Variations in Cell Surface ACE2 Levels Alter Direct Binding of SARS-CoV-2 Spike Protein and Viral Infectivity: Implications for Measuring Spike Protein Interactions with Animal ACE2 Orthologs.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019 (COVID-19), the most severe pandemic in a century. The virus gains access to host cells when the viral spike protein (S-protein) binds to the host cell surface receptor angiotensin-converting enzyme 2 (ACE2). Studies have attempted to understand SARS-CoV-2 S-protein interactions with vertebrate orthologs of ACE2 by expressing ACE2 orthologs in mammalian cells and measuring viral infection or S-protein binding. Often, these cells only transiently express ACE2 proteins, and the levels of ACE2 at the cell surface are not quantified. Here, we describe a cell-based assay that uses stably transfected cells expressing ACE2 proteins in a bicistronic vector with an easy-to-quantify reporter protein, Thy1.1. We found that both the binding of the S-protein receptor-binding domain (RBD) and infection with a SARS-CoV-2 pseudovirus are proportional to the amount of human ACE2 expressed at the cell surface, which can be inferred by quantifying the level of Thy1.1. We also compared different ACE2 orthologs, which were expressed in stably transfected cells expressing equivalent levels of Thy1.1. When ranked for either viral infectivity or RBD binding, mouse ACE2 had a weak to undetectable affinity for S-protein, while human ACE2 had the highest level detected, and feline ACE2 had an intermediate phenotype. The generation of stably transfected cells whose ACE2 level can be normalized for cross-ortholog comparisons allows us to create a reusable cellular library useful for measuring emerging SARS-CoV-2 variants' abilities to potentially infect different animals. IMPORTANCE SARS-CoV-2 is a zoonotic virus responsible for the worst global pandemic in a century. An understanding of how the virus can infect other vertebrate species is important for controlling viral spread and understanding the natural history of the virus. Here, we describe a method to generate cells stably expressing different orthologs of ACE2, the receptor for SARS-CoV-2, on the surface of a human cell line. We find that both the binding of the viral spike protein receptor-binding domain (RBD) and infection of cells with a SARS-CoV-2 pseudovirus are proportional to the ACE2 levels at the cell surface. This method will allow the creation of a library of stably transfected cells expressing similar levels of different vertebrate ACE2 orthologs, which can be used repeatedly for identifying vertebrate species that may be susceptible to infection with SARS-CoV-2 and its many variants.

Variations in cell-surface ACE2 levels alter direct binding of SARS-CoV-2 Spike protein and viral infectivity: Implications for measuring Spike protein interactions with animal ACE2 orthologs.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of COVID-19, the most severe pandemic in a century. The virus gains access to host cells when the viral Spike protein (S-protein) binds to the host cell-surface receptor angiotensin-converting enzyme 2 (ACE2). Studies have attempted to understand SARS-CoV-2 S-protein interaction with vertebrate orthologs of ACE2 by expressing ACE2 orthologs in mammalian cells and measuring viral infection or S-protein binding. Often these cells only transiently express ACE2 proteins and levels of ACE2 at the cell surface are not quantified. Here, we describe a cell-based assay that uses stably transfected cells expressing ACE2 proteins in a bi-cistronic vector with an easy to quantify reporter protein to normalize ACE2 expression. We found that both binding of the S-protein receptor-binding domain (RBD) and infection with a SARS-CoV-2 pseudovirus is proportional to the amount of human ACE2 expressed at the cell surface, which can be inferred by quantifying the level of reporter protein, Thy1.1. We also compared different ACE2 orthologs which were expressed in stably transfected cells expressing equivalent levels of Thy1.1. When ranked for either viral infectivity or RBD binding, mouse ACE2 had a weak to undetectable affinity for S-protein while human ACE2 was the highest level detected and feline ACE2 had an intermediate phenotype. The generation of stably transfected cells whose ACE2 level can be normalized for cross-ortholog comparisons allows us to create a reusable cellular library useful for measuring emerging SARS-CoV-2 variant's ability to potentially infect different animals. IMPORTANCE: SARS-CoV-2 is a zoonotic virus responsible for the worst global pandemic in a century. An understanding of how the virus can infect other vertebrate species is important for controlling viral spread and understanding the natural history of the virus. Here we describe a method to generate cells stably expressing equivalent levels of different ACE2 orthologs, the receptor for SARS-CoV-2, on the surface of a human cell line. We find that both binding of the viral Spike protein receptor binding domain (RBD) and infection of cells with a SARS-CoV-2 pseudovirus are proportional to ACE2 levels at the cell surface. Adaptation of this method will allow for the creation of a library of stable transfected cells expressing equivalent levels of different vertebrate ACE2 orthologs which can be repeatedly used for identifying vertebrate species which may be susceptible to infection with SARS-CoV-2 and its many variants.

