SARS-CoV-2, like other coronaviruses, builds a membrane-bound replication organelle to enable RNA replication 1 . The SARS-CoV-2 replication organelle is composed of double-membrane vesicles (DMVs) that are tethered to the endoplasmic reticulum (ER) by thin membrane connectors 2 , but the viral proteins and the host factors involved remain unknown. Here we identify the viral non-structural proteins (NSPs) that generate the SARS-CoV-2 replication organelle. NSP3 and NSP4 generate the DMVs, whereas NSP6, through oligomerization and an amphipathic helix, zippers ER membranes and establishes the connectors. The NSP6(ΔSGF) mutant, which arose independently in the Alpha, Beta, Gamma, Eta, Iota and Lambda variants of SARS-CoV-2, behaves as a gain-of-function mutant with a higher ER-zippering activity. We identified three main roles for NSP6: first, to act as a filter in communication between the replication organelle and the ER, by allowing lipid flow but restricting the access of ER luminal proteins to the DMVs; second, to position and organize DMV clusters; and third, to mediate contact with lipid droplets (LDs) through the LD-tethering complex DFCP1-RAB18. NSP6 thus acts as an organizer of DMV clusters and can provide a selective means of refurbishing them with LD-derived lipids. Notably, both properly formed NSP6 connectors and LDs are required for the replication of SARS-CoV-2. Our findings provide insight into the biological activity of NSP6 of SARS-CoV-2 and of other coronaviruses, and have the potential to fuel the search for broad antiviral agents. SARS-CoV-2 extensively rearranges host cellular membranes into replication organelles that provide a microenvironment conducive to RNA synthesis and protection from host sensor and defence systems 1,2 . The 16 viral NSPs that are released from polyproteins pp1a and pp1ab by 2 viral proteases include 13 cytosolic proteins, which are involved in RNA replication, and 3 transmembrane proteins, NSP3, NSP4 and NSP6. Studies on other coronaviruses suggest that NSP3 and NSP4, with a hitherto undefined contribution from NSP6, are responsible for generating the replication organelles 3-6 . Despite considerable advances in our understanding of the ultrastructure of the SARS-CoV-2 replication organelle 2,7,8 , mechanistic insights into its biogenesis have so far been limited. In particular, there is at present-to our knowledge-no information on the role of NSP6 in this process. Of note, six SARS-CoV-2 'variants of concern' (VOCs) (Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Eta (B.1.525), Iota (B.1.526) 9 and Lambda (C.37) 10 ) share a three-amino-acid deletion in NSP6 (NSP6(ΔSGF)), in addition to the more noted mutations in the spike protein; this finding adds further impetus to the need to examine the role of NSP6 in the biogenesis of replication organelles and in the replication of SARS-CoV-2. NSP6 induces ER zipperingWe tagged SARS-CoV-2 NSP6 at either the N or the C terminus. C-terminally tagged NSP6 showed a diffuse distribution in the ER (Fig. 1a and Extended Data Fig. ...
Cancer is characterized by pervasive epigenetic alterations with enhancer dysfunction orchestrating the aberrant cancer transcriptional programs and transcriptional dependencies. Here, we epigenetically characterize human colorectal cancer (CRC) using de novo chromatin state discovery on a library of different patient-derived organoids. By exploring this resource, we unveil a tumor-specific deregulated enhancerome that is cancer cell-intrinsic and independent of interpatient heterogeneity. We show that the transcriptional coactivators YAP/TAZ act as key regulators of the conserved CRC gained enhancers. The same YAP/TAZ-bound enhancers display active chromatin profiles across diverse human tumors, highlighting a pan-cancer epigenetic rewiring which at single-cell level distinguishes malignant from normal cell populations. YAP/TAZ inhibition in established tumor organoids causes extensive cell death unveiling their essential role in tumor maintenance. This work indicates a common layer of YAP/TAZ-fueled enhancer reprogramming that is key for the cancer cell state and can be exploited for the development of improved therapeutic avenues.
Background Pregnant women have been identified as a potentially at‐risk group concerning COVID‐19 infection, but little is known regarding the susceptibility of the fetus to infection. Co‐expression of ACE2 and TMPRSS2 has been identified as a prerequisite for infection, and expression across different tissues is known to vary between children and adults. However, the expression of these proteins in the fetus is unknown. Methods We performed a retrospective analysis of a single cell data repository. The data were then validated at both gene and protein level by performing RT‐qPCR and two‐colour immunohistochemistry on a library of second‐trimester human fetal tissues. Findings TMPRSS2 is present at both gene and protein level in the predominantly epithelial fetal tissues analysed. ACE2 is present at significant levels only in the fetal intestine and kidney, and is not expressed in the fetal lung. The placenta also does not co‐express the two proteins across the second trimester or at term. Interpretation This dataset indicates that the lungs are unlikely to be a viable route of SARS‐CoV2 fetal infection. The fetal kidney, despite presenting both the proteins required for the infection, is anatomically protected from the exposure to the virus. However, the gastrointestinal tract is likely to be susceptible to infection due to its high co‐expression of both proteins, as well as its exposure to potentially infected amniotic fluid. Tweetable abstract This work provides detailed mechanistic insight into the relative protection & vulnerabilities of the fetus & placenta to SARS‐CoV‐2 infection by scRNAseq & protein expression analysis for ACE2 & TMPRSS2. The findings help to explain the low rate of vertical transmission.
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