Syndecan-4 Is a Key Facilitator of the SARS-CoV-2 Delta Variant’s Superior Transmission

Emerging SARS-CoV-2 variants pose threats to vaccination campaigns against COVID-19. Being more transmissible than the original virus, the SARS-CoV-2 B.1.617 lineage, named the Delta variant, swept through the world in 2021. The mutations in the Delta’s spike protein shift the protein towards a net positive electrostatic potential. To understand the key molecular drivers of the Delta infection, we investigate the cellular uptake of the Delta spike protein and Delta spike-bearing SARS-CoV-2 pseudoviruses. Specific in vitro modification of ACE2 and syndecan expression enabled us to demonstrate that syndecan-4, the syndecan isoform abundant in the lung, enhances the transmission of the Delta variant by attaching its mutated spike glycoprotein and facilitating its cellular entry. Compared to the wild-type spike, the Delta one shows a higher affinity towards heparan sulfate proteoglycans than towards ACE2. In addition to attachment to the polyanionic heparan sulfate chains, the Delta spike’s molecular interactions with syndecan-4 also involve syndecan-4’s cell-binding domain that mediates cell-to-cell adhesion. Regardless of the complexity of these interactions, exogenously added heparin blocks Delta’s cellular entry as efficiently as syndecan-4 knockdown. Therefore, a profound understanding of the molecular mechanisms underlying Delta infections enables the development of molecularly targeted yet simple strategies to reduce the Delta variant’s spread.

The dengue virus non-structural protein 1 (NS1) uses the scavenger receptor B1 as a cell receptor in cultured cells

The dengue virus NS1 is a multifunctional protein that forms part of replication complexes. NS1 is also secreted, as a hexamer, to the extracellular milieu. Circulating NS1 has been associated with dengue pathogenesis by several mechanisms. Cell binding and internalization of soluble NS1 result in endothelial hyperpermeability and in the downregulation of the innate immune response. In this work, we report that the HDL scavenger receptor B1 (SRB1) in human hepatic cells and a scavenger receptor B1-like in mosquito C6/36 cells act as cell surface binding receptors for dengue virus NS1. The presence of the SRB1 on the plasma membrane of C6/36 cells, as well as in Huh7 cells, was demonstrated by confocal microscopy. The internalization of NS1 can be efficiently blocked by anti-SRB1 antibodies and previous incubation of the cells with HDL significantly reduces NS1 internalization. Significant reduction in NS1 internalization was observed in C6/36 cells transfected with siRNAs specific for SRB1. In addition, the transient expression of SRB1 in Vero cells, which lacks the receptor, allows NS1 internalization in these cells. Direct interaction between soluble NS1 and the SRB1 in Huh7 and C6/36 cells was demonstrated in situ by proximity ligation assays and in vitro by surface plasmon resonance. Finally, results are presented indicating that the SRB1 also acts as a cell receptor for Zika virus NS1. These results demonstrate that dengue virus NS1, a bona fide lipoprotein, usurps the HDL receptor for cell entry and offers explanations for the altered serum lipoprotein homeostasis observed in dengue patients. Importance Dengue is the most common viral disease transmitted to humans by mosquitoes. The dengue virus NS1 is a multifunctional glycoprotein necessary for viral replication. NS1 is also secreted as a hexameric lipoprotein and circulates in high concentrations in the sera of patients. Circulating NS1 has been associated with dengue pathogenesis by several mechanisms, including favoring of virus replication in hepatocytes and dendritic cells and disruption of the endothelial glycocalyx leading to hyperpermeability. Those last actions require NS1 internalization. Here, we identify the scavenger cell receptor B1, as the cell-binding receptor for dengue and Zika virus NS1, in cultured liver and in mosquito cells. The results indicate that flavivirus NS1, a bona fide lipoprotein, usurps the human HDL receptor and may offer explanations for the alterations in serum lipoprotein homeostasis observed in dengue patients.

Crystal Structures of Bat and Human Coronavirus ORF8 Protein Ig-Like Domain Provide Insights Into the Diversity of Immune Responses

ORF8 is a viral immunoglobulin-like (Ig-like) domain protein encoded by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA genome. It tends to evolve rapidly and interfere with immune responses. However, the structural characteristics of various coronavirus ORF8 proteins and their subsequent effects on biological functions remain unclear. Herein, we determined the crystal structures of SARS-CoV-2 ORF8 (S84) (one of the epidemic isoforms) and the bat coronavirus RaTG13 ORF8 variant at 1.62 Å and 1.76 Å resolution, respectively. Comparison of these ORF8 proteins demonstrates that the 62-77 residues in Ig-like domain of coronavirus ORF8 adopt different conformations. Combined with mutagenesis assays, the residue Cys20 of ORF8 is responsible for forming the covalent disulfide-linked dimer in crystal packing and in vitro biochemical conditions. Furthermore, immune cell-binding assays indicate that various ORF8 (SARS-CoV-2 ORF8 (L84), ORF8 (S84), and RaTG13 ORF8) proteins have different interaction capabilities with human CD14+ monocytes in human peripheral blood. These results provide new insights into the specific characteristics of various coronavirus ORF8 and suggest that ORF8 variants may influence disease-related immune responses.

