The spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is required for cell entry and is the major focus for vaccine development. Here, we combine cryo electron tomography, subtomogram averaging and molecular dynamics simulations to structurally analyze S in situ. Compared to recombinant S, the viral S was more heavily glycosylated and occurred mostly in the closed pre-fusion conformation. We show that the stalk domain of S contains three hinges, giving the head unexpected orientational freedom. We propose that the hinges allow S to scan the host cell surface, shielded from antibodies by an extensive glycan coat. The structure of native S contributes to our understanding of SARS-CoV-2 infection and the development of safe vaccines.

Measles (MV) is an aerosol-transmitted virus that affects more than 10 million children each year and accounts for approximately 120,000 deaths1,2. While it was long believed to replicate in the respiratory epithelium before disseminating, it was recently shown to initially infect macrophages and dendritic cells of the airways using the signaling lymphocytic activation molecule (SLAM, CD150) as receptor3-6. These cells then cross the respiratory epithelium and ferry the infection to lymphatic organs where MV replicates vigorously7. How and where the virus crosses back into the airways has remained unknown. Based on functional analyses of surface proteins preferentially expressed on virus-permissive epithelial cell lines, we identified nectin-48 (poliovirus-receptor-like-4) as a candidate host exit receptor. This adherens junction protein of the immunoglobulin superfamily interacts with the viral attachment protein with high affinity through its membrane-distal domain. Nectin-4 sustains MV entry and non-cytopathic lateral spread in well-differentiated primary human airway epithelial sheets infected basolaterally. It is down-regulated in infected epithelial cells, including those of macaque tracheas. While other viruses use receptors to enter hosts or transit through their epithelial barriers, we suggest that MV targets nectin-4 to emerge in the airways. Nectin-4 is a cellular marker of several types of cancer9-11, which has implications for ongoing MV-based clinical trials of oncolysis12.

Retargeting of lentiviral vector entry to cell types of interest is a key factor in improving the safety and efficacy of gene transfer. In this study we show that the retargetable envelope glycoproteins of measles virus (MV), namely, the hemagglutinin (H) responsible for receptor recognition and the fusion protein (F), can pseudotype human immunodeficiency virus 1 (HIV-1) vectors when their cytoplasmic tails are truncated. We then pseudotyped HIV-1 vectors with MV glycoproteins displaying on H either the epidermal growth factor or a single-chain antibody directed against CD20, but without the ability to recognize their native receptors. Gene transfer into cells that expressed the targeted receptor was several orders of magnitude more efficient than into cells that did not. High-target versus nontarget cell discrimination was demonstrated in mixed cell populations, where the targeting vector selectively eliminated CD20-positive cells after suicide gene transfer. Remarkably, primary human CD20-positive B lymphocytes were transduced more efficiently by the CD20-targeted vector than by a vector pseudotyped with the vesicular stomatitis virus G (VSV-G) protein. In addition, the CD20-targeted vector was able to transduce even unstimulated primary B cells, whereas VSV-G pseudotyped vectors were unable to do so. Because MV enters cells through direct fusion at the cell membrane, this novel targeting system should be widely applicable.

The spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is required for cell entry and is the major focus for vaccine development. We combine cryo electron tomography, subtomogram averaging and molecular dynamics simulations to structurally analyze S in situ. Compared to recombinant S, the viral S is more heavily glycosylated and occurs predominantly in a closed pre-fusion conformation. We show that the stalk domain of S contains three hinges that give the globular domain unexpected orientational freedom. We propose that the hinges allow S to scan the host cell surface, shielded from antibodies by an extensive glycan coat. The structure of native S contributes to our understanding of SARS-CoV-2 infection and the development of safe vaccines. The large scale tomography data set of SARS-CoV-2 used for this study is therefore sufficient to resolve structural features to below 5 Ångstrom, and is publicly available at EMPIAR-10453.