The importance of semantic network brain regions in integrating prior knowledge with an ongoing dialogue

To understand a dialogue we need to know the specific topics that are being discussed. This enables us to integrate our knowledge of what was said previously, in order to interpret the current dialogue. Here, we selectively manipulated knowledge about the narrative content of dialogues between two people, presented in short videos. The videos were clips taken from television situation comedies and the speech in the first-half of the clip could either be presented normally (high context) or spectrally rotated in order to render it unintelligible (low context). Knowledge of the preceding narrative boosted memory for the following dialogues as well as increased the inter-subject semantic similarity of recalled descriptions of the dialogues. Sharing knowledge of the preceding narrative across participants had two effects on fMRI markers of neural processing: (1) it strengthened temporal inter-subject correlations in regions including the left angular (AG) and inferior frontal gyri (IFG), and (2) it increased spatial inter-subject pattern similarity in the bilateral anterior temporal lobes (ATL). We argue that these brain regions, which are known to be involved in semantic processing, support the activation and integration of prior knowledge, which helps people to better understand and remember dialogues as they unfold.

Convergent use of phosphatidic acid for Hepatitis C virus and SARS-CoV-2 replication organelle formation

Double membrane vesicles (DMVs) are used as replication organelles by phylogenetically and biologically distant pathogenic RNA viruses such as hepatitis C virus (HCV) and severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Viral DMVs are morphologically analogous to DMVs formed during autophagy, and although the proteins required for DMV formation are extensively studied, the lipids driving their biogenesis are largely unknown. Here we show that production of the lipid phosphatidic acid (PA) by acylglycerolphosphate acyltransferase (AGPAT) 1 and 2 in the ER is important for DMV biogenesis in viral replication and autophagy. Using DMVs in HCV-replicating cells as model, we found that AGPATs are recruited to and critically contribute to HCV replication and DMV formation. AGPAT1/2 double knockout also impaired SARS-CoV-2 replication and the formation of autophagosome-like structures. By using correlative light and electron microscopy, we observed the relocalization of AGPAT proteins to HCV and SARS-CoV-2 induced DMVs. In addition, an intracellular PA sensor accumulated at viral DMV formation sites, consistent with elevated levels of PA in fractions of purified DMVs analyzed by lipidomics. Apart from AGPATs, PA is generated by alternative pathways via phosphotidylcholine (PC) and diacylglycerol (DAG). Pharmacological inhibition of these synthesis pathways also impaired HCV and SARS-CoV-2 replication as well as formation of autophagosome-like DMVs. These data identify PA as an important lipid used for replication organelle formation by HCV and SARS-CoV-2, two phylogenetically disparate viruses causing very different diseases, i.e. chronic liver disease and COVID-19, respectively. In addition, our data argue that host-targeting therapy aiming at PA synthesis pathways might be suitable to attenuate replication of these viruses. One Sentence SummaryPhosphatidic acid is important for the formation of double membrane vesicles, serving as replication organelles of hepatitis C virus and SARS-CoV-2, and offering a possible host-targeting strategy to treat SARS-CoV-2 infection.

The FDA-approved drug cobicistat synergizes with remdesivir to inhibit SARS-CoV-2 replication

Combinations of direct-acting antivirals are needed to minimize drug-resistance mutations and stably suppress replication of RNA viruses. Currently, there are limited therapeutic options against the Severe Acute Respiratory Syndrome Corona Virus 2 (SARS-CoV-2) and testing of a number of drug regimens has led to conflicting results. Here we show that cobicistat, which is an-FDA approved drug-booster that blocks the activity of the drug metabolizing proteins Cytochrome P450-3As (CYP3As) and P-glycoprotein (P-gp), inhibits SARS-CoV-2 replication. Cell-to-cell membrane fusion assays indicated that the antiviral effect of cobicistat is exerted through inhibition of spike protein-mediated membrane fusion. In line with this, incubation with low micromolar concentrations of cobicistat decreased viral replication in three different cell lines including cells of lung and gut origin. When cobicistat was used in combination with the putative CYP3A target and nucleoside analog remdesivir, a synergistic effect on the inhibition of viral replication was observed in cell lines and in a primary human colon organoid. The cobicistat/remdesivir combination was able to potently abate viral replication to levels comparable to mock-infected cells leading to an almost complete rescue of infected cell viability. These data highlight cobicistat as a therapeutic candidate for treating SARS-CoV-2 infection and as a potential building block of combination therapies for COVID-19.

