a-fluorogenic-peptide-cleavage-assay-to-screen-for-proteolytic

A Fluorogenic Peptide Cleavage Assay to Screen for Proteolytic Activity: Applications for coronavirus spike protein activation

Enveloped viruses such as coronaviruses or influenza virus require proteolytic cleavage of their fusion protein to be able to infect the host cell. Often ...

production-pseudotyped-particles-to-study-highly-pathogenic

Production of Pseudotyped Particles to Study Highly Pathogenic Coronaviruses in a Biosafety Level 2 Setting

The protocol aims to generate coronavirus (CoV) spike (S) fusion protein pseudotyped particles with a murine leukemia virus (MLV) core and luciferase ...

Cornell University

INRA

INRA, Virologie et Immunologie Moléculaires

Canine alphacoronaviruses (CCoV) exist in two serotypes, type I and II, both of which can cause severe gastroenteritis. Here, we characterize a canine alphacoronavirus, designated CCoV-A76, first isolated in 1976. Serological studies show that CCoV-A76 is distinct from other CCoVs, such as the prototype CCoV-1-71. Efficient replication of CCoV-A76 is restricted to canine cell lines, in contrast to the prototypical type II strain CCoV-1-71 that more efficiently replicates in feline cells. CCoV-A76 can use canine aminopeptidase N (cAPN) receptor for infection of cells, but was unable to use feline APN (fAPN). In contrast, CCoV-1-71 can utilize both. Genomic analysis shows that CCoV-A76 possesses a distinct spike, which is the result of a recombination between type I and type II CCoV, that occurred between the N- and C-terminal domains (NTD and C-domain) of the S1 subunit. These data suggest that CCoV-A76 represents a recombinant coronavirus form, with distinct host cell tropism.

Characterization of a recombinant canine coronavirus with a distinct receptor-binding (S1) domain.

Coronaviruses are enveloped positive-stranded RNA viruses that replicate in the cytoplasm. To deliver their nucleocapsid into the host cell, they rely on the fusion of their envelope with the host cell membrane. The spike glycoprotein (S) mediates virus entry and is a primary determinant of cell tropism and pathogenesis. It is classified as a class I fusion protein, and is responsible for binding to the receptor on the host cell as well as mediating the fusion of host and viral membranes-A process driven by major conformational changes of the S protein. This review discusses coronavirus entry mechanisms focusing on the different triggers used by coronaviruses to initiate the conformational change of the S protein: receptor binding, low pH exposure and proteolytic activation. We also highlight commonalities between coronavirus S proteins and other class I viral fusion proteins, as well as distinctive features that confer distinct tropism, pathogenicity and host interspecies transmission characteristics to coronaviruses.

Mechanisms of coronavirus cell entry mediated by the viral spike protein.

Virus-like particles are useful materials for studying virus-host interactions in a safe manner. However, the standard production of pseudovirus based on the vesicular stomatitis virus (VSV) backbone is an intricate procedure that requires trained laboratory personnel. In this work, a new strategy for creating virus-like proteoliposomes (VLPLs) and virus-like supported bilayers (VLSBs) is presented. This strategy uses a cell blebbing technique to induce the formation of nanoscale vesicles from the plasma membrane of BHK cells expressing the hemagglutinin (HA) fusion protein of influenza X-31. These vesicles and supported bilayers contain HA and are used to carry out single particle membrane fusion events, monitored using total internal reflection fluorescence microscopy. The results of these studies show that the VLPLs and VLSBs contain HA proteins that are fully competent to carry out membrane fusion, including the formation of a fusion pore and the release of fluorophores loaded into vesicles. This new strategy for creating spherical and planar geometry virus-like membranes has many potential applications. VLPLs could be used to study fusion proteins of virulent viruses in a safe manner, or they could be used as therapeutic delivery particles to transport beneficial proteins coexpressed in the cells to a target cell. VLSBs could facilitate high throughput screening of antiviral drugs or pathogen-host cell interactions.

Membrane fusion-competent virus-like proteoliposomes and proteinaceous supported bilayers made directly from cell plasma membranes.

Feline coronaviruses (FCoV) exist as 2 biotypes: feline enteric coronavirus (FECV) and feline infectious peritonitis virus (FIPV). FECV causes subclinical infections; FIPV causes feline infectious peritonitis (FIP), a systemic and fatal disease. It is thought that mutations in FECV enable infection of macrophages, causing FIP. However, the molecular basis for this biotype switch is unknown. We examined a furin cleavage site in the region between receptor-binding (S1) and fusion (S2) domains of the spike of serotype 1 FCoV. FECV sequences were compared with FIPV sequences. All FECVs had a conserved furin cleavage motif. For FIPV, there was a correlation with the disease and >1 substitution in the S1/S2 motif. Fluorogenic peptide assays confirmed that the substitutions modulate furin cleavage. We document a functionally relevant S1/S2 mutation that arises when FIP develops in a cat. These insights into FIP pathogenesis may be useful in development of diagnostic, prevention, and treatment measures against coronaviruses.

