Vpliv strukturnih proteinov M, N, S in E koronavirusa SARS-CoV-2 na izločanje citokinov in izražanje proteinov APOBEC3 v človeških celicah A549 in HuH-7

Turk, Špela (author), Lovšin, Marija Nika (mentor)

Pandemija koronavirusne bolezni 2019 (COVID-19), ki jo povzroča hudi akutni respiratorni sindrom koronavirus-2 (SARS-CoV-2), je močno vplivala na zdravje ljudi po vsem svetu. Visoka prenosljivost in patogenost virusa je v zadnjih letih vzpodbudila znaten napredek v raziskavah, vključno z velikimi preboji v razvoju cepiv, možnostih zdravljenja, razumevanju prenosa in pojavljanja novih različic. Virus SARS-CoV-2 je RNA virus iz družine Betacoronavirusov, sestavljen iz strukturnih proteinov M, N, S, E, nestrukturnih in pomožnih proteinov. V gostiteljske celice vstopi prek encima angiotenzin konvertaza 2 in se v njih začne razmnoževati, celice gostitelja pa so opremljene z mehanizmi, ki jim omogočajo hitro prepoznavanje virusov in odziv nanje. Mednje sodijo tudi citokini in katalitičnemu polipeptidu podobni encimi, ki preurejajo mRNA apolipoproteina B (APOBEC), ki se začnejo v sklopu imunskega odziva ob okužbi pospešeno izražati in sodelovati pri inaktivaciji virusa. V magistrskem delu smo želeli raziskati, kako prekomerna izraženost strukturnih proteinov M, N, S, E SARS-CoV-2 vpliva na izražanje citokinov in proteinov APOBEC3 v celičnih linijah A549 in Huh-7. Ustrezno pripravljene celice smo najprej transficirali s plazmidi z zapisi za SARS-CoV-2 strukturne, pomožne in kontrolne proteine. Po 48 urah smo z encimskoimunskim testom preverili prisotnost citokinov IL-6 v celičnem gojišču. Iz transficiranih celic smo izolirali mRNA, jo s pomočjo reverzne transkripcije prepisali v stabilnejšo komplementarno DNA in na koncu pridobili kvantitativne rezultate izražanja genov za posamezne citokine in proteine APOBEC3 z verižno reakcijo s polimerazo v realnem času. Ugotovili smo, da v celicah Huh-7 izražanje mRNA citokinov IL-8 in TNF-α ter proteinov A3B in A3F najbolj poviša vnos strukturnih proteinov S, E in M. Vpliv pomožnega proteina ORF6 pri celicah A549 kaže na sorazmerje med izražanjem citokinov in proteinov iz družine APOBEC, raziskava ELISA pa ni pokazala povečanega izločanja citokina IL-6 v supernatantih preiskovanih celičnih vzorcih. Iz naših rezultatov sklepamo, da strukturni proteini S, E in M vplivajo na povišano izražanje citokinov in proteinov APOBEC in izzovejo imunski odziv.

• Language: Slovenian
• Keywords: SARS-CoV-2, proteini APOBEC3, citokini, A549, Huh-7
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2024
• PID: 20.500.12556/RUL-156252
• Publication date in RUL: 16.05.2024

Secondary language

• Language: English
• Title: The effect of M, N, S, and E structural proteins of the SARS-CoV-2 virus on cytokine secretion and expression of APOBEC3 proteins in human A549 and HuH-7 cells

Abstract

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has had a significant impact on global health. The high transmissibility and pathogenicity of the virus has led to significant advances in research in recent years, including major breakthroughs in vaccine development, treatment options, understanding transmission and the emergence of new variants. The SARS-CoV-2 virus is an RNA virus from the Betacoronavirus family, consisting of the structural proteins M, N, S, E, non-structural and accessory proteins. It enters the host cells via the angiotensin-converting enzyme 2 and begins to multiply there. The cells, on the other hand, are equipped with mechanisms that enable them to quickly recognize viruses and react to them. These mechanisms include cytokines and apolipoprotein B mRNA editing catalytic polypeptide-like proteins (APOBEC), which begin to express at an accelerated rate as part of the immune response to infection, and are involved in virus inactivation. In the master thesis, we wanted to investigate how the overexpression of SARS-CoV-2 structural proteins M, N, S, E affects the expression of cytokines and APOBEC3 proteins in the A549 and Huh-7 cell lines. Appropriately prepared cells were first transfected with plasmids containing SARS-CoV-2 gene sequences of structural, accessory and control proteins. After 48 hours, an enzyme-linked immunosorbent assay was performed for the presence of IL-6 cytokines in the cell medium. mRNA was isolated from the transfected cells and transcribed into more stable complementary DNA by reverse transcription. Finally, we obtained quantitative gene expression results for individual cytokines and APOBEC3 proteins by real-time quantitative polymerase chain reaction. We found that in Huh-7 cells, the mRNA expression of cytokines IL-8 and TNF-α, as well as A3B and A3F proteins was most strongly increased by the cell-mediated intake of structural proteins S, E and M. The effect of ORF6 accessory protein in A549 cells indicates a correlation between the expression of cytokines and APOBEC proteins, and the ELISA assay showed no increased IL-6 cytokine excretion in supernatants of test cell samples. From our results, we conclude that structural proteins S, E and M influence the increased expression of cytokines and APOBEC proteins and induce an immune response.

• Keywords: SARS-CoV-2, APOBEC3 proteins, cytokines, A549, Huh-7