The Middle East Respiratory Syndrome Coronavirus (MERS-CoV) causes severe and fatal acute respiratory disease in humans. High fatality rates and continued infectiousness remain a pressing concern for global health preparedness. Antibodies targeted at the receptor-binding domain (RBD) are major countermeasures against human viral infection. Here, we report four potent nanobodies against MERS-CoV, which are isolated from alpaca, and especially the potency of Nb14 is highest in the pseudotyped virus assay. Structural studies show that Nb14 framework regions (FRs) are mainly involved in interactions targeting a novel epitope, which is entirely distinct from all previously reported antibodies, and disrupt the protein-carbohydrate interaction between residue W535 of RBD and hDPP4 N229-linked carbohydrate moiety (hDPP4-N229-glycan). Different from Nb14, Nb9 targets the cryptic face of RBD, which is distinctive from the hDPP4 binding site and the Nb14 epitope, and it induces the β5-β6 loop to inflect towards a shallow groove of the RBD and dampens the accommodation of a short helix of hDPP4. The particularly striking epitopes endow the two Nbs administrate synergistically in the pseudotyped MERS-CoV assays. These results not only character unprecedented epitopes for antibody recognition but also provide promising agents for prophylaxis and therapy of MERS-CoV infection.

Eastern Equine Encephalitis virus (EEEV) is an alphavirus that can cause severe diseases in infected humans. The very low-density lipoprotein receptor (VLDLR) was recently identified as a receptor of EEEV. Herein, we performed cryo-electron microscopy structural and biochemistry studies on the specific interactions between EEEV and VLDLR. Our results show that VLDLR binds EEEV at three different sites A, B and C through its membrane-distal LDLR class A (LA) repeats. Site A is located in the cleft in between the E1-E2 heterodimers. Site B is located near the connecting β ribbon of E2 and is in proximity to site A, while site C is on the domain B of E2. The binding of VLDLR LAs to EEEV is in complex modes, including the LA1-2 and LA3-5 mediated two major modes. Disruption of the LA1-2 mediated binding significantly affect the cell attachment of EEEV. However, the mutation W132G of VLDLR impairs the binding of LA3, drives the switch of the binding modes, and significantly enhances the attachment of EEEV to the cell. The W132G variant of VLDLR could be identified in human genome and SNP sequences, implying that people with similar mutations in VLDLR may be highly susceptible to EEEV infection.

The T cell immunoglobulin and ITAM domain (TIGIT) is a recently discovered synergistic co-suppressor molecule that plays an important role in immune response and tumor immune escape in the context of cancer. Importantly, CD155 acts as a receptor for TIGIT, and CD155 signaling to immune cells is mediated through interactions with the co-stimulatory immune receptor CD226 (DNAM-1) and the inhibitory checkpoint receptors TIGIT and CD96. Aspirin (ASA) has been shown to reduce the growth and survival of colorectal cancer (CRC) cells, but the immunological mechanisms involved have not been sufficiently elucidated. In the present study the effects of aspirin on CRC in mice and on Jurkat cells were investigated. Aspirin may suppress the expression of TIGIT on T cells and Regulatory T cells (Tregs) and inhibit T cell viability, and therefore induce tumor cell apoptosis. TIGIT is expressed at higher levels on infiltrating lymphocytes within CRC tumor tissue than adjacent. Further, aspirin could inhibit Jurkat cell proliferation and induce apoptosis via downregulation of TIGIT expression and the anti-apoptosis B cell lymphoma 2 (BCL2) protein and upregulation of BCL2-associated X protein (BAX) expression. The present study suggests that aspirin can inhibit specific aspects of T cell function by reducing interleukin-10 and transforming growth factor-β1 secretion via the TIGIT-BCL2-BAX signaling pathway, resulting in improved effector T cell function that inhibits tumor progression.

