Group) Belgian pig RVA strains and of all completely characterized pig RVAs from around the globe. In contrast to the large diversity of genotypes found for the outer capsid proteins VP4 and VP7, a relatively conserved genotype constellation (I5-R1-C1-M1-A8-N1-T7-E1-H1) was found for the other 9 genes in most pig RVA strains. VP1, VP2, VP3, NSP2, NSP4, and NSP5 genes of porcine RVAs belonged to genotype 1, which is shared with human Wa-like RVAs. However, for most of these gene segments, pig strains clustered distantly from human Wa-like RVAs, indicating that viruses from both species have entered different evolutionary paths. However, VP1, VP2, and NSP3 genes of some archival human strains were moderately related to pig strains. Phylogenetic analysis of the VP6, NSP1, and NSP3 genes, as well as amino acid analysis of the antigenic regions of VP7, further confirmed this evolutionary segregation. The present results also indicate that the species barrier is less strict for pig P[6] strains but that chances for successful spread of these strains in the human population are hampered by the better adaptation of pig RVAs to pig enterocytes. However, future surveillance of pig and human RVA strains is warranted. IMPORTANCERotaviruses are an important cause of diarrhea in many species, including pigs and humans. Our understanding of the evolutionary relationship between rotaviruses from both species is limited by the lack of genomic data on pig strains. In this study, recent and ancient Belgian pig rotavirus isolates were sequenced, and their evolutionary relationship with human Wa-like strains was investigated. Our data show that Wa-like human and pig strains have entered different evolutionary paths. Our data indicate that pig P[6] strains form the most considerable risk for interspecies transmission to humans. However, efficient spread of pig strains in the human population is most likely hampered by the adaptation of some crucial viral proteins to the cellular machinery of pig enterocytes. These data allow a better understanding of the risk for direct interspecies transmission events and the emergence of pig rotaviruses or pig-human reassortants in the human population. E nteric diseases in pigs are mostly encountered during two critical time points: the suckling period and after weaning. Several pathogens are frequently involved in the pathogenesis of piglet diarrhea, including rotavirus, porcine epidemic diarrhea virus, transmissible gastroenteritis virus, Escherichia coli, Clostridium perfringens, Salmonella spp., Brachyspira spp., and Isospora suis. Furthermore, coinfections between different rotaviruses and other pathogens can be found frequently in diarrheic pigs (1).Rotaviruses are a major cause of diarrhea in many species, including pigs and humans. Five official rotavirus species (A to E) and 3 tentative species (F to H) have been established based on nucleotide similarities of the VP6-encoding genes (2). Species A, B, and C are frequently isolated from feces of diarrheic and nondiarrheic pigs, wh...

Edited by Phyllis I. Hanson Coronavirus M proteins represent the major protein component of the viral envelope. They play an essential role during viral assembly by interacting with all of the other structural proteins. Coronaviruses bud into the endoplasmic reticulum (ER)-Golgi intermediate compartment (ERGIC), but the mechanisms by which M proteins are transported from their site of synthesis, the ER, to the budding site remain poorly understood. Here, we investigated the intracellular trafficking of the Middle East respiratory syndrome coronavirus (MERS-CoV) M protein. Subcellular localization analyses revealed that the MERS-CoV M protein is retained intracellularly in the trans-Golgi network (TGN), and we identified two motifs in the distal part of the C-terminal domain as being important for this specific localization. We identified the first motif as a functional diacidic DxE ER export signal, because substituting Asp-211 and Glu-213 with alanine induced retention of the MERS-CoV M in the ER. The second motif, 199 KxGxYR 204 , was responsible for retaining the M protein in the TGN. Substitution of this motif resulted in MERS-CoV M leakage toward the plasma membrane. We further confirmed the role of 199 KxGxYR 204 as a TGN retention signal by using chimeras between MERS-CoV M and the M protein of infectious bronchitis virus (IBV). Our results indicated that the C-terminal domains of both proteins determine their specific localization, namely TGN and ERGIC/cis-Golgi for MERS-M and IBV-M, respectively. Our findings indicate that MERS-CoV M protein localizes to the TGN because of the combined presence of an ER export signal and a TGN retention motif.

ARS-CoV-2 is the etiologic agent of the coronavirus disease 2019 (COVID-19) pandemic. SARS-CoV-2 is the third highly pathogenic coronavirus to cross the species barrier in the 21st century after SARS-CoV-1 in 2002-2003 (refs. 1-3 ) and MERS-CoV in 2012 (ref. 4 ). Four additional HCoVs (HCoV-229E, HCoV-NL63, HCoV-OC43 and HCoV-HKU1) are known to circulate seasonally in humans, contributing to approximately one-third of common cold infections 5 . Like SARS-CoV-1 and HCoV-NL63, SARS-CoV-2 entry into target cells is mediated by the angiotensin-converting enzyme 2 (ACE2) receptor [6][7][8][9][10] . The cellular serine protease transmembrane protease serine 2 (TMPRSS2) is used by both SARS-CoV-1 and SARS-CoV-2 for Spike protein priming at the plasma membrane 6,11 . Cathepsins are also involved in SARS-CoV spike protein cleavage and fusion peptide exposure upon entry via an endocytic route, in the absence of TMPRSS2 (refs. [12][13][14][15] ).Several whole-genome KO CRISPR screens for the identification of coronavirus regulators have been reported [16][17][18][19][20][21] . These screens used naturally permissive simian Vero E6 cells of kidney origin 20 ; human Huh7 cells (or derivatives) of liver origin (ectopically expressing ACE2 and TMPRSS2, or not) 16,18,19 ; and A549 cells of lung origin, ectopically expressing ACE2 17,21 . Here, we conducted genome-wide, loss-of-function CRISPR KO screens and gain-of-function CRISPRa screens in several cell lines, including physiologically relevant human Calu-3 cells and Caco-2 cells, of lung and colorectal adenocarcinoma origin, respectively, followed by secondary screens in these cell lines and in Huh7.5.1 and A549 cells. Well-known SARS-CoV-2 host-dependency factors were identified among top hits, such as ACE2 and either TMPRSS2 or cathepsin L (depending on the cell type). We characterized the mechanism of action of the top hits and assessed their effect on other coronaviruses and influenza A orthomyxovirus. Altogether, this study provides insights into the coronavirus life cycle by identifying host factors that modulate replication and might lead to pan-coronavirus strategies for host-directed therapies. ResultsMeta-analysis of CRISPR KO screens highlights the importance of multiple models. Vero E6 cells present high levels of cytopathic effects (CPEs) upon SARS-CoV-2 replication, making them ideal to perform whole-genome CRISPR screens for host factor identification. A Chlorocebus sabaeus single-guide RNA (sgRNA) library was previously successfully used to identify host factors regulating SARS-CoV-2 (isolate USA-WA1/2020) replication 20 . Therefore, we initially repeated whole-genome CRISPR KO screens in Vero E6 cells using the SARS-CoV-2 isolate BetaCoV/France/ IDF0372/2020 (Fig. 1a). Importantly, ACE2 was a top hit (Fig. 1b