Search

Mammalian receptor-mediated endocytosis (RME) often involves at least one of three isoforms of the large GTPase dynamin (Dyn). Dyn pinches-off vesicles at the plasma membrane and mediates uptake of many viruses, although some viruses directly penetrate the plasma membrane. RME is classically interrogated by genetic and pharmacological interference, but this has been hampered by undesired effects. Here we studied virus entry in conditional genetic knock-out (KO) mouse embryonic fibroblasts lacking expression of all three dynamin isoforms (Dyn-KO-MEFs). The small canine parvovirus known to use a single receptor, transferrin receptor, strictly depended on dynamin. Larger viruses or viruses known to use multiple receptors, including alphaviruses, influenza, vesicular stomatitis, bunya, adeno, vaccinia, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and rhinoviruses infected Dyn-KO-MEFs, albeit at higher dosage than wild-type MEFs. In absence of the transmembrane protease serine subtype 2 (TMPRSS2), which normally activates the SARS-CoV-2 spike protein for plasma membrane fusion, SARS-CoV-2 infected angiotensin-converting enzyme 2 (ACE2)-expressing MEFs predominantly through dynamin- and actin-dependent endocytosis. In presence of TMPRSS2 the ancestral Wuhan-strain bypassed both dynamin-dependent and -independent endocytosis, and was less sensitive to endosome maturation inhibitors than the Omicron B1 and XBB variants, supporting the notion that the Omicron variants do not efficiently use TMPRSS2. Collectively, our study suggests that dynamin function at endocytic pits can be essential for infection with single-receptor viruses, while it is not essential but increases uptake and infection efficiency of multi-receptor viruses that otherwise rely on a functional actin network for infection. Author summary: To initiate their infection cycle, most viruses first need to enter their target cells, a process called endocytosis. In mammalian cells, endocytosis often involves a class of proteins called dynamins. While numerous viruses, including SARS-CoV-2, efficiently infect cells by dynamin-mediated endocytosis we found that in the absence of these proteins an alternative cell entry mechanism exists, allowing multiple pathogenic human viruses to enter and infect cells. Unlike the dynamin-mediated infection, the efficient internalization of viral particles via dynamin-independent endocytosis seems to always require functional actin fibers, which are structural components of the cell contractile cytoskeleton. Thus, multiple viruses can infect their target cells using at last two entry mechanisms, inhibiting both may provide effective antiviral therapies. [ABSTRACT FROM AUTHOR]

Respiratory infections, such as the flu and common cold, are a major global health issue, causing a significant number of illnesses and deaths worldwide. Recent research has shown that respiratory coinfections, where multiple viruses infect the same person, are more common than previously thought. These coinfections can have both negative and positive interactions between the viruses, affecting their infectivity and replication. Understanding these interactions is crucial for managing and treating respiratory infections. However, there are still many challenges in studying these coinfections, including the need for better experimental models and the complexities of analyzing epidemiological data. [Extracted from the article]

Pairwise compatibility between virus and host proteins can dictate the outcome of infection. During transmission, both inter- and intraspecies variabilities in receptor protein sequences can impact cell susceptibility. Many viruses possess mutable viral entry proteins and the patterns of host compatibility can shift as the viral protein sequence changes. This combinatorial sequence space between virus and host is poorly understood, as traditional experimental approaches lack the throughput to simultaneously test all possible combinations of protein sequences. Here, we created a pseudotyped virus infection assay where a multiplexed target-cell library of host receptor variants can be assayed simultaneously using a DNA barcode sequencing readout. We applied this assay to test a panel of 30 ACE2 orthologs or human sequence mutants for infectability by the original SARS-CoV-2 spike protein or the Alpha, Beta, Gamma, Delta, and Omicron BA1 variant spikes. We compared these results to an analysis of the structural shifts that occurred for each variant spike's interface with human ACE2. Mutated residues were directly involved in the largest shifts, although there were also widespread indirect effects altering interface structure. The N501Y substitution in spike conferred a large structural shift for interaction with ACE2, which was partially recreated by indirect distal substitutions in Delta, which does not harbor N501Y. The structural shifts from N501Y greatly influenced the set of animal orthologs the variant spike was capable of interacting with. Out of the thirteen non-human orthologs, ten exhibited unique patterns of variant-specific compatibility, demonstrating that spike sequence changes during human transmission can toggle ACE2 compatibility and potential susceptibility of other animal species, and cumulatively increase overall compatibilities as new variants emerge. These experiments provide a blueprint for similar large-scale assessments of protein compatibility during entry by diverse viruses. This dataset demonstrates the complex compatibility relationships that occur between variable interacting host and virus proteins. Author summary: The proteins encoded by a viral pathogen must be compatible with the proteins encoded by the host organism to allow the virus to enter and take over their cells. As the SARS-CoV-2 pandemic progressed, various SARS-CoV-2 variants of concern emerged, each altering the sequence of their spike proteins. We developed a new high-throughput infection assay which allowed us to simultaneously test dozens of sequence variants of ACE2 for their ability to allow viral particles coated with the spike proteins from the SARS-CoV-2 variants of concern to enter the cell. We found that the SARS-CoV-2 variant spike proteins only slightly changed in the way they interacted with human ACE2 but exhibited more pronounced changes in their ability to interact with ACE2 versions from other species. The SARS-CoV-2 spike variants generated during the pandemic were collectively compatible with the majority of animal ACE2 sequence versions we tested. We speculate that similar molecular interplays may occur during cross-species transmission, where repeated transmission and escape from immune responses allows the virus to simultaneously explore new molecular compatibilities with different receptor protein versions, thus allowing spread to new species. [ABSTRACT FROM AUTHOR]