Arteriviruses infect a variety of mammalian hosts, but the receptors used by these viruses to enter cells are poorly understood. We identified the neonatal Fc receptor (FcRn) as an important pro-viral host factor via comparative genome-wide CRISPR-knockout screens with multiple arteriviruses. Using a panel of cell lines and divergent arteriviruses, we demonstrate that FcRn is required for the entry step of arterivirus infection and serves as a molecular barrier to arterivirus cross-species infection. We also show that FcRn synergizes with another known arterivirus entry factor, CD163, to mediate arterivirus entry. Overexpression of FcRn and CD163 sensitizes non-permissive cells to infection and enables the culture of fastidious arteriviruses. Treatment of multiple cell lines with a pre-clinical anti-FcRn monoclonal antibody blocked infection and rescued cells from arterivirus-induced death. Altogether, this study identifies FcRn as a novel pan-arterivirus receptor, with implications for arterivirus emergence, cross-species infection, and host-directed pan-arterivirus countermeasure development.

Eastern equine encephalitis virus (EEEV) is the most virulent alphavirus that infects humans, and many survivors develop neurological sequelae, including paralysis and intellectual disability. Alphavirus spike proteins comprise trimers of heterodimers of glycoproteins E2 and E1 that mediate binding to cellular receptors and fusion of virus and host cell membranes during entry. We recently identified very-low density lipoprotein receptor (VLDLR) and apolipoprotein E receptor 2 (ApoER2) as cellular receptors for EEEV and a distantly related alphavirus, Semliki Forest virus (SFV). Here, we use single-particle cryo-electron microscopy (cryo-EM) to determine structures of the EEEV and SFV spike glycoproteins bound to the VLDLR ligand-binding domain and found that EEEV and SFV interact with the same cellular receptor through divergent binding modes. Our studies suggest that the ability of LDLR-related proteins to interact with viral spike proteins through very small footprints with flexible binding modes results in a low evolutionary barrier to the acquisition of LDLR-related proteins as cellular receptors for diverse sets of viruses.

The animal origin of SARS-CoV-2 remains elusive, lacking a plausible evolutionary narrative that may account for its emergence. Its spike protein resembles certain segments of BANAL-236 and RaTG13, two bat coronaviruses considered possible progenitors of SARS-CoV-2. Additionally, its spike contains a furin motif, a common feature of rodent coronaviruses. To explore the possible involvement of rodents in the emergence of SARS-CoV-2 spike, we examined the crystal structures of the spike receptor-binding domains (RBDs) of BANAL-236 and RaTG13 each complexed with mouse receptor ACE2. Both RBDs have residues at positions 493 and 498 that align well with two virus-binding hotspots on mouse ACE2. Our biochemical evidence supports that both BANAL-236 and RaTG13 spikes can use mouse ACE2 as their entry receptor. These findings point to a scenario in which these bat coronaviruses may have coinfected rodents, leading to a recombination of their spike genes and a subsequent acquisition of a furin motif in rodents, culminating in the emergence of SARS-CoV-2.

Sialoglycan-binding enveloped viruses often possess receptor-destroying activity to avoid being immobilized by non-functional decoy receptors. Sialic acid (Sia)-binding paramyxoviruses contain a hemagglutinin-neuraminidase (HN) protein that possesses both Sia-binding and -cleavage activities. The multivalent, dynamic receptor interactions of paramyxovirus particles provide virion motility and are a key determinant of host tropism. However, such multivalent interactions have not been exhaustively analyzed, because such studies are complicated by the low affinity of the individual interactions and the requirement of high titer virus stocks. Moreover, the dynamics of multivalent particle-receptor interactions are difficult to predict from Michaelis-Menten enzyme kinetics. Therefore, we here developed Ni-NTA nanoparticles that multivalently display recombinant soluble HN tetramers via their His tags (HN-NPs). Applying this HN-NP platform to Newcastle disease virus (NDV), we investigated using biolayer interferometry (BLI) the role of important HN residues in receptor-interactions and analyzed long-range effects between the catalytic site and the second Sia binding site (2SBS). The HN-NP system was also applicable to other paramyxoviruses. Comparative analysis of HN-NPs revealed and confirmed differences in dynamic receptor-interactions between type 1 human and murine parainfluenza viruses as well as of lab-adapted and clinical isolates of human parainfluenza virus type 3, which are likely to contribute to differences in tropism of these viruses. We propose this novel platform to be applicable to elucidate the dynamics of multivalent-receptor interactions important for host tropism and pathogenesis, particularly for difficult to grow sialoglycan-binding (paramyxo)viruses.

