Protease-mediated enhancement of SARS infection

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Biological Sciences
Aarti Ashok

BIOD29 Winter 2013: Article - Protease-mediated enhancement of SARS coronavirus infection Introduction  Severe acute respiratory syndrome (SARS) is caused by SARS-associated corona virus (SARS- CoV) which is a fatal respiratory disease in humans  Coronavirus is an enveloped virus with a positive-stranded large genomic RNA with 30 kb.  S protein is a type 1 fusion of an approximate molecular weight of 180 kDa  Spikes exist on the virion surface and resemble solar corona – each composed of a trimer of the spike (S) protein  There are two pathways for SARS infection o Endosome pathway – low pH induces cell fusion o Protease mediated pathway – enters cell via endosomal pathway and S protein is activated for fusion by trypsin-like proteases in an acidic environment  This study shows that various proteases as well as trypsin are effective in inducing the fusion of SARS-CoV infected VeroE6 cells  Protease-mediated nonendosomal pathway is 100 times more efficient that endosome entry infection  Elactase found in lung and other proteases found in the digestive tract show why there are severe illness here as opposed to other areas in the body Results Activation of Cell Fusion and SARS-CoV S protein cleavage by Various Proteases  VeroE6 cells susceptible to SARS-CoV were infected with the Frankfurt strain at a moi of 0.5 and then infected with trypsin 20 hours later, cell fusion was detected 2 H after trypsin introduction  Cell fusion found with trypsin, thermolysin and dispase but little to no fusion occurred after treatment with papain, chymotrypsin, proteinase K or collagenase o Proteases that did not induce cleavage of S protein (as evidenced by S2 protein band in Western Blotting in Figure 1 B) did not induce cell fusion as well  Therefore, S protein cleavage a necessary precursor or marker of subsequent cell fusion which is a vital step towards allowing SARS-CoV infection from happening  Proteases activate cell fusion activity of the SARS-CoV S protein by inducing its cleavage SARS-Cov Entry from Cell Surface Facilitated by Proteases  SARS-CoV can enter cells directly from their surface by attaching the virus there and treating them with trypsin and other proteases to induce cell fusion  Treatment with bafilomycin (BAF) leads to a suppression of the endosomal pathway of cell entry of SARS-CoV in VeroE6 cells  Cells treated with thermolysin and trypsin, which induced cell fusion, also had increased amounts of viral mRNA which suggests greater amounts of virus within the cell  Papain and collagenase did not induce cell fusion and also did not have high levels of viral mRNA  Treatment of cells with trypsin BEFORE virus infection did not facilitate virus entry suggesting that effects of trypsin on cells are not involved in this infection  Therefore, trypsin is vital to facilitating membrane fusion Enhancement of SARS-CoV Infection by Various Proteases BIOD29 Winter 2013: Article - Protease-mediated enhancement of SARS coronavirus infection  Treatment with high concentrations of thermolysin and trypsin increases virus entry by 10 times or higher  Trypsin treated cells were always 10X higher in the level of mRNA9 over other conditions (no BAF therefore having endosomal pathways open, no proteases)  Surface route of nonendosomal protease mediated pathway for cell entry is 1 hour faster leading to greater quantities of infection  Trypsin-treated cells with low moi of SARS-CoV infection had 100 to 1000X higher virus replication as well as viral growth increased by 100X  Virus spreads effectively from cell to cell in the presence of trypsin which cleaves S to S1 and S2 to allow cell entry of SARS-CoV via the cell surface  All those proteases that produce S2 and that induce cell to cell fusion enhanced virus spread. (Thermolysin, Trypsin)  All those proteases that did not generate S2 and that did not induce cell to cell fusion failed to enhance infection  Proteases are likely to be responsible for the high multiplication of SARS-CoV I the major target organs of SARS such as the lungs and bronchus where various proteases are produced as well as in the intestines where a number of proteases are physiologically spread  Elactase, a protease produced by neutrophils in the lungs, which is accumulated in the lungs of SARS patients, enhances SARS-CoV inf
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