Highlights d SARS-CoV-2 spike protein interacts with heparan sulfate and ACE2 through the RBD d Heparan sulfate promotes Spike-ACE2 interaction d SARS-CoV-2 infection is co-dependent on heparan sulfate and ACE2 d Heparin and non-anticoagulant derivatives block SARS-CoV-2 binding and infection

The multifunctional nucleocapsid (N) protein in SARS-CoV-2 binds the ~30 kb viral RNA genome to aid its packaging into the 80–90 nm membrane-enveloped virion. The N protein is composed of N-terminal RNA-binding and C-terminal dimerization domains that are flanked by three intrinsically disordered regions. Here we demonstrate that the N protein’s central disordered domain drives phase separation with RNA, and that phosphorylation of an adjacent serine/arginine rich region modulates the physical properties of the resulting condensates. In cells, N forms condensates that recruit the stress granule protein G3BP1, highlighting a potential role for N in G3BP1 sequestration and stress granule inhibition. The SARS-CoV-2 membrane (M) protein independently induces N protein phase separation, and three-component mixtures of N + M + RNA form condensates with mutually exclusive compartments containing N + M or N + RNA, including annular structures in which the M protein coats the outside of an N + RNA condensate. These findings support a model in which phase separation of the SARS-CoV-2 N protein contributes both to suppression of the G3BP1-dependent host immune response and to packaging genomic RNA during virion assembly.

Topoisomerases are enzymes that use DNA strand scission, manipulation, and rejoining activities to directly modulate DNA topology. These actions provide a powerful means to effect changes in DNA supercoiling levels, and allow some topoisomerases to both unknot and decatenate chromosomes. Since their initial discovery over three decades ago, researchers have amassed a rich store of information on the cellular roles and regulation of topoisomerases, and have delineated general models for their chemical and physical mechanisms. Topoisomerases are now known to be necessary for the survival of cellular organisms and many viruses and are rich clinical targets for anticancer and antimicrobial treatments. In recent years, crystal structures have been obtained for each of the four types of topoisomerases in a number of distinct conformational and substrate-bound states. In addition, sophisticated biophysical methods have been utilized to study details of topoisomerase reaction dynamics and enzymology. A synthesis of these approaches has provided researchers with new physical insights into how topoisomerases employ chemistry and allostery to direct the large-scale molecular motions needed to pass DNA strands through each other.

DNA gyrase is unique among enzymes for its ability to actively introduce negative supercoils into DNA. This function is mediated in part by the C-terminal domain of its A subunit (GyrA CTD). Here, we report the crystal structure of this Ϸ35-kDa domain determined to 1.75-Å resolution. The GyrA CTD unexpectedly adopts an unusual fold, which we term a ␤-pinwheel, that is globally reminiscent of a ␤-propeller but is built of blades with a previously unobserved topology. A large, conserved basic patch on the outer edge of this domain suggests a likely site for binding and bending DNA; fluorescence resonance energy transfer-based assays show that the GyrA CTD is capable of bending DNA by >180°over a 40-bp region. Surprisingly, we find that the CTD of the topoisomerase IV A subunit, which shares limited sequence homology with the GyrA CTD, also bends DNA. Together, these data provide a physical explanation for the ability of DNA gyrase to constrain a positive superhelical DNA wrap, and also suggest that the particular substrate preferences of topoisomerase IV might be dictated in part by the function of this domain.T he topology of cellular DNA is managed by topoisomerases, enzymes that pass DNA strands through each other to relieve excess supercoiling and resolve DNA knots and catenanes (1, 2). Whereas all organisms contain at least one topoisomerase, the bacterium Escherichia coli possesses four, each with distinct roles: topoisomerase (topo) I, topo III, topo IV, and DNA gyrase. Topo I and topo III pass single DNA strands through one another to relax negative supercoils (1) or aid RecQ-family helicases in certain DNA repair processes, respectively (3). DNA gyrase and topo IV use ATP to power the transport of one intact DNA duplex through another, an activity that can alter DNA superhelicity as well as promote chromosome decatenation (4, 5).DNA gyrase and topo IV are members of the type IIA topoisomerase superfamily (6, 7). These enzymes are assembled as oligomeric complexes with distinct domains that coordinate ATP binding and hydrolysis with DNA binding, cleavage, and transport (7,8). The type IIA topo reaction cycle begins when one segment of DNA, termed the G segment, binds across the central region of the enzyme. ATP binding then triggers a series of motions that leads to the capture of a second DNA duplex (the T segment), cleavage and opening of the G segment, and passage of the T segment through the break. Once the T segment is transported, the G segment is resealed, the T segment is expelled from the protein, and ATP is hydrolyzed and released. This enzymatic cycle alters the linking number of the substrate DNA in discrete steps of Ϯ 2.Although all type IIA topos share this basic mechanism, there exist distinct type IIA subtypes that have differing substrate specificities and activities. The eukaryotic enzyme, topo II, relaxes positively and negatively supercoiled DNAs at the same rate (9) and can decatenate chromosomes (5). In contrast, most bacteria possess two somewhat more specialized type IIA topos; DNA ...

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