SARS virus hyödyntää isäntäkehon proteaaseja infektoidessaan solua . Olisiko antiproteaasista hyötyä?

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2773421/

PLoS One. 2009; 4(11): e7870.

Published online 2009 Nov 17. doi: 10.1371/journal.pone.0007870

PMCID: PMC2773421

PMID: 19924243

Cleavage of the SARS Coronavirus Spike Glycoprotein by Airway Proteases Enhances Virus Entry into Human Bronchial Epithelial Cells In Vitro

Yiu-Wing Kam, ^{1 , 2} Yuushi Okumura, ^{3 , 4} Hiroshi Kido, ⁴ Lisa F. P. Ng, ^{2 , 5} Roberto Bruzzone, ^{1 , *} and Ralf Altmeyer ¹

Georges Snounou, Editor  Abstract

Background

Entry of enveloped viruses into host cells requires the activation of viral envelope glycoproteins through cleavage by either intracellular or extracellular proteases. In order to gain insight into the molecular basis of protease cleavage and its impact on the efficiency of viral entry, we investigated the susceptibility of a recombinant native full-length S-protein trimer (triSpike) of the severe acute respiratory syndrome coronavirus (SARS-CoV) to cleavage by various airway proteases.

Methodology/Principal Findings

Purified triSpike proteins were readily cleaved in vitro by three different airway proteases: trypsin, plasmin and TMPRSS11a. High Performance Liquid Chromatography (HPLC) and amino acid sequencing analyses identified two arginine residues (R667 and R797) as potential protease cleavage site(s). The effect of protease-dependent enhancement of SARS-CoV infection was demonstrated with ACE2 expressing human bronchial epithelial cells 16HBE. Airway proteases regulate the infectivity of SARS-CoV in a fashion dependent on previous receptor binding. The role of arginine residues was further shown with mutant constructs (R667A, R797A or R797AR667A). Mutation of R667 or R797 did not affect the expression of S-protein but resulted in a differential efficacy of pseudotyping into SARS-CoVpp. The R667A SARS-CoVpp mutant exhibited a lack of virus entry enhancement following protease treatment.

Conclusions/Significance

These results suggest that SARS S-protein is susceptible to airway protease cleavage and, furthermore, that protease mediated enhancement of virus entry depends on specific conformation of SARS S-protein upon ACE2 binding. These data have direct implications for the cell entry mechanism of SARS-CoV along the respiratory system and, furthermore expand the possibility of identifying potential therapeutic agents against SARS-CoV.

Introduction

Proteolytic cleavage of the viral envelope glycoprotein into a receptor binding and a fusogenic transmembrane subunit is important to regulate virus entry and infectivity [1]. Previous studies showed that viral glycoprotein activation is mediated by secreted proteases recognizing either monobasic or multibasic cleavage sites [2]. Cleavage of viral glycoprotein has been demonstrated in retrovirus, ortho and paramyxoviruses to regulate virus entry and fusion [3], [4], [5]. The extracellular processing of the envelope glycoprotein has a major impact on the infectivity of virulent or avirulent strains of influenza viruses, Sendai virus and Newcastle disease virus [6], [7], [8]. A typical example is influenza A virus, where virus-cell fusion activity is induced by post-translational proteolytic cleavage of the envelope glycoprotein that is mediated by trypsin-like protease in the bronchial epithelium and airway secretion [9]. Several proteases such as tryptase clara, mini-plasmin, ectopic anionic trypsin, mast-cell tryptase and tryptase TC30, which have been isolated from airway epithelial, can selectivity cleave the consensus cleavage motif of human influenza A virus envelope glycoprotein [10], [11], [12], [13] and determine the virus tropism and infectivity. Recent advance of human genome studies identified a large number of transmembrane serine protease (TMPRSS). Various TMPRSS members with known airway localization have been identified from the respiratory tract. TMPRSS11a, one of the newly identified members of type II transmembrane serine proteases, is expressed in upper respiratory tract (pharynx and trachea) (unpublished data). However, less is known about TMPRSS that activate pneumotropic virus under natural infection.

Although enhancement of virus infection has been demonstrated for bovine (BCoV) and rat (RCV) coronaviruses by treatment of cells with trypsin [14], [15], the precise role of trypsin during coronavirus infection is still unknown. Several members of coronavirus possess a protease cleavage site, which is essential for infectivity and virus-cell membrane fusion, and cleavage at these sites yields two non-covalently linked subunits S1 and S2 [16], [17]. The N-terminal S1 subunit is responsible for receptor binding whereas the membrane-anchored S2 subunit is important for fusion between viral and cellular membranes. Evidence from mouse hepatitis virus (MHV) and BCoV suggested the importance of S protein cleavage into two non-covalently linked S1 and S2 subunits that remain on the virus envelope surface during virus maturation [18], [19]. Uncleaved S protein is functional but cleavage may enhance cell fusion activity and/or virus infectivity [20], [21]. Susceptibility of S protein to cleavage depends on virus strains and host cell types. Similar to group I coronaviruses, sequence analysis suggests that S protein from SARS-CoV is not expected to be cleaved since typical amino acid cleavage sites found in coronavirus group II and III (RRFRR, RRSRR, RSRR, RARS and RARR) are not located in the SARS S protein [22].

