SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor

Tracking Changes in SARS-CoV-2 Spike: Evidence that D614G Increases Infectivity of the COVID-19 Virus.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the ongoing coronavirus disease 2019 (COVID-19) pandemic. Alongside investigations into the virology of SARS-CoV-2, understanding the fundamental physiological and immunological processes underlying the clinical manifestations of COVID-19 is vital for the identification and rational design of effective therapies. Here, we provide an overview of the pathophysiology of SARS-CoV-2 infection. We describe the interaction of SARS-CoV-2 with the immune system and the subsequent contribution of dysfunctional immune responses to disease progression. From nascent reports describing SARS-CoV-2, we make inferences on the basis of the parallel pathophysiological and immunological features of the other human coronaviruses targeting the lower respiratory tract - severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV). Finally, we highlight the implications of these approaches for potential therapeutic interventions that target viral infection and/or immunoregulation.

/pdf/the-trinity-of-covid-19-immunity-inflammation-and-10rwiwjpt5.pdf

The trinity of COVID-19: immunity, inflammation and intervention.

A Multibasic Cleavage Site in the Spike Protein of SARS-CoV-2 Is Essential for Infection of Human Lung Cells.

The causative agent of coronavirus disease 2019 (COVID-19) is the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). For many viruses, tissue tropism is determined by the availability of virus receptors and entry cofactors on the surface of host cells. In this study, we found that neuropilin-1 (NRP1), known to bind furin-cleaved substrates, significantly potentiates SARS-CoV-2 infectivity, an effect blocked by a monoclonal blocking antibody against NRP1. A SARS-CoV-2 mutant with an altered furin cleavage site did not depend on NRP1 for infectivity. Pathological analysis of olfactory epithelium obtained from human COVID-19 autopsies revealed that SARS-CoV-2 infected NRP1-positive cells facing the nasal cavity. Our data provide insight into SARS-CoV-2 cell infectivity and define a potential target for antiviral intervention.

https://science.sciencemag.org/content/sci/early/2020/10/19/science.abd2985.full.pdf

Neuropilin-1 facilitates SARS-CoV-2 cell entry and infectivity.

The Middle East respiratory syndrome-related coronavirus (MERS-CoV) can cause severe disease and has pandemic potential. Therefore, development of antiviral strategies is an important task. The activation of the viral spike protein (S) by host cell proteases is essential for viral infectivity and the responsible enzymes are potential therapeutic targets. The cellular proteases furin, cathepsin L and TMPRSS2 can activate MERS-S and may cleave the S protein at two distinct sites, termed S1/S2 and S2′. Moreover, a potential cathepsin L cleavage site in MERS-S has been reported. However, the relative importance of these sites for MERS-S activation is incompletely understood. Here, we used mutagenic analysis and MERS-S-bearing vectors to study the contribution of specific cleavage sites to S protein-driven entry. We found that an intact S1/S2 site was only required for efficient entry into cells expressing endogenous TMPRSS2. In keeping with a previous study, pre-cleavage at the S1/S2 motif (RSVR) was important although not essential for subsequent MERS-S activation by TMPRSS2, and indirect evidence was obtained that this motif is processed by a protease depending on an intact RXXR motif, most likely furin. In contrast, the S2′ site (RSAR) was required for robust viral entry into all cell lines tested and the integrity of one of the two arginines was sufficient for efficient entry. These findings suggest that cleavage at S2′ is carried out by proteases recognizing a single arginine, most likely TMPRSS2 and cathepsin L. Finally, mutation of the proposed cathepsin L site did not impact viral entry and double mutation of S1/S2 and S2′ site was compatible with cathepsin L- but not TMPRSS2-dependent host cell entry, indicating that cathepsin L can process the S protein at auxiliary sites. Collectively, our results indicate a rigid sequence requirement for S protein activation by TMPRSS2 but not cathepsin L.