Mutation in spike protein cleavage site and pathogenesis of feline coronavirus.

Total internal reflection microscopy combined with microfluidics and supported bilayers is a powerful, single particle tracking (SPT) platform for host-pathogen membrane fusion studies. But one major inadequacy of this platform has been capturing the complexity of the cell membrane, including membrane proteins. Because of this, viruses requiring proteinaceous receptors, or other unknown cellular co-factors, have been precluded from study. Here we describe a general method to integrate proteinaceous receptors and cellular components into supported bilayers for SPT fusion studies. This method is general to any enveloped virus-host cell pair, but demonstrated here for feline coronavirus (FCoV). Supported bilayers are formed from mammalian cell membrane vesicles that express feline aminopeptidase N (the viral receptor) using a cell blebbing technique. SPT is then used to identify fusion intermediates and measure membrane fusion kinetics for FCoV. Overall, the fusion results recapitulate what is observed in vivo, that coronavirus entry requires binding to specific receptors, a low-pH environment, and that membrane fusion is receptor- and protease-dependent. But this method also provides quantitative kinetic rate parameters for intermediate steps in the coronavirus fusion pathway, which to our knowledge have not been obtained before. Moreover, the platform offers versatile, precise control over the sequence of triggers for fusion; these triggers may define the fusion pathway, tissue tropism, and pathogenicity of coronaviruses. Systematically varying these triggers in this platform provides a new route to study how viruses rapidly adapt to other hosts, and to identify factors that led to the emergence of zoonotic viruses, such as human SARS-CoV and the newly emerging human MERS-CoV.

Single particle assay of coronavirus membrane fusion with proteinaceous receptor-embedded supported bilayers.

Highly pathogenic human coronaviruses associated with a severe respiratory syndrome, including Middle East respiratory syndrome coronavirus (MERS-CoV), have recently emerged. The MERS-CoV epidemic started in 2012 and is still ongoing, with a mortality rate of approximately 35%. No vaccine is available against MERS-CoV and therapeutic options for MERS-CoV infections are limited to palliative and supportive care. A search for specific antiviral treatments is urgently needed. Coronaviruses are enveloped viruses, with the spike proteins present on their surface responsible for virus entry into the target cell. Lectins are attractive anti-coronavirus candidates because of the highly glycosylated nature of the spike protein. We tested the antiviral effect of griffithsin (GRFT), a lectin isolated from the red marine alga Griffithsia sp. against MERS-CoV infection. Our results demonstrate that while displaying no significant cytotoxicity, griffithsin is a potent inhibitor of MERS-CoV infection. Griffithsin also inhibits entry into host cells of particles pseudotyped with the MERS-CoV spike protein, suggesting that griffithsin inhibits spike protein function during entry. Spike proteins have a dual function during entry, they mediate binding to the host cell surface and also the fusion of the viral envelope with host cell membrane. Time course experiments show that griffithsin inhibits MERS-CoV infection at the binding step. In conclusion, we identify griffithsin as a potent inhibitor of MERS-CoV infection at the entry step.

Middle East respiratory syndrome coronavirus infection is inhibited by griffithsin.

Virus pseudotyping is a useful and safe technique for studying entry of emerging strains of influenza virus. However, few studies have compared different reassortant combinations in pseudoparticle systems, or compared entry kinetics of native viruses and their pseudotyped analogs. Here, vesicular stomatitis virus (VSV)-based pseudovirions displaying distinct influenza virus envelope proteins were tested for fusion activity. We produced VSV pseudotypes containing the prototypical X-31 (H3) HA, either alone or with strain-matched or mismatched N2 NAs. We performed single-particle fusion assays using total internal reflection fluorescence microscopy to compare hemifusion kinetics among these pairings. Results illustrate that matching pseudoparticles behaved very similarly to native virus. Pseudoparticles harboring mismatched HA-NA pairings fuse at significantly slower rates than native virus, and NA-lacking pseudoparticles exhibiting the slowest fusion rates. Relative viral membrane HA density of matching pseudoparticles was higher than in mismatching or NA-lacking pseudoparticles. An equivalent trend of HA expression level on cell membranes of HA/NA co-transfected cells was observed and intracellular trafficking of HA was affected by NA co-expression. Overall, we show that specific influenza HA-NA combinations can profoundly affect the critical role played by HA during entry, which may factor into viral fitness and the emergence of new pandemic influenza viruses.

