive cell lines, which are all openly available to the scientific community. Using these tools, we demonstrate here that the controversial ORF10 protein is expressed in infected cells. Furthermore, we show that the promising repurposed antiviral activity of apilimod is dependent on TMPRSS2 expression. Altogether, our SARS-CoV-2 toolkit, which can be directly accessed via our website at https://mrcppu-covid.bio/, constitutes a resource with considerable potential to advance COVID-19 vaccine design, drug testing, and discovery science

A prenylated dsRNA sensor protects against severe COVID-19

INTRODUCTION Interferons (IFNs) are cytokines that are rapidly deployed in response to invading pathogens. By initiating a signaling cascade that stimulates the expression of hundreds of genes, IFNs create an antiviral state in host cells. Because IFNs heavily influence COVID-19 outcomes, and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replication can be inhibited by the antiviral state, it is important to understand how the individual antiviral effectors encoded by IFN-stimulated genes (ISGs) inhibit SARS-CoV-2. RATIONALE We hypothesized that IFN-stimulated antiviral effectors can inhibit SARS-CoV-2, and that variation at the loci encoding these defenses underlies why some people are more susceptible to severe COVID-19. RESULTS We used arrayed ISG expression screening to reveal that 2′-5′-oligoadenylate synthetase 1 (OAS1) consistently inhibited SARS-CoV-2 in different contexts. Using CRISPR-Cas9, we found that endogenous OAS1 makes a substantial contribution to the antiviral state by recognizing short stretches of double-stranded RNA (dsRNA) and activating RNase L. We globally mapped where OAS1 binds to SARS-CoV-2 viral RNAs and found that OAS1 binding is remarkably specific, with two conserved stem loops in the SARS-CoV-2 5′-untranslated region (UTR) constituting the principal viral target. OAS1 expression was readily detectable at the sites of infection in individuals who died of COVID-19, and specific OAS1 alleles are known to be associated with altered susceptibility to infection and severe disease. It had previously been reported that alleles containing a common splice-acceptor single nucleotide polymorphism in OAS1 (Rs10774671) were associated with less severe COVID-19. We determined that people with at least one allele with a G at this position could express a prenylated form of OAS1 (p46), whereas other individuals could not. Using a series of mutants, we found that C-terminal prenylation was necessary for OAS1 to initiate a block to SARS-CoV-2. Furthermore, confocal microscopy revealed that prenylation targeted OAS1 to perinuclear structures rich in viral dsRNA, whereas non-prenylated OAS1 was diffusely localized and unable to initiate a detectable block to SARS-CoV-2 replication. The realization that prenylation is essential for OAS1-mediated sensing of SARS-CoV-2 allowed us to examine the transcriptome of infected patients and investigate whether there was a link between the expression of prenylated OAS1 and SARS-CoV-2 disease progression. Analysis of the OAS1 transcripts from 499 hospitalized COVID-19 patients revealed that expressing prenylated OAS1 was associated with protection from severe COVID-19. Because prenylated OAS1 was so important in human cases, we wanted to determine whether horseshoe bats, the likely source of SARS-CoV-2, possessed the same defense. When we examined the genomic region where the prenylation signal should reside, retrotransposition of a long terminal repeat sequence had ablated this signal, preventing the expression of prenylated anti-CoV OAS1 in these bats. CONCLUSION C-terminal prenylation targets OAS1 to intracellular sites rich in viral dsRNA, which are likely the SARS-CoV-2 replicative organelles. Once in the right place, OAS1 binds to dsRNA structures in the SARS-CoV-2 5′-UTR and initiates a potent block to SARS-CoV-2 replication. Thus, the correct targeting of OAS1 and the subsequent inhibition of SARS-CoV-2 likely underpins the genetic association of alleles containing a G at Rs10774671 with reduced susceptibility to infection and severe disease in COVID-19. Moreover, the conspicuous absence of this antiviral defense in horseshoe bats potentially explains why SARS-CoV-2 is so sensitive to this defense in humans

A prenylated dsRNA sensor protects against severe COVID-19

Inherited genetic factors can influence the severity of COVID-19, but the molecular explanation underpinning a genetic association is often unclear. Intracellular antiviral defenses can inhibit the replication of viruses and reduce disease severity. To better understand the antiviral defenses relevant to COVID-19, we used interferon-stimulated gene (ISG) expression screening to reveal that OAS1, through RNase L, potently inhibits SARS-CoV-2. We show that a common splice-acceptor SNP (Rs10774671) governs whether people express prenylated OAS1 isoforms that are membrane-associated and sense specific regions of SARS-CoV-2 RNAs, or only express cytosolic, nonprenylated OAS1 that does not efficiently detect SARS-CoV-2. Importantly, in hospitalized patients, expression of prenylated OAS1 was associated with protection from severe COVID-19, suggesting this antiviral defense is a major component of a protective antiviral response

A prenylated dsRNA sensor protects against severe COVID-19

Inherited genetic factors can influence the severity of COVID-19, but the molecular explanation underpinning a genetic association is often unclear. Intracellular antiviral defenses can inhibit the replication of viruses and reduce disease severity. To better understand the antiviral defenses relevant to COVID-19, we used interferon-stimulated gene (ISG) expression screening to reveal that OAS1, through RNase L, potently inhibits SARS-CoV-2. We show that a common splice-acceptor SNP (Rs10774671) governs whether people express prenylated OAS1 isoforms that are membrane-associated and sense specific regions of SARS-CoV-2 RNAs, or only express cytosolic, nonprenylated OAS1 that does not efficiently detect SARS-CoV-2. Importantly, in hospitalized patients, expression of prenylated OAS1 was associated with protection from severe COVID-19, suggesting this antiviral defense is a major component of a protective antiviral response

Development and implementation of a novel assay for L-2-hydroxyglutarate dehydrogenase (L-2-HGDH) in cell lysates: L-2-HGDH deficiency in 15 patients with L-2-hydroxyglutaric aciduria

l-2-hydroxyglutaric aciduria (l-2-HGA) is a rare inherited autosomal recessive neurometabolic disorder caused by mutations in the gene encoding l-2-hydroxyglutarate dehydrogenase. An assay to evaluate l-2-hydroxyglutarate dehydrogenase (l-2-HGDH) activity in fibroblast, lymphoblast and/or lymphocyte lysates has hitherto been unavailable. We developed an l-2-HGDH enzyme assay in cell lysates based on the conversion of stable-isotope-labelled l-2-hydroxyglutarate to 2-ketoglutarate, which is converted into l-glutamate in situ. The formation of stable isotope labelled l-glutamate is therefore a direct measure of l-2-HGDH activity, and this product is detected by liquid chromatography-tandem mass spectrometry. A deficiency of l-2-HGDH activity was detected in cell lysates from 15 out of 15 l-2-HGA patients. Therefore, this specific assay confirmed the diagnosis unambiguously affirming the relationship between molecular and biochemical observations. Residual activity was detected in cells derived from one l-2-HGA patient. The l-2-HGDH assay will be valuable for examining in vitro riboflavin/FAD therapy to rescue l-2-HGDH activity. © 2009 SSIEM and Springer