/pdf/functional-analysis-of-potential-cleavage-sites-in-the-mers-4o5oyhuw86.pdf

Functional analysis of potential cleavage sites in the MERS-coronavirus spike protein

Middle East respiratory syndrome coronavirus (MERS-CoV) poses a threat to public health. The virus is endemic in the Middle East but can be transmitted to other countries by travel activity. The introduction of MERS-CoV into the Republic of Korea by an infected traveler resulted in a hospital outbreak of MERS that entailed 186 cases and 38 deaths. The MERS-CoV spike (S) protein binds to the cellular protein DPP4 via its receptor binding domain (RBD) and mediates viral entry into target cells. During the MERS outbreak in Korea, emergence and spread of viral variants that harbored mutations in the RBD, D510G and I529T, was observed. Counterintuitively, these mutations were found to reduce DPP4 binding and viral entry into target cells. In this study, we investigated whether they also exerted proviral effects. We confirm that changes D510G and I529T reduce S protein binding to DPP4 but show that this reduction only translates into diminished viral entry when expression of DPP4 on target cells is low. Neither mutation modulated S protein binding to sialic acids, S protein activation by host cell proteases, or inhibition of S protein-driven entry by interferon-induced transmembrane proteins. In contrast, changes D510G and I529T increased resistance of S protein-driven entry to neutralization by monoclonal antibodies and sera from MERS patients. These findings indicate that MERS-CoV variants with reduced neutralization sensitivity were transmitted during the Korean outbreak and that the responsible mutations were compatible with robust infection of cells expressing high levels of DPP4.IMPORTANCE MERS-CoV has pandemic potential, and it is important to identify mutations in viral proteins that might augment viral spread. In the course of a large hospital outbreak of MERS in the Republic of Korea in 2015, the spread of a viral variant that contained mutations in the viral spike protein was observed. These mutations were found to reduce receptor binding and viral infectivity. However, it remained unclear whether they also exerted proviral effects. We demonstrate that these mutations reduce sensitivity to antibody-mediated neutralization and are compatible with robust infection of target cells expressing large amounts of the viral receptor DPP4.

Mutations in the Spike Protein of Middle East Respiratory Syndrome Coronavirus Transmitted in Korea Increase Resistance to Antibody-Mediated Neutralization.

Background
Both acute and chronic exercise have profound effects on systemic metabolism and the immune system. While acute exercise transiently disturbs energy homeostasis and elicits acute inflammation, exercise training improves systemic metabolic capacity, lowers basal inflammation, and reduces infection risk. Accordingly, accumulating evidence indicates links between systemic and immune cell metabolism and suggests that cellular metabolism may be an important way exercise influences immune function. Yet, no reviews have systematically surveyed the literature in this area.


Aims
The aims of this scoping review were to collect, summarize, and provide descriptive analysis of literature on the effects of acute exercise, chronic exercise, and physical fitness on peripheral leukocyte energy metabolism of human adults.


Methods
Reports were retrieved from the databases Pubmed, Scopus, and Embase and hierarchically filtered for eligibility. Eligible reports were those that implemented acute or chronic exercise interventions, or assessed physical fitness, in relation to the regulation or function of leukocyte energy metabolism in human adults. Data were charted from eligible reports by two independent reviewers, confirmed by conference, and organized for reporting.


Results & Conclusion
Results suggest acute exercise can influence the regulation and function of leukocyte metabolism, with some similarities to what has been previously documented in skeletal muscle. Data also evidence that exercise training and/ or physical fitness alters cellular metabolic regulation and function. Improvements in markers of cell respiratory function or mitochondrial regulation were frequently observed following training or with greater fitness. However, notable gaps in the literature remain. These gaps include: the effects of acute exercise and exercise training on leukocyte glycolysis, the effects of resistance and concurrent exercise, and potential differences in the effects of exercise between immune cell types and subsets. Future research is encouraged to fill the latter gaps and further delineate how exercise influences the immune system and can be used to support overall health.

A Scoping Review on the Effects of Physical Exercise and Fitness on Peripheral Leukocyte Energy Metabolism in Humans.