Viral fusion efficacy of specific H3N2 influenza virus reassortant combinations at single-particle level.

The conserved transmembrane protein, HAP2/GCS1, has been linked to fertility in a wide range of taxa and is hypothesized to be an ancient gamete fusogen. Using template-based structural homology modeling, we now show that the ectodomain of HAP2 orthologs from Tetrahymena thermophila and other species adopt a protein fold remarkably similar to the dengue virus E glycoprotein and related class II viral fusogens. To test the functional significance of this predicted structure, we developed a flow-cytometry-based assay that measures cytosolic exchange across the conjugation junction to rapidly probe the effects of HAP2 mutations in the Tetrahymena system. Using this assay, alterations to a region in and around a predicted "fusion loop" in T. thermophila HAP2 were found to abrogate membrane pore formation in mating cells. Consistent with this, a synthetic peptide corresponding to the HAP2 fusion loop was found to interact directly with model membranes in a variety of biophysical assays. These results raise interesting questions regarding the evolutionary relationships of class II membrane fusogens and harken back to a long-held argument that eukaryotic sex arose as the byproduct of selection for the horizontal transfer of a "selfish" genetic element from cell to cell via membrane fusion.

Structure-Function Studies Link Class II Viral Fusogens with the Ancestral Gamete Fusion Protein HAP2.

Coronaviruses (CoVs) are a major infectious disease threat and include the pathogenic human pathogens of zoonotic origin: severe acute respiratory syndrome CoV (SARS-CoV) and Middle East respiratory syndrome CoV (MERS-CoV). Entry of CoVs into host cells is mediated by the viral spike (S) protein, which is structurally categorized as a class I viral fusion protein, within the same group as influenza virus and HIV. However, S proteins have two distinct cleavage sites that can be activated by a much wider range of proteases. The exact location of the CoV fusion peptide (FP) has been disputed. However, most evidence suggests that the domain immediately downstream of the S2' cleavage site is the FP (amino acids 798-818 SFIEDLLFNKVTLADAGFMKQY for SARS-CoV, FP1). In our previous electron spin resonance spectroscopic studies, the membrane-ordering effect of influenza virus, HIV, and Dengue virus FPs has been consistently observed. In this study, we used this effect as a criterion to identify and characterize the bona fide SARS-CoV FP. Our results indicate that both FP1 and the region immediately downstream (amino acids 816-835 KQYGECLGDINARDLICAQKF, FP2) induce significant membrane ordering. Furthermore, their effects are calcium dependent, which is consistent with in vivo data showing that calcium is required for SARS-CoV S-mediated fusion. Isothermal titration calorimetry showed a direct interaction between calcium cations and both FPs. This Ca-dependency membrane ordering was not observed with influenza FP, indicating that the CoV FP exhibits a mechanistically different behavior. Membrane-ordering effects are greater and penetrate deeper into membranes when FP1 and FP2 act in a concerted manner, suggesting that they form an extended fusion "platform."

ArticleTotal : 2	Year
Publication title Cited by 24	2019
Publication title Cited by 89	2019

Article	Year
Characterization of a recombinant canine coronavirus with a distinct receptor-binding (S1) domain. Virology\| PubMed ID: 22609354	2012
Mechanisms of coronavirus cell entry mediated by the viral spike protein. Viruses\| PubMed ID: 22816037	2012
Membrane fusion-competent virus-like proteoliposomes and proteinaceous supported bilayers made directly from cell plasma membranes. Langmuir : the ACS journal of surfaces and colloids\| PubMed ID: 23631561	2013
Mutation in spike protein cleavage site and pathogenesis of feline coronavirus. Emerging infectious diseases\| PubMed ID: 23763835	2013
Single particle assay of coronavirus membrane fusion with proteinaceous receptor-embedded supported bilayers. Biomaterials\| PubMed ID: 23886734	2013
Middle East respiratory syndrome coronavirus infection is inhibited by griffithsin. Antiviral research\| PubMed ID: 27424494	2016
Viral fusion efficacy of specific H3N2 influenza virus reassortant combinations at single-particle level. Scientific reports\| PubMed ID: 27752100	2016
Structure-Function Studies Link Class II Viral Fusogens with the Ancestral Gamete Fusion Protein HAP2. Current biology : CB\| PubMed ID: 28238660	2017
The SARS-CoV Fusion Peptide Forms an Extended Bipartite Fusion Platform that Perturbs Membrane Order in a Calcium-Dependent Manner. Journal of molecular biology\| PubMed ID: 29056462	2017

Jean K. Millet

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