SARS-CoV-2 uses metabotropic glutamate receptor subtype 2 as an internalization factor to infect cells

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) uses angiotensin-converting enzyme 2 (ACE2) as a binding receptor to enter cells via clathrin-mediated endocytosis (CME). However, receptors involved in other steps of SARS-CoV-2 infection remain largely unknown. Here, we found that metabotropic glutamate receptor subtype 2 (mGluR2) is an internalization factor for SARS-CoV-2. Our results show that mGluR2 directly interacts with the SARS-CoV-2 spike protein and that knockdown of mGluR2 decreases internalization of SARS-CoV-2 but not cell binding. Further, mGluR2 is uncovered to cooperate with ACE2 to facilitate SARS-CoV-2 internalization through CME and mGluR2 knockout in mice abolished SARS-CoV-2 infection in the nasal turbinates and significantly reduced viral infection in the lungs. Notably, mGluR2 is also important for SARS-CoV spike protein- and Middle East respiratory syndrome coronavirus spike protein-mediated internalization. Thus, our study identifies a novel internalization factor used by SARS-CoV-2 and opens a new door for antiviral development against coronavirus infection.

Cell Entry of Animal Coronaviruses

Coronaviruses (CoVs) are a group of enveloped positive-sense RNA viruses and can cause deadly diseases in animals and humans. Cell entry is the first and essential step of successful virus infection and can be divided into two ongoing steps: cell binding and membrane fusion. Over the past two decades, stimulated by the global outbreak of SARS-CoV and pandemic of SARS-CoV-2, numerous efforts have been made in the CoV research. As a result, significant progress has been achieved in our understanding of the cell entry process. Here, we review the current knowledge of this essential process, including the viral and host components involved in cell binding and membrane fusion, molecular mechanisms of their interactions, and the sites of virus entry. We highlight the recent findings of host restriction factors that inhibit CoVs entry. This knowledge not only enhances our understanding of the cell entry process, pathogenesis, tissue tropism, host range, and interspecies-transmission of CoVs but also provides a theoretical basis to design effective preventive and therapeutic strategies to control CoVs infection.

The Revolving Door of Adenovirus Cell Entry: Not All Pathways Are Equal

Adenoviruses represent exceptional candidates for wide-ranging therapeutic applications, from vectors for gene therapy to oncolytics for cancer treatments. The first ever commercial gene therapy medicine was based on a recombinant adenovirus vector, while most recently, adenoviral vectors have proven critical as vaccine platforms in effectively controlling the global coronavirus pandemic. Here, we discuss factors involved in adenovirus cell binding, entry, and trafficking; how they influence efficiency of adenovirus-based vectors; and how they can be manipulated to enhance efficacy of genetically modified adenoviral variants. We focus particularly on endocytosis and how different adenovirus serotypes employ different endocytic pathways to gain cell entry, and thus, have different intracellular trafficking pathways that subsequently trigger different host antiviral responses. In the context of gene therapy, the final goal of the adenovirus vector is to efficiently deliver therapeutic transgenes into the target cell nucleus, thus allowing its functional expression. Aberrant or inefficient endocytosis can impede this goal, therefore, it should be considered when designing and constructing adenovirus-based vectors.

PET/CT imaging of head-and-neck and pancreatic cancer in humans by targeting the “Cancer Integrin” αvβ6 with Ga-68-Trivehexin

Purpose To develop a new probe for the αvβ6-integrin and assess its potential for PET imaging of carcinomas. Methods Ga-68-Trivehexin was synthesized by trimerization of the optimized αvβ6-integrin selective cyclic nonapeptide Tyr2 (sequence: c[YRGDLAYp(NMe)K]) on the TRAP chelator core, followed by automated labeling with Ga-68. The tracer was characterized by ELISA for activities towards integrin subtypes αvβ6, αvβ8, αvβ3, and α5β1, as well as by cell binding assays on H2009 (αvβ6-positive) and MDA-MB-231 (αvβ6-negative) cells. SCID-mice bearing subcutaneous xenografts of the same cell lines were used for dynamic (90 min) and static (75 min p.i.) µPET imaging, as well as for biodistribution (90 min p.i.). Structure–activity-relationships were established by comparison with the predecessor compound Ga-68-TRAP(AvB6)3. Ga-68-Trivehexin was tested for in-human PET/CT imaging of HNSCC, parotideal adenocarcinoma, and metastatic PDAC. Results Ga-68-Trivehexin showed a high αvβ6-integrin affinity (IC50 = 0.047 nM), selectivity over other subtypes (IC50-based factors: αvβ8, 131; αvβ3, 57; α5β1, 468), blockable uptake in H2009 cells, and negligible uptake in MDA-MB-231 cells. Biodistribution and preclinical PET imaging confirmed a high target-specific uptake in tumor and a low non-specific uptake in other organs and tissues except the excretory organs (kidneys and urinary bladder). Preclinical PET corresponded well to in-human results, showing high and persistent uptake in metastatic PDAC and HNSCC (SUVmax = 10–13) as well as in kidneys/urine. Ga-68-Trivehexin enabled PET/CT imaging of small PDAC metastases and showed high uptake in HNSCC but not in tumor-associated inflammation. Conclusions Ga-68-Trivehexin is a valuable probe for imaging of αvβ6-integrin expression in human cancers.