Identification and Characterization of a Proteolytically Primed Form of the Murine Coronavirus Spike Proteins after Fusion with the Target Cell
ABSTRACT 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.
- SourceAvailable from: Severine Tasker
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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. "
ABSTRACT: Feline coronavirus (FCoV) infections are endemic amongst cats worldwide. The majority of infections are asymptomatic, or result only in mild enteric disease. However, approximately 5% of cases develop feline infectious peritonitis (FIP), a systemic disease that is a frequent cause of death in young cats. In this study, we report the complete coding genome sequences of six FCoVs; three from fecal samples from healthy cats and three from tissue lesion samples from cats with confirmed FIP. The six samples were obtained over a period of eight weeks at a single-site cat rescue and rehoming center in the UK. We found amino acid differences are located at 44 positions across an alignment of the six virus translatomes and, at 21 of these positions, the differences fully or partially discriminate between the genomes derived from the fecal samples and the genomes derived from tissue lesion samples. In this study, two amino acid differences fully discriminate the two classes of genomes; these are both located in the S2 domain of the virus surface glycoprotein gene. We also identified deletions in the 3c protein ORF of genomes from two of the FIP samples. Our results support previous studies that implicate S protein mutations in the pathogenesis of FIP.Journal of General Virology 02/2015; 96(Pt_6). DOI:10.1099/vir.0.000084 · 3.18 Impact Factor
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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. "
ABSTRACT: Porcine epidemic diarrhea virus (PEDV), a member of the genus Alphacoronavirus, is a causative agent of porcine enteric disease characterized by acute watery diarrhea and dehydration in sucking piglet. Similar to other coronaviruses, PEDV spike protein mediates its cell entry by binding to cellular receptors and inducing membrane fusion between viral envelopes and cellular membranes. However, the entry mechanism of PEDV is not studied. Here, we determined the entry mechanism of PEDV into Vero cells. Our data confirmed that PEDV entry followed clathrin-mediated endocytosis independence of caveolae-coated pit assembly. The internalized PEDV was co-localized with the clathrin-mediated endocytic marker, but not with the caveolae-mediated endocytic marker. In addition, cells treated with lysosomotropic agents and serine protease inhibitors were resistant to PEDV. Our data revealed that PEDV entry followed clathrin-mediated endocytosis and was dependent on a low pH and serine proteolysis for successful entry into cells.Virus Research 07/2014; 191(1). DOI:10.1016/j.virusres.2014.07.022 · 2.32 Impact Factor
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ABSTRACT: Canine enteric coronavirus (CCoV) is an alphacoronavirus infecting dogs that is closely related to enteric coronaviruses of cats and pigs. While CCoV has traditionally caused mild gastro-intestinal clinical signs, there are increasing reports of lethal CCoV infections in dogs, with evidence of both gastrointestinal and systemic viral dissemination. Consequently, CCoV is now considered to be an emerging infectious disease of dogs. In addition to the two known serotypes of CCoV, novel recombinant variants of CCoV have been found containing spike protein N-terminal domains (NTDs) that are closely related to those of feline and porcine strains. The increase in disease severity in dogs and the emergence of novel CCoVs can be attributed to the high level of recombination within the spike gene that can occur during infection by more than one CCoV type in the same host.Viruses 08/2014; 6(8):3363-3376. DOI:10.3390/v6083363 · 3.35 Impact Factor