Since the COVID-19 outbreak, raccoon dogs have been suggested as a potential intermediary in transmitting SARS-CoV-2 to humans. To understand their role in the COVID-19 pandemic and the species barrier for SARS-CoV-2 transmission to humans, we analyzed how their ACE2 protein interacts with SARS-CoV-2 spike protein. Biochemical data showed that raccoon dog ACE2 is an effective receptor for SARS-CoV-2 spike protein, though not as effective as human ACE2. Structural comparisons highlighted differences in the virus-binding residues of raccoon dog ACE2 compared to human ACE2 (L24Q, Y34H, E38D, T82M, R353K), explaining their varied effectiveness as receptors for SARS-CoV-2. These variations contribute to the species barrier that exists between raccoon dogs and humans regarding SARS-CoV-2 transmission. Identifying these barriers can help assess the susceptibility of other mammals to SARS-CoV-2. Our research underscores the potential of raccoon dogs as SARS-CoV-2 carriers and identifies molecular barriers that affect the virus's ability to jump between species. Author summary: Raccoon dogs are thought to have been a crucial link in the chain that led to the spread of the COVID-19 virus to humans. The key to a coronavirus moving from one species to another is its spike protein's ability to bind to receptors on the cells of the new host. In our research, we investigated the interaction between the spike protein of the COVID-19 virus and the ACE2 receptor in raccoon dogs, using both structural and biochemical techniques. We found that the ACE2 receptor in raccoon dogs does indeed work as an effective receptor for the virus, but there are critical differences when compared to the human ACE2 receptor, which may act as barriers to the virus making the jump between raccoon dogs and humans. To our knowledge, this study is the first in-depth examination of these cross-species transmission barriers for the COVID-19 virus, shedding light on how other mammals might be impacted by the virus. Our findings indicate that raccoon dogs could potentially carry and pass the COVID-19 virus on to humans, providing foundational knowledge for how the virus can navigate between different species. [ABSTRACT FROM AUTHOR]

Angiotensin-converting enzyme 2 (ACE2), part of the renin-angiotensin system (RAS), serves as an entry point for SARS-CoV-2, leading to viral proliferation in permissive cell types. Using mouse lines in which the Ace2 locus has been humanized by syntenic replacement, we show that regulation of basal and interferon induced ACE2 expression, relative expression levels of different ACE2 transcripts, and sexual dimorphism in ACE2 expression are unique to each species, differ between tissues, and are determined by both intragenic and upstream promoter elements. Our results indicate that the higher levels of expression of ACE2 observed in the lungs of mice relative to humans may reflect the fact that the mouse promoter drives expression of ACE2 in populous airway club cells while the human promoter drives expression in alveolar type 2 (AT2) cells. In contrast to transgenic mice in which human ACE2 is expressed in ciliated cells under the control of the human FOXJ1 promoter, mice expressing ACE2 in club cells under the control of the endogenous Ace2 promoter show a robust immune response after infection with SARS-CoV-2, leading to rapid clearance of the virus. This supports a model in which differential expression of ACE2 determines which cell types in the lung are infected, and this in turn modulates the host response and outcome of COVID-19. Author summary: SARS-CoV-2, the virus responsible for COVID-19, infects the human respiratory tract by binding to angiotensin-converting enzyme 2 (ACE2), a protein on the outer surface of cells that is exposed to the air we inhale. Differences in the structure of ACE2 between mouse and man prevent the virus from infecting cells lining the airways of the mouse, limiting the usefulness of wildtype mice as a model system for studying COVID-19. To circumvent this problem, we have created two mouse lines in which the chromosomal segment that encodes the mouse ACE2 protein has been replaced with the equivalent segment of human DNA. In the first of these lines, expression of human ACE2 is regulated by the mouse upstream promoter, while in the second line it is regulated by the human upstream promoter. Using these mice, we show both qualitative and quantitative differences between ACE2 expression driven by the mouse and human promoters that are likely to impact disease progression in the two species. Mice expressing the full-length human ACE2 gene under the control of its own promoter should provide a useful model system for understanding the complex pathological processes and immune responses associated with COVID-19 in humans. [ABSTRACT FROM AUTHOR]

