Location: Foreign Animal Disease Research
Project Number: 8064-32000-063-016-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Jun 1, 2020
End Date: May 31, 2023
Using the viral proteome as a starting point, this collaborative research project seeks to identify and characterize possible viroporin candidates as incorporated into the African Swine Fever Virus (ASFV) particle, which could assist in the processes of viral entry and/or assembly. We hypothesize that membrane cholesterol could play a role in this process. In addition, low pH-activated viroporins could confine the assembly of new particles to cell compartments with a propitious neutral pH environment, and even assist in the pinching-off of the new particles. several research activities to establish the membrane-permeabilizing activity of the candidates and characterize them structurally, as a function of cholesterol and low pH. Specifically, several successive activities will be performed for the detection and characterization of possible structural viroporins in ASFV, which may be involved in the regulation of both viral entry and assembly processes during the replicative cycle: 1. Bioinformatics prediction 2. Design of sequences and synthesis and/or expression 3. Screening for active viroporin-like sequences 4. Functional characterization of selected candidates in vitro 5. Localization and functional characterization of selected candidates in cells 6. Mutation design and evaluation in vitro 7: Structural characterization of ASFV proteins and their mutants 8: Mining non-structural genes for identification of additional viroporin candidates
1: Bioinformatics –prediction: mining the proteome Among the identified proteins that take part in ASFV particle composition we will search for regulatory, viroporin-like proteins that are: incorporation at low quantities; small size; bearing predicted membrane-spanning domains. Candidates will be selected and analyzed to determine positions of conserved residues potentially implied in connecting transmembrane helices or filtering solutes in the lumen of the permeating pores. 2: Design of sequences and synthesis and/or expression Using the information generated in the previous section, synthetic peptides will be devised so that they span the different domains that compose the selected protein sequences. In some instances, full proteins will be synthesized. 3: Screening for active viroporin-like sequences Three to five viroporin candidates among the members of the ASFV virion proteome will be identified. It is estimated that about 3 peptides per protein will be required for an initial screening. Candidates will be screened and tested. Two lipid compositions will be compared, containing or not containing cholesterol, and measurements will be carried out at neutral and low-pH. 4: Functional characterization of selected candidates in vitro Characterization of the mechanism of membrane-porating activity of the sequences identified in the screening step. 5: Localization and functional characterization of selected candidates in cells. The preferential localization in the cell and possible permeabilization of organelles to calcium will be tested. The goal of these studies is to establish whether a preferential localization exists in the cell for a given candidate. From the available lipidomes, we will then infer the lipid environment in which the protein should perform its function. In the case of endoplasmic reticulum or mitochondria permeabilization, we can also establish in living cells if these proteins operate following a viroporin-like mechanism. 6: Mutation design and evaluation in vitro The aforementioned activities will allow mapping key regions involved in ion conductance and solute permeability, such that the effect of mutations directed to these regions can be determined. Thus, in parallel, new sequences bearing the selected mutations will be tested in the functional assays described above. 7: Structural characterization of ASFV proteins and their mutants Structural characterization will be conducted through both low and high-resolution techniques. 8: Mining non-structural genes for identification of additional viroporin candidates Apart from the viroporins incorporated to the particle, during the early stages of the replication cycle the virus genome is likely to express viroporins involved in the permeabilization of organelles.