Dottorato in SCIENZE DELLA VITA

Titolo della tesi: Influenza virus induces oxidative stress by modulating G6PD enzyme to promote its replication

Respiratory viruses use different strategies to manipulate host cell machinery to their advantage, including the modulation of redox state. Among respiratory viruses, influenza virus replication is favored by pro-oxidant conditions mainly determined by an increase of Reactive Oxygen Species (ROS) production and decrease of intracellular glutathione (GSH) content. Glucose-6-phosphate dehydrogenase (G6PD) activity is responsible for the production of reducing equivalents of nicotinamide adenine dinucleotide phosphate (NADPH), used for regenerating GSH. It has been reported that G6PD-deficiency condition increases the susceptibility to viral infections but the modulation of G6PD enzyme during the infection and the mechanisms by which the viruses induce oxidative stress through this pathway are not well elucidated. G6PD enzyme is regulated at different levels into cell. Nrf2 transcription factor is activated under a stressful condition and transcribes antioxidant genes including G6PD, playing a central role in the antioxidant response. At the same time G6PD is also regulated at the activity level by the deacetylase SIRT2, a NADPH-dependent deacetylase, through the deacetylation of lysine regulatory site of the enzyme. In this study I evaluated the modulation of G6PD expression and activity during influenza virus infection. I analysed the role of G6PD down-regulation during influenza virus replication and its correlation with oxidative stress induction. Afterwards, I verified the mechanisms by which influenza virus modulates the enzyme and the possible effects of reducing compounds on virus replication. With the beginning of COVID19 outbreak my studies were focused on SARS-CoV-2 pathogenesis. Therefore, I analysed whether the new coronavirus modulates the same cell pathways for its replication comparing to influenza virus infection. Finally, I evaluated the possibility to use antioxidant compounds able to modulate specific redox-related pathways, against respiratory viruses. The results showed that G6PD expression and activity were reduced after the infection and their down-regulation correlated with the reduction of Nrf2 and SIRT2 expression, respectively. The acetylation of G6PD increased during influenza virus infection and its activity was strictly dependent on SIRT2 expression. The use of SIRT2 activator induced a rescue of reducing conditions in terms of GSH production, Nrf2 and G6PD expression that correlated with a decrease of viral replication. SARS-CoV-2 infection determined the same modulation of the antioxidant response compared to influenza virus. The use of specific antioxidant molecules was able to impair the replication of influenza virus by recovering the intracellular redox balance in terms of increased expression of Nrf2 and G6PD, or by modulating ACE2, that is involved in the SARS-CoV-2 infection and in regulating the intracellular redox state. Overall these data identify a new cellular pathway used by influenza virus to induce oxidative stress and to favor its replication in host cells. Furthermore, the use of molecules able to restore reducing conditions into cell and able to manipulate specific redox-related pathways may open to new cell-targeting strategies against respiratory viruses infection.