Identification and Characterization of a Proteolytically Primed Form of the Murine Coronavirus Spike Proteins after Fusion with the Target Cell

Journal of Virology (Impact Factor: 4.44). 02/2014; 88(9). DOI: 10.1128/JVI.03451-13
Source: PubMed


Enveloped viruses carry highly specialized glycoproteins that catalyze membrane fusion under strict spatial and temporal control. To prevent premature activation after biosynthesis, viral class I fusion proteins adopt a locked conformation and require proteolytic cleavage to render them fusion-ready. This priming step may occur during virus exit from the infected cell, in the extracellular milieu or during entry at or in the next target cell. Proteolytic processing of coronavirus spike (S) fusion proteins during virus entry has been suggested but not yet formally demonstrated, while the nature and functionality of the resulting subunit is still unclear. We used the prototype coronavirus - mouse hepatitis virus (MHV) - to develop a conditional biotinylation assay that enables the specific identification and biochemical characterization of viral S proteins on virions that mediated membrane fusion with the target cell. We demonstrate that MHV S proteins are indeed cleaved upon virus endocytosis and we identified a novel processing product S2* with characteristics of a fusion-active subunit. The precise cleavage site and the enzymes involved remain to be elucidated.
Virus entry determines the tropism and is a crucial step in the virus life cycle. We developed an approach to characterize structural components of virus particles after entering new target cells. A prototype coronavirus was used to illustrate how the virus fusion machinery can be controlled.

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Available from: B.J. Bosch, Nov 11, 2015
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    • "Alternatively, it could be argued that once the virus has acquired a tropism for the monocyte/ macrophage, cleavage at the furin recognition motif may no longer be relevant to virus entry and mutations may accumulate due to a lack of selection pressure. For coronaviruses such as mouse hepatitis virus, cleavage at the canonical furin motif does not seem to be essential, at least for in vitro infectivity (Bos et al., 1997), and recent results suggest that activation of the coronavirus S protein fusion activity requires proteolytic cleavage at a different position in the S2 subunit (Millet & Whittaker, 2014; Wicht et al., 2014). Finally, Table 1 shows that two of the three translatomes derived from the FIP samples had a deletion in the 3c protein gene, which was not found in any of the non-FIP samples. "
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    • "In addition, our finding that trypsin treatment is not bypass the infection inhibition with lysosomotropic agents and CPZ as shown in Fig. 7, clearly indicates that PEDV entry requires pH-dependent step rather than the presence of pH-dependent proteolytic processing. Recently, similar possibilities were proposed by others in MHV-A59 S mediated infection study (Wicht et al., 2014). Another question is how trypsin sufficiently mediates cell–cell fusion at neutral pH. "
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