Targeting of HER/ErbB family proteins using broad spectrum Sec61 inhibitors coibamide A and apratoxin A.

Coibamide A is a potent cancer cell toxin and one of a select group of natural products that inhibit protein entry into the secretory pathway via a direct inhibition of the Sec61 protein translocon. Many Sec61 client proteins are clinically relevant drug targets once trafficked to their final destination in or outside the cell, however the use of Sec61 inhibitors to block early biosynthesis of specific proteins is at a pre-clinical stage. In the present study we evaluated the action of coibamide A against human epidermal growth factor receptor (HER, ErbB) proteins in representative breast and lung cancer cell types. HERs were selected for this study as they represent a family of Sec61 clients that is frequently dysregulated in human cancers, including coibamide-sensitive cell types. Although coibamide A inhibits biogenesis of a broad range of Sec61 substrate proteins in a presumed substrate-nonselective manner, endogenous HER3 (ErbB-3) and EGFR (ErbB-1) proteins were more sensitive to coibamide A, and the related Sec61 inhibitor apratoxin A, than HER2 (ErbB-2). Despite this rank order of sensitivity (HER3 > EGFR > HER2), Sec61-dependent inhibition by coibamide A was sufficient to decrease cell surface expression of HER2. We report that coibamide A- or apratoxin A-mediated block of HER3 entry into the secretory pathway is unlikely to be mediated by the HER3 signal peptide alone. HER3 (G11L/S15L), that is fully resistant to the highly substrate-selective cotransin analogue CT8, was more resistant than wild-type HER3 but only at low coibamide A (3 nM) concentrations; HER3 (G11L/S15L) expression was inhibited by higher concentrations of either natural product. Time- and concentration-dependent decreases in HER protein expression induced a commensurate reduction in AKT/MAPK signaling in breast and lung cancer cell types and loss in cell viability. Coibamide A potentiated the cytotoxic efficacy of small molecule kinase inhibitors lapatinib and erlotinib in breast and lung cancer cell types, respectively. These data indicate that natural product modulators of Sec61 function have value as chemical probes to interrogate HER/ErbB signaling in treatment-resistant human cancers.

Coibamide A Targets Sec61 to Prevent Biogenesis of Secretory and Membrane Proteins.

Coibamide A (CbA) is a marine natural product with potent antiproliferative activity against human cancer cells and a unique selectivity profile. Despite promising antitumor activity, the mechanism of cytotoxicity and specific cellular target of CbA remain unknown. Here, we develop an optimized synthetic CbA photoaffinity probe (photo-CbA) and use it to demonstrate that CbA directly targets the Sec61α subunit of the Sec61 protein translocon. CbA binding to Sec61 results in broad substrate-nonselective inhibition of ER protein import and potent cytotoxicity against specific cancer cell lines. CbA targets a lumenal cavity of Sec61 that is partially shared with known Sec61 inhibitors, yet profiling against resistance conferring Sec61α mutations identified from human HCT116 cells suggests a distinct binding mode for CbA. Specifically, despite conferring strong resistance to all previously known Sec61 inhibitors, the Sec61α mutant R66I remains sensitive to CbA. A further unbiased screen for Sec61α resistance mutations identified the CbA-resistant mutation S71P, which confirms nonidentical binding sites for CbA and apratoxin A and supports the susceptibility of the Sec61 plug region for channel inhibition. Remarkably, CbA, apratoxin A, and ipomoeassin F do not display comparable patterns of potency and selectivity in the NCI60 panel of human cancer cell lines. Our work connecting CbA activity with selective prevention of secretory and membrane protein biogenesis by inhibition of Sec61 opens up possibilities for developing new Sec61 inhibitors with improved drug-like properties that are based on the coibamide pharmacophore.

IKVAV-linked cell membrane-spanning peptide treatment induces neuronal reactivation following spinal cord injury.

Spinal cord regeneration following treatment with a novel membrane-spanning peptide (MSP) expressing the isoleucine-lysine-valine-alanine-valine (IKVAV) epitope was assessed in Balb-c mice. After hemilaminectomy and compression injury, mice were treated with IKVAV, IKVAV-MSP, peptide or vehicle control. Functional improvement was assessed using modified Basso, Beattie, and Bresnahan Scale (mBBB) and spinal cord segments were studied histologically 28 days after injury. IKVAV-MSP group scores increased significantly compared with control groups after 4 weeks of observation (p < 0.05). The number of protoplasmic astrocytes, neurons and muscle bundle size in the IKVAV-MSP mice were significantly increased (p < 0.001; p < 0.05 and p < 0.007; respectively). This study demonstrates that it is possible to promote functional recovery after SCI using bioactive IKVAV presenting cell membrane-spanning peptides.