Global analysis of protein-RNA interactions in SARS-CoV-2 infected cells reveals key regulators of infection

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes COVID-19. SARS-CoV-2 relies on cellular RNA-binding proteins (RBPs) to replicate and spread, although which RBPs control SARS-CoV-2 infection remains largely unknown. Here, we employ a multi-omic approach to identify systematically and comprehensively which cellular and viral RBPs are involved in SARS-CoV-2 infection. We reveal that the cellular RNA-bound proteome is remodelled upon SARS-CoV-2 infection, having widespread effects on RNA metabolic pathways, non-canonical RBPs and antiviral factors. Moreover, we apply a new method to identify the proteins that directly interact with viral RNA, uncovering dozens of cellular RBPs and six viral proteins. Amongst them, several components of the tRNA ligase complex, which we show regulate SARS-CoV-2 infection. Furthermore, we discover that available drugs targeting host RBPs that interact with SARS-CoV-2 RNA inhibit infection. Collectively, our results uncover a new universe of host-virus interactions with potential for new antiviral therapies against COVID-19.

Challenges for targeting SARS-CoV-2 proteases as a therapeutic strategy for COVID-19

Two proteases produced by the SARS-CoV-2 virus, Mpro and PLpro, are essential for viral replication and have become the focus of drug development programs for treatment of COVID-19. We screened a highly focused library of compounds containing covalent warheads designed to target cysteine proteases to identify new lead scaffolds for both Mpro and PLpro proteases. These efforts identified a small number of hits for the Mpro protease and no viable hits for the PLpro protease. Of the Mpro hits identified as inhibitors of the purified recombinant protease, only two compounds inhibited viral infectivity in cellular infection assays. However, we observed a substantial drop in antiviral potency upon expression of TMPRSS2, a transmembrane serine protease that acts in an alternative viral entry pathway to the lysosomal cathepsins. This loss of potency is explained by the fact that our lead Mpro inhibitors are also potent inhibitors of host cell cysteine cathepsins. To determine if this is a general property of Mpro inhibitors, we evaluated several recently reported compounds and found that they are also effective inhibitors of purified human cathepsin L and B and showed similar loss in activity in cells expressing TMPRSS2. Our results highlight the challenges of targeting Mpro and PLpro proteases and demonstrate the need to carefully assess selectivity of SARS-CoV-2 protease inhibitors to prevent clinical advancement of compounds that function through inhibition of a redundant viral entry pathway.

SARS-CoV-2 infection induces a pro-inflammatory cytokine response through cGAS-STING and NF-κB

SARS-CoV-2 is a novel virus that has rapidly spread, causing a global pandemic. In the majority of infected patients, SARS-CoV-2 leads to mild disease; however, in a significant proportion of infections, individuals develop severe symptoms that can lead to permanent lung damage or death. These severe cases are often associated with high levels of pro-inflammatory cytokines and low antiviral responses which can lead to systemic complications. We have evaluated transcriptional and cytokine secretion profiles from infected cell cultures and detected a distinct upregulation of inflammatory cytokines that parallels samples taken from infected patients. Building on these observations, we found a specific activation of NF-{kappa}B and a block of IRF3 nuclear translocation in SARS-CoV-2 infected cells. This NF-{kappa}B response is mediated by cGAS-STING activation and could be attenuated through STING targeting drugs. Our results show that SARS-CoV-2 curates a cGAS-STING mediated NF-{kappa}B driven inflammatory immune response in epithelial cells that likely contributes to inflammatory responses seen in patients and might be a target to suppress severe disease symptoms.

