Viruses are defined as obligate intracellular parasites that rely entirely on host cell metabolic machinery during replication. The viral replication causes various alterations in cellular metabolic pathways to provide energy and macromolecules required for the production of viral progeny. Alterations in virus-infected cells often resemble those occurring in cancer cells as both are characterized by a high rate of anabolic reactions.
Over the last few decades, there have been published many studies investigating the virus-host interactions in cells infected with both RNA and DNA viruses. However, not much is known about this kind of interaction in the case of the Arenaviridae family, which comprises dangerous pathogens causing hemorrhagic fever, such as the Lassa, Junín, and Machupo virus. Our research focuses on the alterations in cells infected with lymphocytic choriomeningitis virus (LCMV), a prototypic arenavirus. Since lipids play an important role during infection with almost all viruses, we chose to analyze the impact of LCMV infection on lipid metabolism. The first objective was to analyze the gene expression of genes encoding lipogenic enzymes in mock and LCMV-infected cells. Then, we used pharmacological inhibitors to inhibit two of the analyzed enzymes and examined the impact they had on the production of new virions. Lastly, we investigated the mechanisms responsible for regulating the observed changes.
The transcriptional analysis revealed that LCMV infection increases the expression of genes coding for lipogenic enzymes at later stages of infection. This suggests that lipids may play a role during the LCMV life cycle. When we pharmacologically inhibited acetyl-CoA carboxylase (ACC), a rate-limiting enzyme catalyzing fatty acid synthesis, we observed a 20-fold reduction in LCMV titer. Additionally, inhibition of fatty acid synthase (FASN) showed similar results, which highlights the importance of de novo synthesized fatty acids during the synthesis of new LCMV infectious virions. The analysis of regulation on the transcriptional level showed that the expression of genes encoding lipogenic enzymes is at least partially regulated by sterol regulated element-binding protein (SREBP) as we observed an increase in its activity at later stages of the infection. Furthermore, we discovered that LCMV regulates ACC activity by impairing AMP-activated protein kinase (AMPK) phosphorylation.
Our results shed light on the importance of lipids, particularly fatty acids, during LCMV replication in human cells. Fatty acids have many roles, such as posttranslational modifications, energy storage, and phospholipid membrane synthesis. Therefore, it is important to conduct further analysis and acquire a more detailed understanding of the requirements for the completion of the arenavirus life cycle.
This project was supported by VEGA 2/0030/19 grant.
Vaša téma je naozaj veľmi
Vaša téma je naozaj veľmi pútavá a prezentácia veľmi pekne spracovaná. Chcel by som sa Vás preto spýtať na jednu vec z Vašej prezentácie. Na slide 13 popisujete výsledky Western blotu, na ktorých je vidieť pokles hladiny p-AMPK a naopak nárast hladiny p-ACC. Nakoľko je tento postupný pokles/rast možné sledovať nielen u ARM (LCMV infikovaných buniek), ale aj u negatívnej kontroly ("mock"), zaujímalo by ma, či tento výsledok naozaj možno s určitosťou prisúdiť aktivite LCMV vírusu alebo sa môže jednať aj o prozaickejšie vysvetlenie - a to také, že hladina p-AMPK sa jednoducho znižuje v dôsledku toho, že táto kinázy iba fosforyluje ACC, ktorého fosforylovaná forma v korelácii na pokles p-AMPK narastá (hlavne keď tento jav môžeme pozorovať aj u zmienenej negatívnej kontroly - "mock")? A to tiež aj s ohľadom na fakt, že sa nefosforylovaná a teda aktívna forma ACC po 72 hod. podľa výsledkov blotu nijak výrazne nemení. Vedeli by Ste poskytnúť aj k tomuto komentár. Dúfam, že som problematike dobre porozumel a moja otázka nie je zmätočná. Ďakujem vopred za odpoveď.
Veľmi pekne ďakujem za
Veľmi pekne ďakujem za komentár a otázku. Je pravda, že zmeny v hladinách p-ACC a p-AMPK súvisia aj s inými procesmi v bunke, avšak my sme sa pozerali konkrétne na rozdiely medzi infikovanými a neinfikovanými bunkami (mock a ARM) vo vybraných časových intervaloch. Tieto rozdiely sú viditeľné 24 a 72 hpi, kedy hladina p-AMPK v LCMV infikovaných bunkách klesá v porovnaní s kontrolou (mock). Podobný výsledok sa dá pozorovať aj pri p-ACC, avšak je pravda že rozdiely sú menšie. Výsledky vpravo (ACC a AMPK) predstavujú aj fosforylovanú aj nefosforylovanú formu (total), ospravedlňujem sa za nepresné označenie.
Ďakujem veľmi pekne za
Ďakujem veľmi pekne za odpoveď aj ozrejmenie problematiky. Prajem veľa zdaru do ďalšieho experimentálneho bádania.
Hello,
Hello,
very interesting work. I am not an expert in this field and highly informed. However, my question is: Could be used your results in Covid19 and to reduce / prevent virus replication in the human?
Thanks
Hello, thank you very much
Hello, thank you very much for the question. SARS-CoV-2 is a virus belonging to a different family, meaning that it alters cellular metabolism in different ways. However, a few articles describe how inhibiting lipid metabolism affects SARS-CoV-2 replication.
Chu et al. (2021) showed that de novo fatty acid synthesis is crucial for SARS-CoV-2 replication by inhibiting FASN, which resulted in a decrease in viral levels and an increase in the survival of infected mice.
Williams et al. (2021) showed that pharmacological inhibition of fatty acid and lipid droplet synthesis inhibits SARS-CoV-2 propagation. Moreover, they showed that inhibition of fatty acid oxidation, known as β-oxidation, also reduces SARS-CoV-2 titer. These results correlate with the previously published study by da Silva Gomes Dias et al. (2020), which shows that SARS-CoV-2 infection increases levels of lipid droplets. These are often used by viruses as storage for viral proteins during replication or as a source of energy since their oxidation yields much more ATP than glycolysis.
Most of the aforementioned research was done on mice, however, a recently published article (Farley et al., 2022) showed that SARS-CoV-2-infected human cells undergo a very similar reprogramming as mice and that inhibition of enzymes catalyzing de novo fatty acid synthesis, as well as lipid droplet synthesis, suppress SARS-CoV-2 propagation in human cells.