The binding of the SARS-CoV-2 spike to angiotensin-converting enzyme 2 (ACE2) promotes virus entry into the cell. Targeting this interaction represents a promising strategy to generate antivirals. By screening a phage-display library of biosynthetic protein sequences build on a rigid alpha-helicoidal HEAT-like scaffold (named αReps), we selected candidates recognizing the spike receptor binding domain (RBD). Two of them (F9 and C2) bind the RBD with affinities in the nM range, displaying neutralisation activity in vitro and recognizing distinct sites, F9 overlapping the ACE2 binding motif. The F9-C2 fusion protein and a trivalent αRep form (C2-foldon) display 0.1 nM affinities and EC50 of 8–18 nM for neutralization of SARS-CoV-2. In hamsters, F9-C2 instillation in the nasal cavity before or during infections effectively reduced the replication of a SARS-CoV-2 strain harbouring the D614G mutation in the nasal epithelium. Furthermore, F9-C2 and/or C2-foldon effectively neutralized SARS-CoV-2 variants (including delta and omicron variants) with EC50 values ranging from 13 to 32 nM. With their high stability and their high potency against SARS-CoV-2 variants, αReps provide a promising tool for SARS-CoV-2 therapeutics to target the nasal cavity and mitigate virus dissemination in the proximal environment. Author summary: The entry of SARS-CoV-2 in permissive cells is mediated by the binding of its spike to angiotensin-converting enzyme 2 (ACE2) on the cell surface. To select ligands able to block this interaction, we screened a library of phages encoding biosynthetic proteins (named αReps) for binding to its receptor binding domain (RBD). Two of them were able to bind the RBD with high affinity and block efficiently the virus entry in cultured cells. Assembled αReps through covalent or non-covalent linkages blocked virus entry at lower concentration than their precursors (with around 20-fold activity increase for a trimeric αRep). These αReps derivates neutralize efficiently SARS-CoV-2 β, γ, δ and Omicron virus variants. Instillation of an αRep dimer in the nasal cavity effectively reduced virus replication in the hamster model of SARS-CoV-2 and pathogenicity. [ABSTRACT FROM AUTHOR]

SARS-CoV and SARS-CoV-2 encode spike proteins that bind human ACE2 on the cell surface to enter target cells during infection. A small fraction of humans encode variants of ACE2, thus altering the biochemical properties at the protein interaction interface. These and other ACE2 coding mutants can reveal how the spike proteins of each virus may differentially engage the ACE2 protein surface during infection. We created an engineered HEK 293T cell line for facile stable transgenic modification, and expressed the major human ACE2 allele or 28 of its missense mutants, 24 of which are possible through single nucleotide changes from the human reference sequence. Infection with SARS-CoV or SARS-CoV-2 spike pseudotyped lentiviruses revealed that high ACE2 cell-surface expression could mask the effects of impaired binding during infection. Drastically reducing ACE2 cell surface expression revealed a range of infection efficiencies across the panel of mutants. Our infection results revealed a non-linear relationship between soluble SARS-CoV-2 RBD binding to ACE2 and pseudovirus infection, supporting a major role for binding avidity during entry. While ACE2 mutants D355N, R357A, and R357T abrogated entry by both SARS-CoV and SARS-CoV-2 spike proteins, the Y41A mutant inhibited SARS-CoV entry much more than SARS-CoV-2, suggesting differential utilization of the ACE2 side-chains within the largely overlapping interaction surfaces utilized by the two CoV spike proteins. These effects correlated well with cytopathic effects observed during SARS-CoV-2 replication in ACE2-mutant cells. The panel of ACE2 mutants also revealed altered ACE2 surface dependencies by the N501Y spike variant, including a near-complete utilization of the K353D ACE2 variant, despite decreased infection mediated by the parental SARS-CoV-2 spike. Our results clarify the relationship between ACE2 abundance, binding, and infection, for various SARS-like coronavirus spike proteins and their mutants, and inform our understanding for how changes to ACE2 sequence may correspond with different susceptibilities to infection. Author summary: SARS-like coronaviruses, such as SARS-CoV-2, use their spike proteins to bind a common surface on the human ACE2 protein to gain entry and subsequently infect cells. We used site-specific genomic integration and expression of WT ACE2 or its missense variants, many of them previously observed in human exomes, to determine how ACE2 sequence and abundance correspond to infectability by SARS-CoV or SARS-CoV-2. We found that reduced binding only partially corresponded to infection, and mainly only at lower ACE2 abundance levels. We observed some human ACE2 variants differentially affect SARS-CoV, SARS-CoV-2, or SARs-CoV-2 N501Y spike variant pseudovirus entry, showing that each viral spike binds ACE2 in a unique manner. Our results provide improved quantitative understanding for how ACE2 sequence and abundance correlate with infectivity, with implications for how natural human ACE2 variants, or variants observed in related species, may impact susceptibility to infection. These genetic tools can be repurposed to characterize future SARS-CoV-2 spike variants, or to better understand how receptor protein sequences correspond with entry by zoonotic viruses during cross-species transmission events. [ABSTRACT FROM AUTHOR]

