COVID-19 has become a global pandemic caused by the novel coronavirus SARS-CoV-2. Understanding the origins of SARS-CoV-2 is critical for deterring future zoonosis, discovering new drugs, and developing a vaccine. We show evidence of strong purifying selection around the receptor binding motif (RBM) in the spike and other genes among bat, pangolin, and human coronaviruses, suggesting similar evolutionary constraints in different host species. We also demonstrate that SARS-CoV-2’s entire RBM was introduced through recombination with coronaviruses from pangolins, possibly a critical step in the evolution of SARS-CoV-2’s ability to infect humans. Similar purifying selection in different host species, together with frequent recombination among coronaviruses, suggests a common evolutionary mechanism that could lead to new emerging human coronaviruses.

Mimivirus is the largest known virus whose genome and physical size are comparable to some small bacteria, blurring the boundary between a virus and a cell. Structural studies of Mimivirus have been difficult because of its size and long surface fibers. Here we report the use of enzymatic digestions to remove the surface fibers of Mimivirus in order to expose the surface of the viral capsid. Cryo-electron microscopy (cryoEM) and atomic force microscopy were able to show that the 20 icosahedral faces of Mimivirus capsids have hexagonal arrays of depressions. Each depression is surrounded by six trimeric capsomers that are similar in structure to those in many other large, icosahedral double-stranded DNA viruses. Whereas in most viruses these capsomers are hexagonally close-packed with the same orientation in each face, in Mimivirus there are vacancies at the systematic depressions with neighboring capsomers differing in orientation by 60°. The previously observed starfish-shaped feature is well-resolved and found to be on each virus particle and is associated with a special pentameric vertex. The arms of the starfish fit into the gaps between the five faces surrounding the unique vertex, acting as a seal. Furthermore, the enveloped nucleocapsid is accurately positioned and oriented within the capsid with a concave surface facing the unique vertex. Thus, the starfish-shaped feature and the organization of the nucleocapsid might regulate the delivery of the genome to the host. The structure of Mimivirus, as well as the various fiber components observed in the virus, suggests that the Mimivirus genome includes genes derived from both eukaryotic and prokaryotic organisms. The three-dimensional cryoEM reconstruction reported here is of a virus with a volume that is one order of magnitude larger than any previously reported molecular assembly studied at a resolution of equal to or better than 65 Å.

Dengue virus (DENV) is a significant human pathogen that causes millions of infections and results in about 24,000 deaths each year. Dendritic cell-specific ICAM3 grabbing nonintegrin (DC-SIGN), abundant in immature dendritic cells, was previously reported as being an ancillary receptor interacting with the surface of DENV. The structure of DENV in complex with the carbohydrate recognition domain (CRD) of DC-SIGN was determined by cryo-electron microscopy at 25 A resolution. One CRD monomer was found to bind to two glycosylation sites at Asn67 of two neighboring glycoproteins in each icosahedral asymmetric unit, leaving the third Asn67 residue vacant. The vacancy at the third Asn67 site is a result of the nonequivalence of the glycoprotein environments, leaving space for the primary receptor binding to domain III of E. The use of carbohydrate moieties for receptor binding sites suggests a mechanism for avoiding immune surveillance.

Icosahedral double-stranded DNA viruses use a single portal for genome delivery and packaging. The extensive structural similarity revealed by such portals in diverse viruses, as well as their invariable positioning at a unique icosahedral vertex, led to the consensus that a particular, highly conserved vertex-portal architecture is essential for viral DNA translocations. Here we present an exception to this paradigm by demonstrating that genome delivery and packaging in the virus Acanthamoeba polyphaga mimivirus occur through two distinct portals. By using high-resolution techniques, including electron tomography and cryo-scanning electron microscopy, we show that Mimivirus genome delivery entails a large-scale conformational change of the capsid, whereby five icosahedral faces open up. This opening, which occurs at a unique vertex of the capsid that we coined the “stargate”, allows for the formation of a massive membrane conduit through which the viral DNA is released. A transient aperture centered at an icosahedral face distal to the DNA delivery site acts as a non-vertex DNA packaging portal. In conjunction with comparative genomic studies, our observations imply a viral packaging pathway akin to bacterial DNA segregation, which might be shared by diverse internal membrane–containing viruses.

