Characterisation of protease activity during SARS-CoV-2 infection identifies novel viral cleavage sites and cellular targets with therapeutic potential

Meyer, Björn; Chiaravalli, Jeanne; Gellenoncourt, Stacy; Brownridge, Philip; Bryne, Dominic P.; Daly, Leonard A.; Grauslys, Arturas; Walter, Marius; Agou, Fabrice; Chakrabarti, Lisa A.; Craik, Charles S.; Eyers, Claire E.; Eyers, Patrick A.; Gambin, Yann; Jones, Andrew R.; Sierecki, Emma; Verdin, Eric; Vignuzzi, Marco; Emmott, Edward

doi:10.1101/2020.09.16.297945

Cited by 16 publications

(31 citation statements)

References 81 publications

(117 reference statements)

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“…26,27,28 New evidence reveals proteolytic cleavage of multiple viral proteins during SARS-CoV-2 infection, including extensive cleavage of the N protein. 29 Our results share striking similarities with the cleavage sites observed for both SARS-CoV and N protein from SARS-CoV-2 infected cells 29 and therefore elucidating the role of N proteoforms represents a necessary endeavor for targeting cellular processes involved in viral proliferation.…”

Section: Resultssupporting

confidence: 70%

Proteoforms of the SARS-CoV-2 nucleocapsid protein are primed to proliferate the virus and attenuate the antibody response

Lutomski

El‐Baba

Bolla

et al. 2020

Preprint

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The SARS-CoV-2 nucleocapsid (N) protein is the most immunogenic of the structural proteins and plays essential roles in several stages of the virus lifecycle. It is comprised of two major structural domains: the RNA binding domain, which interacts with viral and host RNA, and the oligomerization domain which assembles to form the viral core. Here, we investigate the assembly state and RNA binding properties of the full-length nucleocapsid protein using native mass spectrometry. We find that dimers, and not monomers, of full-length N protein bind RNA, implying that dimers are the functional unit of ribonucleoprotein assembly. In addition, we find that N protein binds RNA with a preference for GGG motifs which are known to form short stem loop structures. Unexpectedly, we found that N undergoes autoproteolytic processing within the linker region, separating the two major domains. This process results in the formation of at least five proteoforms that we sequenced using electron transfer dissociation, higher-energy collision induced dissociation and corroborated by peptide mapping. The cleavage sites identified are in highly conserved regions leading us to consider the potential roles of the resulting proteoforms. We found that monomers of N-terminal proteoforms bind RNA with the same preference for GGG motifs and that the oligomeric state of a C-terminal proteoform (N156-419) is sensitive to pH. We used mass spectrometry to show that N binds to a monoclonal antibody raised against full-length N. No antibody interactions were detected for N proteoforms without C-terminal residues, therefore locating antigenic regions towards the C-terminus. We then tested interactions of the proteoforms with the immunophilin cyclophilin A, a key component in coronavirus replication. We found that N1-209 and N1-273 bind directly to cyclophilin A, an interaction that is abolished by the approved immunosuppressant drug cyclosporin A. We propose that the proteoforms generated via autoproteolysis evade antibody detection through removal of the antigenic C-terminus and facilitate interactions with structured RNA or cyclophilin thereby enabling the virus to proliferate.

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Section: Resultssupporting

confidence: 70%

Proteoforms of the SARS-CoV-2 nucleocapsid protein are primed to proliferate the virus and attenuate the antibody response

Lutomski

El‐Baba

Bolla

et al. 2020

Preprint

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“…Data type Raw data Identification/ feature data Code Bezstarosti et al [8] proteomics yes partial NA Bojkova et al [12] proteomics yes partial no Bouhaddou et al [13] proteomics yes partial no Cai et al [16] metabolomics yes partial partial Cardozo et al [17] proteomics no no no Cazares et al [18] proteomics no no NA D'Alessandro et al [21] proteomics yes no no Davidson et al [22] proteomics yes no no Gordon et al [40] proteomics yes partial partial Gao et al [35] glycoproteomics no no no Gouveia et al [41] proteomics yes yes NA Gouveia et al [42] proteomics partial partial NA Grenga et al [43] proteomics yes yes no Ihling et al [55] proteomics yes yes NA Iles et al [56] proteomics no no NA Kimhofer et al [63] metabolomics no no no Klann et al [64] proteomics yes partial no Laurent et al [67] proteomics no no no Li et al [68] proteomics no no no Messner et al [76] proteomics no no no Meyer et al [77] proteomics yes partial yes Nachtigall et al [81] proteomics partial partial no Nikolaev et al [84] proteomics yes partial NA Overmyer et al [89] metabolomics, proteomics, lipidomics yes partial no…”