Structures, conformations and distributions of SARS-CoV-2 spike protein trimers on intact virions

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virions are surrounded by a lipid bilayer from which spike (S) protein trimers protrude. Heavily glycosylated S trimers bind the ACE2 receptor and mediate entry of virions into target cells. S exhibits extensive conformational flexibility: it modulates the exposure of its receptor binding site and later undergoes complete structural rearrangement to drive fusion of viral and cellular membranes. The structures and conformations of soluble, overexpressed, purified S proteins have been studied in detail using cryo-electron microscopy. The structure and distribution of S on the virion surface, however, has not been characterised. Here we applied cryo-electron microscopy and tomography to image intact SARS-CoV-2 virions, determining the high-resolution structure, conformational flexibility and distributions of S trimers in situ on the virion surface. These results provide a basis for understanding the conformations of S present on the virion, and for studying their interactions with neutralizing antibodies.

SARS-CoV-2 structure and replication characterized by in situ cryo-electron tomography

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the COVID19 pandemic, is a highly pathogenic {beta}-coronavirus. As other coronaviruses, SARS-CoV-2 is enveloped, replicates in the cytoplasm and assembles at intracellular membranes. Here, we structurally characterize the viral replication compartment and report critical insights into the budding mechanism of the virus, and the structure of extracellular virions close to their native state by in situ cryo-electron tomography and subtomogram averaging. We directly visualized RNA filaments inside the double membrane vesicles, compartments associated with viral replication. The RNA filaments show a diameter consistent with double-stranded RNA and frequent branching likely representing RNA secondary structures. We found that assembled S trimers in lumenal cisternae do not alone induce membrane bending but laterally reorganize on the envelope during virion assembly. The viral ribonucleoprotein complexes (vRNPs) are accumulated at the curved membrane characteristic for budding sites suggesting that vRNP recruitment is enhanced by membrane curvature. Subtomogram averaging shows that vRNPs are distinct cylindrical assemblies. We propose that the genome is packaged around multiple separate vRNP complexes, thereby allowing incorporation of the unusually large coronavirus genome into the virion while maintaining high steric flexibility between the vRNPs.

Microscopy-based assay for semi-quantitative detection of SARS-CoV-2 specific antibodies in human sera

Emergence of the novel pathogenic coronavirus SARS-CoV-2 and its rapid pandemic spread presents numerous questions and challenges that demand immediate attention. Among these is the urgent need for a better understanding of humoral immune response against the virus as a basis for developing public health strategies to control viral spread. For this, sensitive, specific and quantitative serological assays are required. Here we describe the development of a semi-quantitative high-content microscopy-based assay for detection of three major classes (IgG, IgA and IgM) of SARS-CoV-2 specific antibodies in human samples. The possibility to detect antibodies against the entire viral proteome together with a robust semi-automated image analysis workflow resulted in specific, sensitive and unbiased assay which complements the portfolio of SARS-CoV-2 serological assays. The procedure described here has been used for clinical studies and provides a general framework for the application of quantitative high-throughput microscopy to rapidly develop serological assays for emerging virus infections.

Critical role of type III interferon in controlling SARS-CoV-2 infection, replication and spread in primary human intestinal epithelial cells

SARS-CoV-2 is an unprecedented worldwide health problem that requires concerted and global approaches to better understand the virus in order to develop novel therapeutic approaches to stop the COVID-19 pandemic and to better prepare against potential future emergence of novel pandemic viruses. Although SARS-CoV-2 primarily targets cells of the lung epithelium causing respiratory infection and pathologies, there is growing evidence that the intestinal epithelium is also infected. However, the importance of the enteric phase of SARS-CoV-2 for virus-induced pathologies, spreading and prognosis remains unknown. Here, using both colon-derived cell lines and primary non-transformed colon organoids, we engage in the first comprehensive analysis of SARS-CoV-2 lifecycle in human intestinal epithelial cells. Our results demonstrate that human intestinal epithelial cells fully support SARS-CoV-2 infection, replication and production of infectious de-novo virus particles. Importantly, we identified intestinal epithelial cells as the best culture model to propagate SARS-CoV-2. We found that viral infection elicited an extremely robust intrinsic immune response where, interestingly, type III interferon mediated response was significantly more efficient at controlling SARS-CoV-2 replication and spread compared to type I interferon. Taken together, our data demonstrate that human intestinal epithelial cells are a productive site of SARS-CoV-2 replication and suggest that the enteric phase of SARS-CoV-2 may participate in the pathologies observed in COVID-19 patients by contributing in increasing patient viremia and by fueling an exacerbated cytokine response.