While VLPs will be useful components of VINCEnT, we wanted to focus on development of a novel non-immunogenic PNC with RNA packaging capabilities. Such a tool could enable simpler transfection of mammalian cell lines for fellow iGEMers and other researchers.
To do this, we identified the Rattus norvegicus Arc protein as a candidate for modelling a minimal packaging protein. Arc is an activity-regulated cytoskeletal-associated protein that has recently been recognized as a repurposed Ty3/Gypsy retrotransposon. A bi-lobar domain within Arc has significant homology to Gag proteins, which are the major capsid proteins of many viruses including Human Immunodeficiency Virus type 1 (HIV-1), Rous-Sarcoma Virus (RSV), and Bovine Leukemia Virus (BLV). In response to synaptic activity in neurons, Arc proteins self-assemble via this Gag domain (similar to the related viral particles) to encapsulate Arc mRNA and shuttle it to neighbouring cells (Pastuzyn et al., 2018; Ashley et al., 2018).
To ensure we would not retain any native Arc functionality that might impact cellular activity, we designed a “minimal” Arc Gag protein based on homology with other known Gag domains. We used template-based structural predictions to model this minimal Arc Gag and its predicted assembly into higher-order structures.
Using the EMBL-EBI Clustal Omega tool,we aligned the protein sequences of HIV-1 (GenBank BAF32552.1), BLV (GenBank BAA00543.1), and RSV (PDB 5A9E) Gag proteins to R. norvegicus Arc (NCBI Ref NP_062234.1). The most closely conserved sequences are shown below:
Based on HIV-1 homology to Arc, Zhang et al. (2015) similarly predicted a conserved Gag domain to span R. norvegicus Arc amino acids 207-278 (Gag N-lobe) and 278-370 (Gag C-lobe).
We then compared this conserved bi-lobar Gag region with the predicted RNA binding region for Gag gene products to ensure RNA packaging functionality would be maintained (Clever, Sassetti, & Parslow, 1995). A portion of the 4 stem loop RNA secondary structure in HIV-1 strongly aligns with a sequence located within the Arc Gag N-lobe:
Structural Model of Minimal Arc
Using I-TASSER(Roy, Kucukural, & Zhang, 2010; Yang et al., 2015; Zhang, 2008), we then predicted and modeled the secondary and tertiary structure of this minimal Arc Gag protein (where a higher score indicates a more confident prediction of secondary structure):
Figure 1: Minimal Arc Gag (rainbow ribbon indicating red N-terminus to blue C-terminus). Model produced with I-TASSER.
Figure 2: Minimal Arc Gag (rainbow ribbon indicating red N-terminus to blue C-terminus) overlaid with HIV-1 Gag (purple backbone; PDB ID: 3DIK). Model produced with I-TASSER using the TM-align structural alignment algorithm.
This Arc Gag subunit was predicted to form a hexamer from C-terminal subunit interactions and confirmed using GalaxyHomomer (a GalaxyWEB server for prediction of homomeric protein structures; Ko et al., 2012; Shin et al., 2014). HIV-1 and RSV Gag proteins similarly interact at the C-terminus to form hexamers for assembly of higher-order spherical multi-subunit structures (de Marco et al., 2010). Thus, we were confident that the minimal Arc Gag would similarly be capable of forming a spherical or pseudo-icosahedral nanocompartment.
Figure 3: Based on structural homology with RSV (PDB ID: 3TIR), minimal Arc Gag proteins are predicted to form a hexameric subunit (each monomer indicated by a different colour). Model produced with GalaxyWEB using template-based oligomer modeling.
- Ashley, J., Cordy, B., Lucia, D., Fradkin, L. G., Budnik, V., & Thomson, T. (2018). Retrovirus-like Gag protein Arc1 binds RNA and traffics across synaptic boutons. Cell, 172, 262-274.
- Clever, J., Sassetti, C., & Parslow, T. G. (1995). RNA secondary structure and binding sites for gag gene products in the 5' packaging signal of human immunodeficiency virus type 1. J Virol, 69, 2101-2109.
- de Marco, A., Davey, N. E., Ulbrich, P., Phillips, J. M., Lux, V., Riches, J. D., Fuzik, T., Ruml, T., Kräusslich, H.-G., Vogt, V. M., & Briggs, J. A. G. (2010). Conserved and variable features of Gag structure and arrangement in immature retrovirus particles. J Virol, 84, 11729-11736.
- Ko, J., Park, H., Heo, L., & Seok, C. (2012). GalaxyWEB server for protein structure prediction and refinement. Nucleic Acids Res., 40, W294-W297.
- Pastuzyn, E. D., Day, C. E., Kearns, R. B., Kyrke-Smith, M., Taibi, A. V., McCormick, J., Yoder, N., Belnap, D. M., Erlendsson, S., Morado, D. R., Briggs, J. A. G., Feschotte, C., & Shepherd, J. D. (2018). The neuronal gene Arc encodes a repurposed retrotransposon Gag protein that mediates intercellular RNA transfer. Cell, 172, 275-288.
- Roy, A., Kucukural, A., Zhang, Y. (2010). I-TASSER: a unified platform for automated protein structure and function prediction. Nature Protocols, 5, 725-738.
- Shin, W.-H., Lee, G. R., Heo, L., Lee, H., & Seok, C. (2014). Prediction of protein structure and interaction by GALAXY protein modeling programs. Bio Design, 2, 1-11.
- Yang, J., Yan, R., Roy, A., Xu, D., Poisson, J., Zhang, Y. (2015). The I-TASSER Suite: Protein structure and function prediction. Nature Methods, 12, 7-8.
- Zhang, W., Wu, J., Ward, M. D., Yang, S., Chuang, Y.-A., Xiao, M., Li, R., Leahy, D. J., & Worley, P. F. (2015). Structural basis of Arc binding to synaptic proteins: implications for cognitive disease. Neuron, 86, 490-500.
- Zhang, Y. (2008). I-TASSER server for protein 3D structure prediction. BMC Bioinformatics, 9, 40.