Recent findings have suggested the importance of trypsin treatment in activating SARS spike glycoprotein mediated cell-cell fusion [23]. Syncytia formation was observed between SARS spike glycoprotein expressing 293T cells and VeroE6 cells after brief trypsin treatment [24]; trypsin has been shown to induce cleavage of monobasic cleavage site and activate influenza viruses in cell culture system [4], [25]. It is not known whether the functionality of spike glycoproteins is dependent on the activity of trypsin inducing their proteolytic cleavage. Nothing is known about the role of proteases that cleave/modify SARS spike glycoprotein under natural infection.

Conformational reorganization of SARS spike glycoprotein has been demonstrated from cryo-electron microscopic analysis whereby structural transition of the spike glycoprotein has been observed when irradiated SARS-CoV virion binds to the virus receptor, angiotensin-converting enzyme (ACE2) [26]. These experiments showed that receptor-binding and subsequent membrane fusion occur with different phases of structural re-arrangements. Possibly, the protease-modified SARS spike glycoprotein is de facto the glycoprotein responsible for virus entry. To address this possibility we have investigated whether cleavage has any significant effect on SARS-CoV entry into airway epithelial cells by using S-pseudotyped lentiviral vectors (SARS-CoVpp) encoding a luciferase reporter gene to mimic SARS-CoV entry. We observed that SARS-CoV spike glycoprotein can be efficiently cleaved by several airway proteases and that this processing enhances entry of SARS-CoVpp. Furthermore, we have identified the putative cleavage sites of airway proteases and, by site-directed mutagenesis, have determined the role of specific amino acid residue for proteolytic processing of the envelope glycoprotein, and for SARS-CoVpp entry into human airway epithelial cells (16HBE) in vitro. While this manuscript was still in progress, one of the two natural cleavage sites described here, at position 797, was reported in a separate independent study using only trypsin for cleavage [27]. This study further supports and strengthens the demonstration of the critical role of receptor-dependent cleavage of spike protein by airway proteases, providing deeper insights into the exact mechanism of virus entry enhancement.

 Discussion..

Current studies have suggested that SARS-CoV enters and exits preferentially via the apical surface of the epithelium [31], and co-localization of airway proteases with SARS-CoV along the respiratory tract supports the positive feedback loop of virus infection in vivo. To conclude, we have found that cleavage of the receptor-bound spike glycoprotein by airway proteases enhances in vitro virus entry and fusion. Therefore, identification of reagents that are able to suppress in vivo activity of airway proteases might provide additional antiviral strategy against SARS-CoV infection [57], and possibly other viral respiratory infections such as human influenza A virus in the face of the current flu epidemic threat.

Muita artikkeleita:

TMPRSS2 and ADAM17 cleave ACE2 differentially and only proteolysis by TMPRSS2 augments entry driven by the severe acute respiratory syndrome coronavirus spike protein.

Heurich A, Hofmann-Winkler H, Gierer S, Liepold T, Jahn O, Pöhlmann S.

J Virol. 2014 Jan;88(2):1293-307. doi: 10.1128/JVI.02202-13. Epub 2013 Nov 13.

The type II transmembrane serine proteases TMPRSS2 and HAT can cleave and activate the spike protein (S) of the severe acute respiratory syndrome coronavirus (SARS-CoV) for membrane fusion. In addition, these proteases cleave the viral receptor, the carboxypeptidase angiotensin-converting enzyme 2 (ACE2), and it was proposed that ACE2 cleavage augments viral infectivity. However, no mechanistic insights into this process were obtained and the relevance of ACE2 cleavage for SARS-CoV S protein (SARS-S) activation has not been determined. Here, we show that arginine and lysine residues within ACE2 amino acids 697 to 716 are essential for cleavage by TMPRSS2 and HAT and that ACE2 processing is required for augmentation of SARS-S-driven entry by these proteases. In contrast, ACE2 cleavage was dispensable for activation of the viral S protein. Expression of TMPRSS2 increased cellular uptake of soluble SARS-S, suggesting that protease-dependent augmentation of viral entry might be due to increased uptake of virions into target cells. Finally, TMPRSS2 was found to compete with the metalloprotease ADAM17 for ACE2 processing, but only cleavage by TMPRSS2 resulted in augmented SARS-S-driven entry. Collectively, our results in conjunction with those of previous studies indicate that TMPRSS2 and potentially related proteases promote SARS-CoV entry by two separate mechanisms: ACE2 cleavage, which might promote viral uptake, and SARS-S cleavage, which activates the S protein for membrane fusion. These observations have interesting implications for the development of novel therapeutics. In addition, they should spur efforts to determine whether receptor cleavage promotes entry of other coronaviruses, which use peptidases as entry receptors.Free PMC Article

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