Section: Referencementioning

confidence: 99%

Open Science Resources for the Mass Spectrometry-Based Analysis of SARS-CoV-2

et al. 2021

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The SARS-CoV-2 virus is the causative agent of the 2020 pandemic leading to the COVID-19 respiratory disease. With many scientific and humanitarian efforts ongoing to develop diagnostic tests, vaccines, and treatments for COVID-19, and to prevent the spread of SARS-CoV-2, mass spectrometry research, including proteomics, is playing a role in determining the biology of this viral infection. Proteomics studies are starting to lead to an understanding of the roles of viral and host proteins during SARS-CoV-2 infection, their protein-protein interactions, and post-translational modifications. This is beginning to provide insights into potential therapeutic targets or diagnostic strategies that can be used to reduce the long-term burden of the pandemic. However, the extraordinary situation caused by the global pandemic is also highlighting the need to improve mass spectrometry data and workflow sharing. We therefore describe freely available data and computational resources that can facilitate and assist the mass spectrometry-based analysis of SARS-CoV-2. We exemplify this by reanalyzing a virus-host interactome dataset to detect protein-protein interactions and identify host proteins that could potentially be used as targets for drug repurposing.

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“…4A). The S1 coding sequence in the segment 7 ORF includes an Nterminal signal sequence which, in SARS-CoV-2 infected cells, is cleaved from the S1 protein during synthesis on the endoplasmic reticulum (ER) (29,38). Cleavage of the signal sequence may have removed the upstream 3x FLAG tag from a S1 product, preventing its detection by the FLAG antibody.…”

Section: Expression Of S Coding Sequences By Rsa11 Rotavirusesmentioning

confidence: 99%

Rotavirus as an Expression Platform of the SARS-CoV-2 Spike Protein

Philip

Patton

2021

Preprint

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Rotavirus, a segmented double-stranded RNA virus, is a major cause of acute gastroenteritis in young children. The introduction of live oral rotavirus vaccines has reduced the incidence of rotavirus disease in many countries. To explore the possibility of establishing a combined rotavirus-SARS-CoV-2 vaccine, we generated recombinant (r)SA11 rotaviruses with modified segment 7 RNAs that contained coding sequences for NSP3 and FLAG-tagged portions of the SARS-CoV-2 spike (S) protein. A 2A translational element was used to drive separate expression of NSP3 and the S product. rSA11 viruses were recovered that encoded the S-protein S1 fragment, N-terminal domain (NTD), receptor-binding domain (RBD), extended receptor-binding domain (ExRBD), and S2 core (CR) domain (rSA11/NSP3-fS1, -fNTD, -fRBD, -fExRBD, and -fCR, respectively). Generation of rSA11/fS1 required a foreign-sequence insertion of 2.2-kbp, the largest such insertion yet made into the rotavirus genome. Based on isopycnic centrifugation, rSA11 containing S sequences were denser than wildtype virus, confirming the capacity of the rotavirus to accommodate larger genomes. Immunoblotting showed that rSA11/-fNTD, -fRBD, -fExRBD, and -fCR viruses expressed S products of expected size, with fExRBD expressed at highest levels. These rSA11 viruses were genetically stable during serial passage. In contrast, rSA11/NSP3-fS1 failed to express its expected 80-kDa fS1 product, for unexplained reasons. Moreover, rSA11/NSP3-fS1 was genetically unstable, with variants lacking the S1 insertion appearing during serial passage. Nonetheless, these results emphasize the potential usefulness of rotavirus vaccines as expression vectors of portions of the SARS-CoV-2 S protein (e.g., NTD, RBD, ExRBD, and CR) with sizes smaller than the S1 fragment.

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Characterisation of protease activity during SARS-CoV-2 infection identifies novel viral cleavage sites and cellular targets with therapeutic potential

Cited by 16 publications

References 81 publications

Proteoforms of the SARS-CoV-2 nucleocapsid protein are primed to proliferate the virus and attenuate the antibody response

Proteoforms of the SARS-CoV-2 nucleocapsid protein are primed to proliferate the virus and attenuate the antibody response

Open Science Resources for the Mass Spectrometry-Based Analysis of SARS-CoV-2

Rotavirus as an Expression Platform of the SARS-CoV-2 Spike Protein

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