Western equine encephalitis virus (WEEV) is an arthropod-borne virus (arbovirus) that frequently caused major outbreaks of encephalitis in humans and horses in the early twentieth century, but the frequency of outbreaks has since decreased markedly, and strains of this alphavirus isolated in the past two decades are less virulent in mammals than strains isolated in the 1930s and 1940s1-3. The basis for this phenotypic change in WEEV strains and coincident decrease in epizootic activity (known as viral submergence3) is unclear, as is the possibility of re-emergence of highly virulent strains. Here we identify protocadherin 10 (PCDH10) as a cellular receptor for WEEV. We show that multiple highly virulent ancestral WEEV strains isolated in the 1930s and 1940s, in addition to binding human PCDH10, could also bind very low-density lipoprotein receptor (VLDLR) and apolipoprotein E receptor 2 (ApoER2), which are recognized by another encephalitic alphavirus as receptors4. However, whereas most of the WEEV strains that we examined bind to PCDH10, a contemporary strain has lost the ability to recognize mammalian PCDH10 while retaining the ability to bind avian receptors, suggesting WEEV adaptation to a main reservoir host during enzootic circulation. PCDH10 supports WEEV E2-E1 glycoprotein-mediated infection of primary mouse cortical neurons, and administration of a soluble form of PCDH10 protects mice from lethal WEEV challenge. Our results have implications for the development of medical countermeasures and for risk assessment for re-emerging WEEV strains.

Canine distemper virus (CDV) belongs to morbillivirus, including measles virus (MeV) and rinderpest virus, which causes serious immunological and neurological disorders in carnivores, including dogs and rhesus monkeys, as recently reported, but their vaccines are highly effective. The attachment glycoprotein hemagglutinin (CDV-H) at the CDV surface utilizes signaling lymphocyte activation molecule (SLAM) and Nectin-4 (also called poliovirus-receptor-like-4; PVRL4) as entry receptors. Although fusion models have been proposed, the molecular mechanism of morbillivirus fusion entry is poorly understood. Here, we determined the crystal structure of the globular head domain of CDV-H vaccine strain at 3.2 Å resolution, revealing that CDV-H exhibits a highly tilted homodimeric form with a six-bladed β-propeller fold. While the predicted Nectin-4-binding site is well conserved with that of MeV-H, that of SLAM is similar but partially different, which is expected to contribute to host specificity. Five N-linked sugars covered a broad area of the CDV-H surface to expose receptor-binding sites only, supporting the effective production of neutralizing antibodies. These features are common to MeV-H, although the glycosylation sites are completely different. Furthermore, real-time observation using high-speed atomic force microscopy revealed highly mobile features of the CDV-H dimeric head via the connector region. These results suggest that sugar-shielded tilted homodimeric structure and dynamic conformational changes are common characteristics of morbilliviruses and ensure effective fusion entry and vaccination.

Human noroviruses (HuNoVs) are the leading etiological agent causing the worldwide outbreaks of acute epidemic non-bacterial gastroenteritis. Histo-blood group antigens (HBGAs) are commonly acknowledged as cellular receptors or co-receptors for HuNoVs. However, certain genotypes of HuNoVs cannot bind with any HBGAs, suggesting potential additional co-factors and attachment receptors have not been identified yet. In addition, food items, such as oysters and lettuce, play an important role in the transmission of HuNoVs. In the past decade, a couple of attachment factors other than HBGAs have been identified and analyzed from foods and microbiomes. Attachment factors exhibit potential as inhibitors of viral binding to receptors on host cells. Therefore, it is imperative to further characterize the attachment factors for HuNoVs present in foods to effectively control the spread of HuNoVs within the food chain. This review summarizes the potential attachment factors/receptors of HuNoVs in humans, foods, and microbiome.