Coronavirus interaction with its viral receptor is a primary genetic determinant of host range and tissue tropism. SARS-CoV-2 utilizes ACE2 as the receptor to enter host cell in a species-specific manner. We and others have previously shown that ACE2 orthologs from New World monkey, koala and mouse cannot interact with SARS-CoV-2 to mediate viral entry, and this defect can be restored by humanization of the restrictive residues in New World monkey ACE2. To better understand the genetic determinants behind the ability of ACE2 orthologs to support viral entry, we compared koala and mouse ACE2 sequences with that of human and identified the key residues in koala and mouse ACE2 that restrict viral receptor activity. Humanization of these critical residues rendered both koala and mouse ACE2 capable of binding the spike protein and facilitating viral entry. Our study shed more lights into the genetic determinants of ACE2 as the functional receptor of SARS-CoV-2, which facilitates our understanding of viral entry. Author summary: SARS-CoV-2 spike protein could bind cellular receptor ACE2 for cell entry in a species-specific manner. A diverse of mammalian ACE2 proteins could be used by SARS-CoV-2 for entry, but ACE2 proteins of koala or mouse cannot bind with viral spike protein. We compared the koala or mouse ACE2 with human ACE2, and found Thr at 31 position of koala ACE2 or His at 353 position of mouse ACE2 as the restrictive residue which limits its function as the viral receptor, respectively. Interestingly, koala or mouse ACE2 could gain the receptor function once the restrictive reside was replaced by human counterpart by genetic engineering. This study could facilitate our understanding of the genetic basis of ACE2 as the functional receptor of SARS-CoV-2, which could inform the animal model development. [ABSTRACT FROM AUTHOR]

The trimeric SARS coronavirus (SARS-CoV) surface spike (S) glycoprotein consisting of three S1-S2 heterodimers binds the cellular receptor angiotensin-converting enzyme 2 (ACE2) and mediates fusion of the viral and cellular membranes through a pre- to postfusion conformation transition. Here, we report the structure of the SARS-CoV S glycoprotein in complex with its host cell receptor ACE2 revealed by cryo-electron microscopy (cryo-EM). The complex structure shows that only one receptor-binding domain of the trimeric S glycoprotein binds ACE2 and adopts a protruding “up” conformation. In addition, we studied the structures of the SARS-CoV S glycoprotein and its complexes with ACE2 in different in vitro conditions, which may mimic different conformational states of the S glycoprotein during virus entry. Disassociation of the S1-ACE2 complex from some of the prefusion spikes was observed and characterized. We also characterized the rosette-like structures of the clustered SARS-CoV S2 trimers in the postfusion state observed on electron micrographs. Structural comparisons suggested that the SARS-CoV S glycoprotein retains a prefusion architecture after trypsin cleavage into the S1 and S2 subunits and acidic pH treatment. However, binding to the receptor opens up the receptor-binding domain of S1, which could promote the release of the S1-ACE2 complex and S1 monomers from the prefusion spike and trigger the pre- to postfusion conformational transition. [ABSTRACT FROM AUTHOR]