https://www.biorxiv.org/content/biorxiv/early/2020/02/20/2020.02.19.956581.full.pdf

Structure, Function, and Antigenicity of the SARS-CoV-2 Spike Glycoprotein.

Evolution of Protein Molecules

Middle East respiratory syndrome coronavirus (MERS-CoV) is a lineage C betacoronavirus that since its emergence in 2012 has caused outbreaks in human populations with case-fatality rates of ∼36%. As in other coronaviruses, the spike (S) glycoprotein of MERS-CoV mediates receptor recognition and membrane fusion and is the primary target of the humoral immune response during infection. Here we use structure-based design to develop a generalizable strategy for retaining coronavirus S proteins in the antigenically optimal prefusion conformation and demonstrate that our engineered immunogen is able to elicit high neutralizing antibody titers against MERS-CoV. We also determined high-resolution structures of the trimeric MERS-CoV S ectodomain in complex with G4, a stem-directed neutralizing antibody. The structures reveal that G4 recognizes a glycosylated loop that is variable among coronaviruses and they define four conformational states of the trimer wherein each receptor-binding domain is either tightly packed at the membrane-distal apex or rotated into a receptor-accessible conformation. Our studies suggest a potential mechanism for fusion initiation through sequential receptor-binding events and provide a foundation for the structure-based design of coronavirus vaccines.

https://www.pnas.org/content/pnas/114/35/E7348.full.pdf

Immunogenicity and structures of a rationally designed prefusion MERS-CoV spike antigen

Coronaviruses (CoVs) traffic frequently between species resulting in novel disease outbreaks, most recently exemplified by the newly emerged SARS-CoV-2, the causative agent of COVID-19. Here, we show that the ribonucleoside analog β-d-N4-hydroxycytidine (NHC; EIDD-1931) has broad-spectrum antiviral activity against SARS-CoV-2, MERS-CoV, SARS-CoV, and related zoonotic group 2b or 2c bat-CoVs, as well as increased potency against a CoV bearing resistance mutations to the nucleoside analog inhibitor remdesivir. In mice infected with SARS-CoV or MERS-CoV, both prophylactic and therapeutic administration of EIDD-2801, an orally bioavailable NHC prodrug (β-d-N4-hydroxycytidine-5'-isopropyl ester), improved pulmonary function and reduced virus titer and body weight loss. Decreased MERS-CoV yields in vitro and in vivo were associated with increased transition mutation frequency in viral, but not host cell RNA, supporting a mechanism of lethal mutagenesis in CoV. The potency of NHC/EIDD-2801 against multiple CoVs and oral bioavailability highlights its potential utility as an effective antiviral against SARS-CoV-2 and other future zoonotic CoVs.

/pdf/an-orally-bioavailable-broad-spectrum-antiviral-inhibits-akz1d8qlou.pdf

An orally bioavailable broad-spectrum antiviral inhibits SARS-CoV-2 in human airway epithelial cell cultures and multiple coronaviruses in mice.

Recent history is punctuated by the emergence of highly pathogenic coronaviruses such as SARS- and MERS-CoV into human circulation. Upon infecting host cells, coronaviruses assemble a multi-subunit RNA-synthesis complex of viral non-structural proteins (nsp) responsible for the replication and transcription of the viral genome. Here, we present the 3.1 A resolution structure of the SARS-CoV nsp12 polymerase bound to its essential co-factors, nsp7 and nsp8, using single particle cryo-electron microscopy. nsp12 possesses an architecture common to all viral polymerases as well as a large N-terminal extension containing a kinase-like fold and is bound by two nsp8 co-factors. This structure illuminates the assembly of the coronavirus core RNA-synthesis machinery, provides key insights into nsp12 polymerase catalysis and fidelity and acts as a template for the design of novel antiviral therapeutics. The pathogenic human coronaviruses SARS- and MERS-CoV can cause severe respiratory disease. Here the authors present the 3.1A cryo-EM structure of the SARS-CoV RNA polymerase nsp12 bound to its essential co-factors nsp7 and nsp8, which is of interest for antiviral drug development.

/pdf/structure-of-the-sars-cov-nsp12-polymerase-bound-to-nsp7-and-55oy8lsy65.pdf

Structure of the SARS-CoV nsp12 polymerase bound to nsp7 and nsp8 co-factors.

The degree of evolutionary conservation of an amino acid in a protein or a nucleic acid in DNA/RNA reflects a balance between its natural tendency to mutate and the overall need to retain the structural integrity and function of the macromolecule. The ConSurf web server (http://consurf.tau.ac.il), established over 15 years ago, analyses the evolutionary pattern of the amino/nucleic acids of the macromolecule to reveal regions that are important for structure and/or function. Starting from a query sequence or structure, the server automatically collects homologues, infers their multiple sequence alignment and reconstructs a phylogenetic tree that reflects their evolutionary relations. These data are then used, within a probabilistic framework, to estimate the evolutionary rates of each sequence position. Here we introduce several new features into ConSurf, including automatic selection of the best evolutionary model used to infer the rates, the ability to homology-model query proteins, prediction of the secondary structure of query RNA molecules from sequence, the ability to view the biological assembly of a query (in addition to the single chain), mapping of the conservation grades onto 2D RNA models and an advanced view of the phylogenetic tree that enables interactively rerunning ConSurf with the taxa of a sub-tree.

/pdf/consurf-2016-an-improved-methodology-to-estimate-and-4e3p549tv3.pdf

ConSurf 2016: an improved methodology to estimate and visualize evolutionary conservation in macromolecules.

