Manipulation of Human Immunodeficiency Virus Restriction Factors: RPRD2, SERINC3 and SERINC5
AbstractBackground: There is a need for a newer approach to tackle human immunodeficiency virus (HIV) due to limitations of the current antiretrovirals, innate cell restriction factors offer such opportunity. This study seeks to characterise three novel HIV restriction factors and explore their therapeutic potentials. Methods: Jurkat cell line was treated with polyinosinic-polycytidylic acid (poly I:C), a toll-like receptor-3 agonist (TLR3), at different concentrations (5 µg/ml, 10 µg/ml, 20 µg/ml) and (4 µg/ml, 8 µg/ml, 12 µg/ml) and untreated controls. Its effects on cellular proliferation were observed over many hours. A bioinformatic search of Regulation of nuclear pre-mRNA domain-containing 2 (RPRD2) and Serine incorporator 3 and 5 (SERINC3 and SERINC5) were conducted to predict for nuclear localisation signal (NLS) and potential ubiquitination and Sumoylation sites in the proteins using online NLS Mapper, UbPred and GPS-SUMO tools respectively. Small interference RNA (siRNA) transfection of Jurkat cell line was done to knockdown karyopherin alpha 2 (KPNA2) using Lipofectamine 3000. Western blot analysis was done to assess transfection efficiency. Results: Jurkat cells treated with 5 µg/ml, 10 µg/ml and 20 µg/ml proliferated more than the control while those treated with 4 µg/ml, 8 µg/ml and 12 µg/ml proliferated less with statistical significance between the untreated and 4 µg/ml concentration at 72 hours (p = 0.021).RPRD2 was the only protein that has NLS (RDPFHSLKRPRPPFARGPPFFAPKRPFFP)at position 1430 with a score of 9.8. RPRD2 has a site predicted each for SUMO interaction and sumoylation consensus (p = 0.022), SERINC3 had six sites for SUMO interactions and 2 for sumoylation non-consensus, all with no statistical significance and SERINC5 has a significant SUMO interaction site at position 44-48 (p = 0.049). The predicted ubiquitination sites for RPRD2 were 44 (ten with high, 27 with medium and seven with low confidence respectively), SERINC3 had four sites (two with medium and low confidence each) and SERINC5 had three (one with high, medium and low confidence each). The KPNA2 knockdown was not successful. Conclusion: HIV restriction factors present a potential therapeutic target; adequate characterisation of these proteins is important towards fashioning drugs in this regard. Keywords: Toll-like receptor 3, Polyinosinic-polycytidylic acid (poly I:C), Nuclear localisation signal, Sumoylation, Ubiquitination, Karyopherin alpha 2 (KPNA2)
CitationAriyo, O. E. (2020). Manipulation of Human Immunodeficiency Virus Restriction Factors: RPRD2, SERINC3 and SERINC5. (Masters thesis). University of Chester, United Kingdom.
PublisherUniversity of Chester
TypeThesis or dissertation
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