Vpliv prekomernega izražanja strukturnih proteinov M, N, S in E ter pomožnega proteina ORF6 virusa SARS-CoV-2 na izražanje genov iz družine APOBEC v človeških celičnih linijah HuH-7 in HeLa

Levačić, Patrik (avtor), Lovšin, Marija Nika (mentor)

Proteini APOBEC so citidinske deaminaze, skupina encimov, ki deaminirajo citidine v uridine na enojnoverižnih polinukleotidih. Njihova osnovna funkcija je urejanje RNA molekul, vendar lahko nekateri od teh proteinov urejajo tudi DNA molekule in s tem sodelujejo v procesih, ki vplivajo na genomsko variabilnost. Proteini APOBEC so protivirusni proteini in preprečijo razmnoževanje različnim RNA virusom, DNA virusom in retroelementom. Povezava med proteini APOBEC in virusom SARS-CoV-2 izhaja iz raziskav, ki so razkrile, da ti proteini lahko vplivajo na genom virusa. Med virusno okužbo se lahko izražanje APOBEC genov spremeni, kar lahko privede do nastanka mutacij v genomu SARS-CoV-2. Te mutacije lahko vplivajo na lastnosti virusa, kot je npr. sposobnost razmnoževanja, izmikanje imunskemu sistemu ali odpornost na zdravila. Poleg tega mutacije v genomu virusa prispevajo k njegovi evoluciji in pospešijo nastanek novih sevov ter s tem vplivajo na razširjenost virusa v populaciji. Izražanje genov iz družine APOBEC je običajno povišano med virusno okužbo, vendar ni popolnoma jasno, kakšen je vpliv na izražanje teh genov pri virusu SARS-CoV-2. Zato smo se v nalogi osredotočili na preučevanje vpliva prekomernega izražanja proteinov SARS-CoV-2 na izražanje genov APOBEC. V ta namen smo uporabili sesalski celični liniji, Huh-7 (izvorno izhaja iz hepatocelularnega tumorja) in HeLa (izvorno izhaja iz raka materničnega vratu). Sesalske celice smo transfecirali z različnimi plazmidnimi vektorji, ki so vsebovali zapis za strukturne proteine M, N, S, E in pomožni protein ORF6 virusa SARS-CoV-2 in s kvantitativno verižno reakcijo s polimerazo v realnem času (qPCR) izmerili nivo izražanja genov A1, A3A, A3B, A3C, A3D, A3F in A3G. Pri celični liniji Huh-7 je pri prekomernem izražanju strukturnega proteina N in pomožnega proteina ORF6 prišlo do znižanja izražanja gena A1, pri ostalih genih vpliv prekomernega izražanja proteinov SARS-CoV-2 na izražanje genov APOBEC nismo uspeli dokazati. Pri celični liniji HeLa vpliv prekomernega izražanja proteinov SARS-CoV-2 na izražanje genov iz družine APOBEC prav tako nismo uspeli dokazati. Rezultati raziskovalnega dela so pokazali, da prekomerno izražanje strukturnega proteina N in pomožnega proteina ORF6 zniža izražanje gena A1 in so v nasprotju s pričakovanji, saj naj bi se izražanje genov APOBEC med virusno okužbo zvišalo. Za boljše razumevanje vpliva na izražanje teh genov so potrebne nadaljnje raziskave.

• Jezik: Slovenski jezik
• Ključne besede: Proteini APOBEC, COVID-19, HeLa, Huh-7, SARS-CoV-2.
• Vrsta gradiva: Magistrsko delo/naloga
• Organizacija: FFA - Fakulteta za farmacijo
• Leto izida: 2023
• PID: 20.500.12556/RUL-150739
• Datum objave v RUL: 22.09.2023

Sekundarni jezik

• Jezik: Angleški jezik
• Naslov: The effect of SARS-CoV-2 virus structural proteins M, N, S, E and accessory protein ORF6 overexpression on the APOBEC gene family expression in human HuH-7 and HeLa cell lines

Izvleček

APOBEC proteins are cytidine deaminases, a group of enzymes that deaminate cytidines into uridines on single-stranded polynucleotides. Their primary function is editing RNA molecules, but some of these proteins can also edit DNA molecules, participating in processes that influence genomic variability. APOBEC proteins are antiviral and inhibit the replication of various RNA viruses, DNA viruses, and retroelements. The connection between APOBEC proteins and the SARS-CoV-2 virus arises from research that has revealed these proteins can impact the virus's genome. During a viral infection, the expression of APOBEC genes can change, potentially leading to mutations in the SARS-CoV-2 genome. These mutations can affect the virus's characteristics, such as its replication ability, evasion of the immune system, or drug resistance. Furthermore, mutations in the viral genome contribute to its evolution, speeding up the emergence of new strains and influencing the virus's spread within the population. Gene expression from the APOBEC family is typically elevated during viral infections. However, it is not entirely clear how this affects the expression of these genes in the case of the SARS-CoV-2 virus. Therefore, our study focused on investigating how the overexpression of SARS-CoV-2 proteins affects the expression of APOBEC genes. To achieve this, we used mammalian cell lines, Huh-7 (originating from hepatocellular carcinoma) and HeLa (originating from cervical cancer). We transfected mammalian cells with various plasmid vectors containing the coding sequences for the structural proteins M, N, S, E, and the accessory protein ORF6 of the SARS-CoV-2 virus. Using quantitative real-time polymerase chain reaction (qPCR), we measured the expression levels of genes A1, A3A, A3B, A3C, A3D, A3F, and A3G. In the Huh-7 cell line, overexpression of the structural protein N and accessory protein ORF6 resulted in a decrease in the expression of gene A1. For the remaining genes, we were unable to confirm the impact of the overexpression of SARS-CoV-2 proteins on the expression of APOBEC genes. In the HeLa cell line, we also could not confirm the impact of the overexpression of SARS-CoV-2 proteins on the expression of genes from the APOBEC family. The results of our research demonstrated that the overexpression of the structural protein N and accessory protein ORF6 of SARS-CoV-2 reduced the expression of gene A1, which contradicts expectations since the expression of APOBEC genes was anticipated to increase during viral infection. Further research is needed to better understand the influence on the expression of these genes.

• Ključne besede: APOBEC proteins, COVID-19, HeLa, Huh-7, SARS-CoV-2.