T-cell subsets, including naïve (NA), central memory (CM), transitional memory (TM), effector memory (EM), and RA + effector memory (EMRA), differ in phenotype and function. T-cells are mobilized by exercise, with differences in the magnitude of mobilization between subsets. However, the response of TM T-cells to exercise has not yet been described. Further, T-cells expressing the late differentiation marker CD57 are known to be highly responsive to exercise, but the relative response of CD57 + and CD57- within T-cell subsets is unknown. We therefore aimed to characterize the exercise-induced mobilization of TM T-cells, as well as to compare the exercise response of CD57 + and CD57- cells within T-cell subsets. Methods Seventeen participants (7 female; aged 18–40 years) cycled 30 min at 80% of their estimated maximum heart rate. Venous blood obtained pre, post, and 1H post-exercise was analyzed by flow cytometry. CD45RA, CCR7, and CD28 expression within CD4 + and CD8+ T-cells identified NA, CM, TM, EM, and EMRA subsets. CD57 expression within EM, EMRA, and CD28+ T-cells was also quantified. The relative mobilization of each subset was compared by calculating fold change in cell concentration during (ingress, post/pre) and after exercise (egress,1H post/post). Cytomegalovirus (CMV) serostatus was determined by ELISA and was considered in models. Results TM CD8+ T-cell concentration was greater post-exercise than pre-exercise (138.59 ± 56.42 cells/µl vs. 98.51 ± 39.68 cells/µl, p < 0.05), and the proportion of CD8 + with a TM phenotype was elevated 1H post-exercise (1H: 32.44 ± 10.38% vs. Pre: 30.15 ± 8.77%, p < 0.05). The relative mobilization during and after exercise of TM T-cells did not differ from NA and CM but was less than EM and EMRA subsets. Similar results were observed within CD4+ T-cells. CD57 + subsets of CD28+ T-cells and of EM and EMRA CD8+ T-cells exhibited a greater relative mobilization than CD57- subsets (all p < 0.05). Conclusion These results indicate TM CD4 + and CD8+ T-cells are transiently mobilized into the blood with exercise, but not to as great of an extent as later differentiated EM and EMRA T-cells. Results also indicate CD57 identifies highly exercise responsive cells within CD8+ T-cell subsets.

Characterization of transitional memory CD4+ and CD8+ T-cell mobilization during and after an acute bout of exercise

Exercise preferentially mobilizes later differentiated T-cell subsets, such as effector memory T-cells. Separately, a preferential mobilization of T-cells expressing CD57 has also been shown. CD57 is a surface protein associated with a decrease in both telomere length and proliferative potential. However, earlier differentiated T-cells, including naïve cells, may also express CD57. Whether there are differences in exercise-induced mobilization of CD57 expressing cells within both earlier and later differentiated subsets has not yet been determined. PURPOSE: To determine whether CD57 expression indicates lymphocyte responsiveness to exercise, independently of differentiation status. METHODS: Healthy adults (N=17, 7 female, 27 ± 4.5 yrs) exercised on a cycle ergometer for 30 min at 80% of age-predicted maximum HR. Blood was collected Pre-, Post-, and 1H Post-exercise, and lymphocyte subsets in blood were identified by flow cytometry. Within CD8+ T-cells, CD45RA and CCR7 identified Naïve (NA; CCR7+CD45RA+), Central Memory (CM; CCR7+CD45RA-), Effector Memory (EM; CCR7-CD45RA-), and RA+Effector Memory (EMRA; CCR7-CD45RA+) subsets. Within each subset, numbers of CD57+ and CD57- cells were determined. The fold change in cell number due to exercise was calculated Post-exercise relative to Pre-exercise (ingress) and 1H Post-exercise relative to Post-exercise (egress). The ingress and egress of CD57+ vs CD57- cells within each subset was compared by paired T-tests. RESULTS: CD57+ cells exhibited a larger ingress compared to CD57- cells within NA, EM, and EMRA subsets of CD8+ T-cells (fold change: NA: 3.60 vs 1.12; EM: 2.13 vs 1.44; EMRA: 2.54 vs 1.74; all p<0.01). CD57+ cells also exhibited a larger egress compared to CD57- within NA, EM, CM, and EMRA subsets (fold change: NA: 0.52 vs 0.84; CM: 0.60 vs 0. 84; EM: 0.49 vs 0.71; EMRA: 0.39 vs 0.56; all p<0.05). CONCLUSION: Even within earlier differentiated CD8+ T-cell subsets, such as NA, the expression of CD57 identifies exercise-responsive cells. Whether CD57 expression on exercise-mobilized NA cells relates to differences in cell function or proliferative capacity requires further study. 

Mahmoud Tarek Elzayat

Papers

Functional analysis of potential cleavage sites in the MERS-coronavirus spike protein

Mutations in the Spike Protein of Middle East Respiratory Syndrome Coronavirus Transmitted in Korea Increase Resistance to Antibody-Mediated Neutralization.

A Scoping Review on the Effects of Physical Exercise and Fitness on Peripheral Leukocyte Energy Metabolism in Humans.

Characterization of transitional memory CD4+ and CD8+ T-cell mobilization during and after an acute bout of exercise

CD57 Expression Identifies Highly Exercise Responsive Cells Even Among Early Differentiated CD8 T Cells