For nearly a century, biologists, and botanists in particular, have been interested in the determination and documentation of chromosome numbers for extant taxa (reviewed in Goldblatt & Lowry, 2011) as well as extinct ones (Laane & Hoiland, 1986; Masterson, 1994). These data have beenwidely used to evaluate the evolutionary pattern of chromosome number change and to estimate the base chromosome number of clades of interest. Chromosome numbers have also been extensively utilized as an important phylogenetic character in the context of cytotaxonomy (Chatterjee & Kumar Sharma, 1969; Schlarbaum & Tsuchiya, 1984; Guerra, 2012). Perhaps the most influential use of chromosome number data has been in the inference of major genomic events such aswhole genomeduplications (polyploidy), as well as changes in single chromosome numbers (e.g. dysploidy). Early researchers analyzed the distribution of chromosome numbers within a group of interest and employed various threshold techniques to estimate ploidy levels for the analyzed taxa (Stebbins, 1938; Grant, 1963; Goldblatt, 1980).More recently, phylogenetic information was incorporated into the analyses, allowing researchers to infer transitions in chromosome numbers along branches of the tree using either the maximum parsimony principle (Schultheis, 2001; Hansen et al., 2006; Ohi-Toma et al., 2006; Wood et al., 2009) or by using a probabilistic evolutionary model within the likelihood paradigm (Mayrose et al., 2010; Cusimano et al., 2012; Glick & Mayrose, 2014). Due to their significance and the relative ease by which chromosome numbers can be obtained, it is not surprising that chromosome number is the most extensively and consistently recorded cytological property in most plant families and genera (Guerra, 2008). These data have been documented along the years in an array of journal manuscripts, printed books (L€ove & L€ove, 1948; Darlington & Wylie, 1955; Fedorov, 1969) and, more recently, in the form of online databases (Goldblatt & Johnson, 1979; Watanabe, 2002; Bennett & Leitch, 2011). To date, the most comprehensive data source is the Index to PlantChromosome Numbers (IPCN; Goldblatt & Johnson, 1979), which provides reference point to original chromosome counts reported in the literature. IPCN was initially established at the University of California Berkeley in the 1950s and was later maintained by Canada Department of Agriculture, Missouri Botanical Garden, and currently by the International Association for Plant Taxonomy (IAPT). A large portion of the counts referenced during 1979– 2006, the years that IPCN has been housed in the Missouri Botanical Garden, can be accessed and searched online. Counts reported in more recent years are currently published under IAPT/ IOPBChromosomeData series (Marhold, 2006) but are not stored within a central, easily searched, database. In addition to IPCN, several other online data sources are available, most of which are dedicated to either a specific geographical region (Slovakia – Marhold et al., 2007; Poland –G oralski et al., 2009 onwards) or to a certain taxonomic group (e.g. Hieracium – Schuhwerk, 1996; Asteraceae –Watanabe, 2002). The amount of chromosome counts that exist to date is extensive, and searching the large number of resources that contain such information is a daunting task, particularly when a large number of taxa is examined. Consequently, many researchers search for chromosome number information only through the largest online database(s), while smaller but nonetheless valuable sources are ignored.This usually results inmissing data for someof the species in question, which may lead to erroneous conclusions drawn from the analysis. Obviously, a large accessible database that unifies all currently known databases, including both printed and online sources, would be of great value to the botanical community and wouldmake the task of data collectionmuch easier. In addition, such a central resource would enable researchers to add new counts as soon as they are being reported, facilitating the task of data sharing. Here, we present the Chromosome Counts Database (CCDB), as a community resource of plant chromosome numbers. The database incorporates data from dozens of sources, more than doubling the amount of data available within any single resource. The online database additionally enables researchers to add new counts or to comment on existing data entries, thereby facilitating data sharing. The extensive amount of data currently available in CCDB further allowed us to analyze the patterns of chromosome number distribution among major plant groups. We estimate the percentage of plant species exhibiting intraspecific variation in chromosome numbers as well as in their ploidy levels.

https://nph.onlinelibrary.wiley.com/doi/pdf/10.1111/nph.13191

The Chromosome Counts Database (CCDB) – a community resource of plant chromosome numbers

Phylogeny reconstruction is a key instrument in numerous biological analyses, ranging from evolutionary and ecology research, to conservation and systems biology. The increasing accumulation of genomic data makes it possible to reconstruct phylogenies with both high accuracy and at increasingly finer resolution. Yet, taking advantage of the enormous amount of sequence data available requires the use of computational tools for efficient data retrieval and processing, or else the process could quickly become an error-prone endeavour. Here, we present OneTwoTree (http://onetwotree.tau.ac.il/), a Web-based tool for tree reconstruction based on the supermatrix paradigm. Given a list of taxa names of interest as the sole input requirement, OneTwoTree retrieves all available sequence data from NCBI GenBank, clusters these into orthology groups, identifies the most informative set of markers, searches for an appropriate outgroup, and assembles a partitioned sequence matrix that is then used for the final phylogeny reconstruction step. OneTwoTree further allows users to control various steps of the process, such as the merging of sequences from similar clusters, or phylogeny reconstruction based on markers from a specific genome type. By comparing the performance of OneTwoTree to a manually reconstructed phylogeny of the Antirrhineae tribe, we show that the use of OneTwoTree resulted in substantially higher data coverage in terms of both taxon sampling and the number of informative markers assembled. OneTwoTree provides a flexible online tool for species-tree reconstruction, aimed to assist researchers ranging in their level of prior expertise in the task of phylogeny reconstruction.

Ofer Chay

Papers

ConSurf 2016: an improved methodology to estimate and visualize evolutionary conservation in macromolecules.

The Chromosome Counts Database (CCDB) – a community resource of plant chromosome numbers

OneTwoTree: An online tool for phylogeny reconstruction.