Akt Interacts with Usutu Virus Polymerase, and Its Activity Modulates Viral Replication

Abstract

Usutu virus (USUV) is a flavivirus that mainly infects wild birds through the bite of Culex mosquitoes. Recent outbreaks have been associated with an increased number of cases in humans. Despite being a growing source of public health concerns, there is yet insufficient data on the virus or host cell targets for infection control. In this work we have investigated whether the cellular kinase Akt and USUV polymerase NS5 interact and co-localize in a cell. To this aim, we performed co-immunoprecipitation (Co-IP) assays, followed by confocal microscopy analyses. We further tested whether NS5 is a phosphorylation substrate of Akt in vitro. Finally, to examine its role in viral replication, we chemically silenced Akt with three inhibitors (MK-2206, honokiol and ipatasertib). We found that both proteins are localized (confocal) and pulled down (Co-IP) together when expressed in different cell lines, supporting the fact that they are interacting partners. This possibility was further sustained by data showing that NS5 is phosphorylated by Akt. Treatment of USUV-infected cells with Akt-specific inhibitors led to decreases in virus titers (>10-fold). Our results suggest an important role for Akt in virus replication and stimulate further investigations to examine the PI3K/Akt/mTOR pathway as an antiviral target.

Akt Kinase Intervenes in Flavivirus Replication by Interacting with Viral Protein NS5

“…We confirmed that all these recombinant proteins were phosphorylated by Akt in vitro ( Figure 2 ). The phosphorylated positions of band-purified NS5s, including the corresponding to USUV previously described [ 11 ], were determined by mass spectrometry, with sequence coverage values of 93%, 95% and 96% for WNV, ZIKV and USUV NS5 proteins, respectively ( Supplementary Figure S1 ). The analysis revealed that the same residue, a Ser contained in the catalytic motif SGDD, was phosphorylated in all flaviviral NS5 proteins tested: S664, S669 and S670 in ZIKV, USUV, and WNV, respectively ( Figure 3 ).…”

“…The expected sequence was confirmed by DNA sequencing for all the constructs. Plasmids encoding the entire NS5 protein and the RdRpD of USUV have been previously described [ 11 ].…”

“…pET21b and pRSET plasmid constructs were used to transform the BL21(DE3) pLysS Rosetta Escherichia coli strain. Protein purification procedures have been previously described [ 10 , 11 , 12 , 13 ].…”

“…For Western blot (WB) and co-immunoprecipitation (Co-IP) assays we used human cell lines Huh7.5 and Hek-293T. WB and Co-IP procedures have been previously described [ 11 ]. As a control prior to the co-immunoprecipitation experiment we used anti-GFP antibodies to check that we did not rescue Akt, NS5 or the RdRp domain from the immunoprecipitate.…”

“…Cytotoxicity was evaluated as previously described [ 11 ]. Briefly, we used 96-well plates seeded with Vero cells as above to determine the relative number of viable cells after treatment with MK-2206, ipatasertib and honokiol.…”