Abstract Title
Cross-Species Profiling of MNV Protease Cleavage Events Using Quantitative Proteomics
Presenter
Frazer Buchanan, University of Liverpool
Co-Author(s)
Frazer J T Buchanan - Centre for Proteome Research, Institute of Systems, Molecular and Integrative Biology.
Leandro X Neves - Centre for Proteome Research, Institute of Systems, Molecular and Integrative Biology.
Edward Emmott - Centre for Proteome Research, Institute of Systems, Molecular and Integrative Biology.
Abstract Category
Entry and Replication
Abstract
Murine Norovirus (MNV) has long been used as a model virus for human noroviruses to aid in research. Noroviruses encode for highly conserved 3C like (3CL) viral proteases which are critical to regulate polyprotein processing. Emerging evidence suggests that these proteases cleave host proteins in a manner to benefit viral replication, and that in some cases the substrates themselves are pro-viral. Due to the high conservation across Caliciviridae, these enzymes and potentially their substrates offer excellent targets for the development of antiviral therapeutics. To identify these cleavage events, we undertook an unbiased proteomics approach, N-terminomics for identification and quantification of protein N-termini, including neo-N-termini resulting from (viral) proteolytic cleavage.
As an experimental model of natural infection, BV2 cells were infected with MNV (MOI=3) and cells were collected at 0, 4, 8 and 12 h post-infection (hpi) and processed for LC-MS-based proteomics. Additionally, we generated a transgenic HEK293T cell line expressing the MNV receptor CD300lf, allowing us to investigate MNV-mediated proteolysis in a human cell line. Cells were harvested and processed for N-terminomics analysis.
In BV2 cells, over 3,700 neo-N-termini were detected and quantified in the infection time-courses revealing the regulation of proteolysis during MNV infection. Motif analysis showed over 40 cleavage events after Q or E residues, matching the consensus motif for 3CL viral protease. We are currently repeating this analysis for MNV-infected HEK293T-CD300lf cells. which will expand this list and reveal 3CL-substrates conserved between murine and human cells.
This approach enables the cross-species evaluation of a conserved Calicivirus protease in both murine and human cells. Through unbiased, high-throughput profiling of viral cleavage events, we can identify host protein targets with potential relevance to infection. Our integrated methodology enables downstream validation via siRNA knockdown and microscopy, allowing investigation of these host factors in in the context of Calicivirus infection.
As an experimental model of natural infection, BV2 cells were infected with MNV (MOI=3) and cells were collected at 0, 4, 8 and 12 h post-infection (hpi) and processed for LC-MS-based proteomics. Additionally, we generated a transgenic HEK293T cell line expressing the MNV receptor CD300lf, allowing us to investigate MNV-mediated proteolysis in a human cell line. Cells were harvested and processed for N-terminomics analysis.
In BV2 cells, over 3,700 neo-N-termini were detected and quantified in the infection time-courses revealing the regulation of proteolysis during MNV infection. Motif analysis showed over 40 cleavage events after Q or E residues, matching the consensus motif for 3CL viral protease. We are currently repeating this analysis for MNV-infected HEK293T-CD300lf cells. which will expand this list and reveal 3CL-substrates conserved between murine and human cells.
This approach enables the cross-species evaluation of a conserved Calicivirus protease in both murine and human cells. Through unbiased, high-throughput profiling of viral cleavage events, we can identify host protein targets with potential relevance to infection. Our integrated methodology enables downstream validation via siRNA knockdown and microscopy, allowing investigation of these host factors in in the context of Calicivirus infection.