ESCPE-1 mediates retrograde endosomal sorting of the SARS-CoV-2 host factor Neuropilin-1.

Endosomal sorting maintains cellular homeostasis by recycling transmembrane proteins and associated proteins and lipids (termed "cargoes") from the endosomal network to multiple subcellular destinations, including retrograde traffic to the trans-Golgi network (TGN). Viral and bacterial pathogens subvert retrograde trafficking machinery to facilitate infectivity. Here, we develop a proteomic screen to identify retrograde cargo proteins of the endosomal SNX-BAR sorting complex promoting exit 1 (ESCPE-1). Using this methodology, we identify Neuropilin-1 (NRP1), a recently characterized host factor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, as a cargo directly bound and trafficked by ESCPE-1. ESCPE-1 mediates retrograde trafficking of engineered nanoparticles functionalized with the NRP1-interacting peptide of the SARS-CoV-2 spike (S) protein. CRISPR-Cas9 deletion of ESCPE-1 subunits reduces SARS-CoV-2 infection levels in cell culture. ESCPE-1 sorting of NRP1 may therefore play a role in the intracellular membrane trafficking of NRP1-interacting viruses such as SARS-CoV-2.

Evaluation of Host Protein Biomarkers by ELISA From Whole Lysed Peripheral Blood for Development of Diagnostic Tests for Active Tuberculosis.

Tuberculosis (TB) remains a significant global health crisis and the number one cause of death for an infectious disease. The health consequences in high-burden countries are significant. Barriers to TB control and eradication are in part caused by difficulties in diagnosis. Improvements in diagnosis are required for organisations like the World Health Organisation (WHO) to meet their ambitious target of reducing the incidence of TB by 50% by the year 2025, which has become hard to reach due to the COVID-19 pandemic. Development of new tests for TB are key priorities of the WHO, as defined in their 2014 report for target product profiles (TPPs). Rapid triage and biomarker-based confirmatory tests would greatly enhance the diagnostic capability for identifying and diagnosing TB-infected individuals. Protein-based test methods e.g. lateral flow devices (LFDs) have a significant advantage over other technologies with regard to assay turnaround time (minutes as opposed to hours) field-ability, ease of use by relatively untrained staff and without the need for supporting laboratory infrastructure. Here we evaluate the diagnostic performance of nine biomarkers from our previously published biomarker qPCR validation study; CALCOCO2, CD274, CD52, GBP1, IFIT3, IFITM3, SAMD9L, SNX10 and TMEM49, as protein targets assayed by ELISA. This preliminary evaluation study was conducted to quantify the level of biomarker protein expression across latent, extra-pulmonary or pulmonary TB groups and negative controls, collected across the UK and India, in whole lysed blood samples (WLB). We also investigated associative correlations between the biomarkers and assessed their suitability for ongoing diagnostic test development, using receiver operating characteristic/area under the curve (ROC) analyses, singly and in panel combinations. The top performing single biomarkers for pulmonary TB versus controls were CALCOCO2, SAMD9L, GBP1, IFITM3, IFIT3 and SNX10. TMEM49 was also significantly differentially expressed but downregulated in TB groups. CD52 expression was not highly differentially expressed across most of the groups but may provide additional patient stratification information and some limited use for incipient latent TB infection. These show therefore great potential for diagnostic test development either in minimal configuration panels for rapid triage or more complex formulations to capture the diversity of disease presentations.

SNX27 suppresses SARS-CoV-2 infection by inhibiting viral lysosome/late endosome entry.

After binding to its cell surface receptor angiotensin converting enzyme 2 (ACE2), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) enters the host cell through directly fusing with plasma membrane (cell surface pathway) or undergoing endocytosis traveling to lysosome/late endosome for membrane fusion (endocytic pathway). However, the endocytic entry regulation by host cell remains elusive. Recent studies show ACE2 possesses a type I PDZ binding motif (PBM) through which it could interact with a PDZ domain-containing protein such as sorting nexin 27 (SNX27). In this study, we determined the ACE2-PBM/SNX27-PDZ complex structure, and, through a series of functional analyses, we found SNX27 plays an important role in regulating the homeostasis of ACE2 receptor. More importantly, we demonstrated SNX27, together with retromer complex (the core component of the endosomal protein sorting machinery), prevents ACE2/virus complex from entering lysosome/late endosome, resulting in decreased viral entry in cells where the endocytic pathway dominates. The ACE2/virus retrieval mediated by SNX27-retromer could be considered as a countermeasure against invasion of ACE2 receptor-using SARS coronaviruses.

PDZ-Containing Proteins Targeted by the ACE2 Receptor.

Angiotensin-converting enzyme 2 (ACE2) is a main receptor for SARS-CoV-2 entry to the host cell. Indeed, the first step in viral entry is the binding of the viral trimeric spike (S) protein to ACE2. Abundantly present in human epithelial cells of many organs, ACE2 is also expressed in the human brain. ACE2 is a type I membrane protein with an extracellular N-terminal peptidase domain and a C-terminal collectrin-like domain that ends with a single transmembrane helix and an intracellular 44-residue segment. This C-terminal segment contains a PDZ-binding motif (PBM) targeting protein-interacting domains called PSD-95/Dlg/ZO-1 (PDZ). Here, we identified the human PDZ specificity profile of the ACE2 PBM using the high-throughput holdup assay and measuring the binding intensities of the PBM of ACE2 against the full human PDZome. We discovered 14 human PDZ binders of ACE2 showing significant binding with dissociation constants' values ranging from 3 to 81 µM. NHERF, SHANK, and SNX27 proteins found in this study are involved in protein trafficking. The PDZ/PBM interactions with ACE2 could play a role in ACE2 internalization and recycling that could be of benefit for the virus entry. Interestingly, most of the ACE2 partners we identified are expressed in neuronal cells, such as SHANK and MAST families, and modifications of the interactions between ACE2 and these neuronal proteins may be involved in the neurological symptoms of COVID-19.