Abstract
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.
Highlights
The family Coronaviridae comprises enveloped, positive sense RNA viruses that infect mammals, birds (Coronavirinae) and fish (Torovirinae)[1]
We first investigated whether the S1/S2, the cathepsin L and the S2′ cleavage sites (Fig. 1) were universally required for Middle East respiratory syndrome (MERS)-S-driven entry or were only required for entry into certain target cells
Immunoblot analysis revealed that all S protein mutants studied were efficiently incorporated into vesicular stomatitis virus (VSV) particles, some variation of incorporation efficiency was observed between experiments, and that mutation of the S1/S2 site largely abrogated proteolytic processing of the S protein in particle producing cells (Fig. 2), as expected[14,16,21]
Summary
The family Coronaviridae comprises enveloped, positive sense RNA viruses that infect mammals (members of the subfamilies Coronavirinae and Torovirinae), birds (Coronavirinae) and fish (Torovirinae)[1]. Cleavage of the S protein in the Golgi apparatus of infected cells has been proposed to be essential for subsequent MERS-S activation by TMPRSS2 or furin during entry into target cells because it may endow the S protein with sufficient structural flexibility to engage these proteases for processing[16]. The contribution of furin to MERS-S activation in the host remains to be determined In this context, it is noteworthy that one study questioned whether cleavage by furin contributes to MERS-CoV infectivity in cell culture[21] while another showed that furin is not involved in S protein activation during host cell entry[22]. An AFNH motif located between the S1/S2 site and the S2′ site was reported to be
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