The main viral protease (3CL pro ) is indispensable for SARS-CoV-2 replication. We delineate the human protein substrate landscape of 3CL pro by TAILS substrate-targeted N-terminomics. We identify >100 substrates in human lung and kidney cells supported by analyses of SARS-CoV-2-infected cells. Enzyme kinetics and molecular docking simulations of 3CL pro engaging substrates reveal how noncanonical cleavage sites, which diverge from SARS-CoV, guide substrate specificity. Cleaving the interactors of essential effector proteins, effectively stranding them from their binding partners, amplifies the consequences of proteolysis. We show that 3CL pro targets the Hippo pathway, including inactivation of MAP4K5, and key effectors of transcription, mRNA processing, and translation. We demonstrate that Spike glycoprotein directly binds galectin-8, with galectin-8 cleavage disengaging CALCOCO2/NDP52 to decouple antiviral-autophagy. Indeed, in post-mortem COVID-19 lung samples, NDP52 rarely colocalizes with galectin-8, unlike healthy lung cells. The 3CL pro substrate degradome establishes a foundational substrate atlas to accelerate exploration of SARS-CoV-2 pathology and drug design.

The upside-down jellyfish Cassiopea xamachana (Scyphozoa: Rhizostomeae) has been predominantly studied to understand its interaction with the endosymbiotic dinoflagellate algae Symbiodinium. As an easily culturable and tractable cnidarian model, it is an attractive alternative to stony corals to understanding the mechanisms driving establishment and maintenance of symbiosis. Cassiopea is also unique in requiring the symbiont in order to complete its transition to the adult stage, thereby providing an excellent model to understand symbiosis-driven development and evolution. Recently, the Cassiopea research system has gained interest beyond symbiosis in fields related to embryology, climate ecology, behavior, and more. With these developments, resources Ohdera et al. Cassiopea xamachana System Review including genomes, transcriptomes, and laboratory protocols are steadily increasing. This review provides an overview of the broad range of interdisciplinary research that has utilized the Cassiopea model and highlights the advantages of using the model for future research.

RNA viruses, particularly genetically diverse members of thePicornavirales, are widespread and abundant in the ocean. Gene surveys suggest that there are spatial and temporal patterns in the composition of RNA virus assemblages, but data on their diversity and genetic variability in different oceanographic settings are limited. Here, we show that specific RNA virus genomes have widespread geographic distributions and that the dominant genotypes are under purifying selection. Genomes from three previously unknown picorna-like viruses (BC-1, -2, and -3) assembled from a coastal site in British Columbia, Canada, as well as marine RNA viruses JP-A, JP-B, andHeterosigma akashiwoRNA virus exhibited different biogeographical patterns. Thus, biotic factors such as host specificity and viral life cycle, and not just abiotic processes such as dispersal, affect marine RNA virus distribution. Sequence differences relative to reference genomes imply that virus quasispecies are under purifying selection, with synonymous single-nucleotide variations dominating in genomes from geographically distinct regions resulting in conservation of amino acid sequences. Conversely, sequences from coastal South Africa that mapped to marine RNA virus JP-A exhibited more nonsynonymous mutations, probably representing amino acid changes that accumulated over a longer separation. This biogeographical analysis of marine RNA viruses demonstrates that purifying selection is occurring across oceanographic provinces. These data add to the spectrum of known marine RNA virus genomes, show the importance of dispersal and purifying selection for these viruses, and indicate that closely related RNA viruses are pathogens of eukaryotic microbes across oceans.IMPORTANCEVery little is known about aquatic RNA virus populations and genome evolution. This is the first study that analyzes marine environmental RNA viral assemblages in an evolutionary and broad geographical context. This study contributes the largest marine RNA virus metagenomic data set to date, substantially increasing the sequencing space for RNA viruses and also providing a baseline for comparisons of marine RNA virus diversity. The new viruses discovered in this study are representative of the most abundant family of marine RNA viruses, theMarnaviridae, and expand our view of the diversity of this important group. Overall, our data and analyses provide a foundation for interpreting marine RNA virus diversity and evolution.

Metagenomics has altered our understanding of microbial diversity and ecology. This includes its applications to viruses in marine environments that have demonstrated their enormous diversity. Within these are RNA viruses, many of which share genetic features with members of the order Picornavirales; yet, very few of these have been taxonomically classified. The only recognized family of marine RNA viruses is the Marnaviridae, which was founded based on discovery and characterization of the species Heterosigma akashiwo RNA virus. Two additional genera of marine RNA viruses, Labyrnavirus (one species) and Bacillarnavirus (three species), were subsequently defined within the order Picornavirales but not assigned to a family. We have defined a sequence-based framework for taxonomic classification of twenty marine RNA viruses into the family Marnaviridae. Using RNA-dependent RNA polymerase (RdRp) phylogeny and distance-based analyses, we assigned the genera Labyrnavirus and Bacillarnavirus to the family Marnaviridae and created four additional genera in the family: Locarnavirus (four species), Kusarnavirus (one species), Salisharnavirus (four species) and Sogarnavirus (six species). We used pairwise capsid protein comparisons to delineate species within families, with 75 per cent identity as the species demarcation threshold. The family displays high sequence diversities and Jukes–Cantor distances for both the RdRp and capsid genes, suggesting that the classified viruses are not representative of all of the virus diversity within the family and that there are many more extant taxa. Our proposed taxonomic framework provides a sound classification system for this group of viruses that will have broadly applicable principles for other viral groups. It is based on sequence data alone and provides a robust taxonomic framework to include viruses discovered via metagenomic studies, thereby greatly expanding the realm of viruses subject to taxonomic classification.

Charophyte algae, not chlorophyte algae, are the ancestors of ‘higher plants’; hence, viruses infecting charophytes may be related to those that first infected higher plants. Streamwaters from British Columbia, Canada, yielded single-stranded RNA metagenomes of Charavirus canadensis (CV-Can), that are similar in genomic architecture, length (9593 nt), nucleotide identity (63.4%), and encoded amino-acid sequence identity (53.0%) to those of Charavirus australis (CV-Aus). The sequences of their RNA-dependent RNA-polymerases (RdRp) resemble those found in benyviruses, their helicases those of hepaciviruses and hepegiviruses, and their coat-proteins (CP) those of tobamoviruses; all from the alphavirus/flavivirus branch of the ‘global RNA virome’. The 5’-terminus of the CV-Can genome, but not that of CV-Aus, is complete and encodes a methyltransferase domain. Comparisons of CP sequences suggests that Canadian and Australian charaviruses diverged 29–46 million years ago (mya); whereas, the CPs of charaviruses and tobamoviruses last shared a common ancestor 212 mya, and the RdRps of charaviruses and benyviruses 396 mya. CV-Can is sporadically abundant in low-nutrient freshwater rivers in British Columbia, where Chara braunii, a close relative of C. australis, occurs, and which may be its natural host. Charaviruses, like their hosts, are ancient and widely distributed, and thus provide a window to the viromes of early eukaryotes and, even, Archaea.