Proteome-wide cross-linking mass spectrometry to identify specific virus capsid-host interactions between tick-borne encephalitis virus and neuroblastoma cells

Abstract

Virus-host protein-protein interactions are central to viral infection, but are challenging to identify and characterise, especially in complex systems involving intact viruses and cells. In this work, we demonstrate a proteome-wide approach to identify virus-host interactions using chemical cross-linking coupled with mass spectrometry. We adsorbed tick-borne encephalitis virus onto metabolically-stalled neuroblastoma cells, covalently cross-linked interacting virus-host proteins, and performed limited proteolysis to release primarily the surface-exposed proteins for identification by mass spectrometry. Using the intraviral protein cross-links as an internal control to assess cross-link confidence levels, we identified 22 high confidence unique intraviral cross-links and 59 high confidence unique virus-host protein-protein interactions. The identified host proteins were shown to interact with eight distinct sites on the outer surface of the virus. Notably, we identified an interaction between the substrate-binding domain of heat shock protein family A member 5, an entry receptor for four related flaviviruses, and the hinge region of the viral envelope protein. We also identified host proteins involved in endocytosis, cytoskeletal rearrangement, or located in the cytoskeleton, suggesting that entry mechanisms for tick-borne encephalitis virus could include both clathrin-mediated endocytosis and macropinocytosis. Additionally, cross-linking of the viral proteins showed that the capsid protein forms dimers within tick-borne encephalitis virus, as previously observed with purified C proteins for other flaviviruses. This method enables the identification and mapping of transient virus-host interactions, under near-physiological conditions, without the need for genetic manipulation.Author summaryTick-borne encephalitis virus is an important human pathogen that can cause severe infection often resulting in life-long neurological complications or even death. As with other viruses, it fully relies on the host cells, and any successful infection starts with interactions between the viral structural proteins and cellular surface proteins. Mapping these interactions is essential both for the fundamental understanding of viral entry mechanisms, and for guiding the design of new antiviral drugs and vaccines. Here, we stabilise the interactions between tick-borne encephalitis virus and human proteins by chemical cross-linking. We then detect the interactions using mass spectrometry and analyse the data to identify protein-protein complexes. We demonstrate that we can visualise the protein interaction interfaces by mapping the cross-linked sites onto the host and viral protein structures. We reveal that there are eight distinct sites on the outer surface of the viral envelope protein that interact with host. Using this approach, we mapped interactions between the tick-borne encephalitis virus envelope protein, and 59 host proteins, identifying a possible new virus receptor. These results highlight the potential of chemical cross-linking coupled with mass spectrometry to identify and map interactions between viral and host proteins.

Simultaneous Membrane and RNA Binding by Tick-Borne Encephalitis Virus Capsid Protein

“…The first 17 residues of the C have been reported to be dispensable for TBEV particle assembly, which is consistent with our in vitro results, and supports that the N-terminus primarily has a role in modulating the host response (Kofler et al , 2002; Yang et al , 2002; Limjindaporn et al , 2007; Colpitts et al , 2011; Bhuvanakantham & Ng, 2013; Katoh et al , 2013; Urbanowski & Hobman, 2013; Samuel et al , 2016; Slomnicki et al , 2017; Fontaine et al , 2018). As the initial membrane binding and RNA recruitment are charge-mediated and the charged N-terminus is not needed for either of these functions, it seems like the positively-charged α 4 –α 4 interface would bind both, unless conformational changes occur during membrane insertion expose additional binding sites (Figure S1) (Pulkkinen et al , 2018; Barrass et al , 2021). Based on the QCM-D results, the binding followed two different kinetics, and after binding and insertion, only half of the C protein could be removed by a high NaCl wash.…”

“…These additional molecules would more likely be removed by the high NaCl wash, as they would not be inserted into the membrane due to steric hindrance from the first layer of protein. The only evidence for regular interactions in the virion is that of a dimer from cross-linking mass spectrometry, but in order to form the NC, it is highly likely that there are additional, higher-order C protein interactions (Barrass et al , 2021). The results support that C binding to negatively-charged surfaces is an important step in RNA recruitment, just as in dengue virus (DENV) (Mebus-Antunes et al , 2022).…”

“…The figure was created with the homology model from Barrass et al . (2021) and the electrostatic potential of the surfaces were calculated in ChimeraX (Goddard et al , 2018)…”

“…The TBEV C protein is a dimer both in solution, and within the virion, although higher-order oligomerisation has been suggested to occur in other flaviviruses such as Zika virus (Kiermayr et al , 2004; Kaufman et al , 2020; Tan et al , 2020; Barrass et al , 2021). The C protein contains a positively-charged, flexible N-terminus (17 aa) and four α-helices (α 1 –α 4 ), with dimerization occurring via antiparallel interactions of the α 2 and α 4 helices (Ma et al , 2004; Dokland et al , 2004; Shang et al , 2018; Boon et al , 2018; Pulkkinen et al , 2018; Poonsiri et al , 2019; Barrass et al , 2021). The α 4 helix contains multiple positively-charged residues, thought to form a charged surface responsible for nucleic acid binding (Samuel et al , 2016; Shang et al , 2018; Kaufman et al , 2020).…”

Molecular Organisation of Tick-Borne Encephalitis Virus

“…The heterotetramers pack tightly together to form the icosahedrally symmetric lattice of the virion surface. The E protein is the major TBEV antigen, and is responsible for receptor binding and membrane fusion [5][6][7][8][9][10]. The M Viruses 2022, 14, 792 2 of 17 protein is initially synthesised as its precursor prM, which interacts with E and protects its fusion loop from premature activation.…”