Human enterovirus D68 (EV-D68) is a causative agent of childhood respiratory diseases and has now emerged as a global public health threat. Nevertheless, knowledge of the tissue tropism and pathogenesis of EV-D68 has been hindered by a lack of studies on the receptor-mediated EV-D68 entry into host cells. Here we demonstrate that cell surface sialic acid is essential for EV-D68 to bind to and infect susceptible cells. Crystal structures of EV-D68 in complex with sialylated glycan receptor analogues show that they bind into the ‘canyon' on the virus surface. The sialic acid receptor induces a cascade of conformational changes in the virus to eject a fatty-acid-like molecule that regulates the stability of the virus. Thus, virus binding to a sialic acid receptor and to immunoglobulin-like receptors used by most other enteroviruses share a conserved mechanism for priming viral uncoating and facilitating cell entry.

One-component homopolymers of cationic monomers (polycations) and diblock copolymers comprising poly(ethylene glycol) (PEG) and a polycation block have been the most widely used types of polymers for formulation of polymer-based gene delivery systems. In this study, we incorporate a hydrophobic middle block into the conventional PEG-polycation architecture, and investigate the effects of this hydrophobic modification on the physicochemical and cell-level biological properties of the polymer-DNA complexes that are relevant to gene delivery applications. The ABC-type triblock copolymer used in this study consists of (A) PEG, (B) hydrophobic poly(n-butyl acrylate) (PnBA) and (C) cationic poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA) component polymers. The properties of the triblock copolymer/DNA complexes are compared with those of two other, more conventional DNA carriers derived, respectively, using a PDMAEMA homopolymer and a PEG-PDMAEMA diblock copolymer having comparable molecular weights for individual blocks. The PEG-PnBA-PDMAEMA polymer forms, in aqueous solution, positively-charged spherical micelles. The electrostatic complexation of these micelles with plasmid DNA molecules results in the formation of stable small-size DNA particles coated with a micelle monolayer, as confirmed by agarose gel electrophoresis, dynamic light scattering (DLS) and cryogenic transmission electron microscopy (cryo-TEM). Proton nuclear magnetic resonance (1H NMR) spectroscopy measurements indicate that the whole micelle-DNA assembly (named for convenience as “micelleplex”) is shielded predominantly by the PEG chains. DLS and optical microscopy imaging measurements indicate that in comparison with PDMAEMA/DNA polyplexes, the micelleplexes have a significantly lower tendency to aggregate under physiological salt concentrations, and show reduced interactions with negatively-charged components in serum such as albumin and erythrocytes. While the micelleplexes are comparable with the PEG-PDMAEMA-based DNA polyplexes in terms of their stability against aggregation under high salt concentrations and in the presence of the albumin protein, they have a slightly higher tendency to interact with erythrocytes than the diblock copolymer polyplexes. Agarose gel electrophoresis measurements indicate that relative to the PEG-PDMAEMA polyplexes, the micelleplexes provide better protection of the encapsulated DNA from enzymatic degradation, and also exhibit greater stability against disintegration induced by polyanionic additives; in these respects, the PDMAEMA homopolymer-based polyplexes show the best performance. In vitro studies in HeLa cells indicate that the PDMAEMA polyplexes show the highest gene transfection efficiency among the three different gene delivery systems. Between the micelleplexes and PEG-PDMAEMA polyplexes, a higher gene transfection efficiency is observed with the latter system. All three formulations show comparable levels of cytotoxicity in HeLa cells.

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The program RIVEM (Radial Interpretation of Viral Electron density Maps) was developed to project density radially onto a sphere that is then presented as a stereographic diagram. This permits features resulting from an asymmetric reconstruction to be projected and positioned onto an icosahedral virus surface. The features that constitute the viral surface can also be simultaneously represented in terms of atoms, amino acid residues, potential charge distribution, and surface topology. The procedure can also be adapted for the investigation of various molecular interactions.