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.
Highlights
Flaviviriruses are obligate intracellular parasites with a single-stranded RNA genome of positive polarity ((+)ssRNA viruses) that are grouped into a single family (Flaviviridae) divided into four genera (Flavivirus, Hepacivirus, Pegivirus, and Pestivirus)
We found that a purified USUV NS5 protein is phosphorylat by Akt
We demonstrate a connection between the activity of the Akt kinase and USUV polymerase which is important for viral replication in the infected cell
Summary
Flaviviriruses are obligate intracellular parasites with a single-stranded RNA genome of positive polarity ((+)ssRNA viruses) that are grouped into a single family (Flaviviridae) divided into four genera (Flavivirus, Hepacivirus, Pegivirus, and Pestivirus). The genus Flavivirus is the one that groups the largest number of species that cause disease in humans [1,2]. Some relevant members of the genus Flavivirus to humans are dengue virus (DENV), Japanese encephalitis virus (JEV), yellow fever virus (YFV), West Nile virus (WNV), Zika virus (ZIKV), Usutu virus (USUV), tick-borne encephalitis virus (TBEV), and Powassan virus (POWV). Most viruses belonging to this genus are arboviruses, as they are transmitted to humans and other vertebrate hosts through the bite of arthropods, either mosquitoes or ticks [3]. Vaccines have been developed against some of them, providing high levels of protection in controlling infection (JEV, WNV, YFV, TBEV) [3]. There are some drugs approved for the treatment of flaviviral disease [5,6], most treatments available aim to mitigate the symptoms rather than inhibit virus replication and infection [7]
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