Tick-Borne Flaviviruses Depress AKT Activity during Acute Infection by Modulating AKT1/2.

Tick-borne flaviviruses (TBFVs) are reemerging public health threats. To develop therapeutics against these pathogens, increased understanding of their interactions with the mammalian host is required. The PI3K-AKT pathway has been implicated in TBFV persistence, but its role during acute virus infection remains poorly understood. Previously, we showed that Langat virus (LGTV)-infected HEK 293T cells undergo a lytic crisis with a few surviving cells that become persistently infected. We also observed that AKT2 mRNA is upregulated in cells persistently infected with TBFV. Here, we investigated the virus-induced effects on AKT expression over the course of acute LGTV infection and found that total phosphorylated AKT (pAKT), AKT1, and AKT2 decrease over time, but AKT3 increases dramatically. Furthermore, cells lacking AKT1 or AKT2 were more resistant to LGTV-induced cell death than wild-type cells because they expressed higher levels of pAKT and antiapoptotic proteins, such as XIAP and survivin. The differential modulation of AKT by LGTV may be a mechanism by which viral persistence is initiated, and our results demonstrate a complicated manipulation of host pathways by TBFVs.

Conserved Active-Site Residues Associated with OAS Enzyme Activity and Ubiquitin-Like Domains Are Not Required for the Antiviral Activity of goOASL Protein against Avian Tembusu Virus.

Interferon (IFN)-induced 2'-5'-oligoadenylate synthetase (OAS) proteins exhibit an extensive and efficient antiviral effect against flavivirus infection in mammals and birds. Only the 2'-5'-oligoadenylate synthetase-like (OASL) gene has been identified thus far in birds, except for ostrich, which has both OAS1 and OASL genes. In this study, we first investigated the antiviral activity of goose OASL (goOASL) protein against a duck-origin Tembusu virus (DTMUV) in duck embryo fibroblast cells (DEFs). To investigate the relationship of conserved amino acids that are related to OAS enzyme activity and ubiquitin-like (UBL) domains with the antiviral activity of goOASL, a series of mutant goOASL plasmids was constructed, including goOASL-S64C/D76E/D78E/D144T, goOASL∆UBLs and goOASL∆UBLs-S64C/D76E/D78E/D144T. Interestingly, all these mutant proteins significantly inhibited the replication of DTMUV in DEFs in a dose-dependent manner. Immunofluorescence analysis showed that the goOASL, goOASL-S64C/D76E/D78E/D144T, goOASL∆UBLs and goOASL∆UBLs-S64C/D76E/D78E/D144T proteins were located not only in the cytoplasm where DTMUV replicates but also in the nucleus of DEFs. However, the goOASL and goOASL mutant proteins were mainly colocalized with DTMUV in the cytoplasm of infected cells. Our data indicated that goOASL could significantly inhibit DTMUV replication in vitro, while the active-site residues S64, D76, D78 and D144, which were associated with OAS enzyme activity, the UBL domains were not required for the antiviral activity of goOASL protein.

Expression of flavivirus capsids enhance the cellular environment for viral replication by activating Akt-signalling pathways.

Flaviviruses depend on multiple host pathways during their life cycles and have evolved strategies to avoid the innate immune response. Previously, we showed that the West Nile virus capsid protein plays a role in this process by blocking apoptosis. In this study, we examined how expression of capsid proteins from several flaviviruses affects apoptosis and other host processes that impact virus replication. All of the tested capsid proteins protected cells from Fas-dependent apoptosis through a mechanism that requires activated Akt. Capsid expression upregulated other Akt-dependent cellular processes including expression of glucose transporter 1 and mitochondrial metabolism. Protein phosphatase 1, which is known to inactivate Akt, was identified as a DENV capsid interacting protein. This suggests that DENV capsid expression activates Akt by sequestering phosphatases that downregulate phospho-Akt. Capsid-dependent upregulation of Akt would enhance downstream signalling pathways that affect cell survival and metabolism, thus providing a favourable environment for virus replication.

Standing your ground to exoribonucleases: Function of Flavivirus long non-coding RNAs.

Members of the Flaviviridae (e.g., Dengue virus, West Nile virus, and Hepatitis C virus) contain a positive-sense RNA genome that encodes a large polyprotein. It is now also clear most if not all of these viruses also produce an abundant subgenomic long non-coding RNA. These non-coding RNAs, which are called subgenomic flavivirus RNAs (sfRNAs) or Xrn1-resistant RNAs (xrRNAs), are stable decay intermediates generated from the viral genomic RNA through the stalling of the cellular exoribonuclease Xrn1 at highly structured regions. Several functions of these flavivirus long non-coding RNAs have been revealed in recent years. The generation of these sfRNAs/xrRNAs from viral transcripts results in the repression of Xrn1 and the dysregulation of cellular mRNA stability. The abundant sfRNAs also serve directly as a decoy for important cellular protein regulators of the interferon and RNA interference antiviral pathways. Thus the generation of long non-coding RNAs from flaviviruses, hepaciviruses and pestiviruses likely disrupts aspects of innate immunity and may directly contribute to viral replication, cytopathology and pathogenesis.

Structure of the methyltransferase domain from the Modoc virus, a flavivirus with no known vector.

The Modoc virus (MODV) is a flavivirus with no known vector (NKV). Evolutionary studies have shown that the viruses in the MODV group have evolved in association with mammals (bats, rodents) without transmission by an arthropod vector. MODV methyltransferase is the first enzyme from this evolutionary branch to be structurally characterized. The high-resolution structure of the methyltransferase domain of the MODV NS5 protein (MTase(MODV)) was determined. The protein structure was solved in the apo form and in complex with its cofactor S-adenosyl-L-methionine (SAM). Although it belongs to a separate evolutionary branch, MTase(MODV) shares structural characteristics with flaviviral MTases from the other branches. Its capping machinery is a relatively new target in flaviviral drug development and the observed structural conservation between the three flaviviral branches indicates that it may be possible to identify a drug that targets a range of flaviviruses. The structural conservation also supports the choice of MODV as a possible model for flavivirus studies.

The flaviviral methyltransferase is a substrate of Casein Kinase 1.

Serine/Threonine phosphorylation of the nonstructural protein 5 (NS5) is a conserved feature of flaviviruses, but the identity and function(s) of the responsible kinase(s) remain unknown. Serine 56 in the methyltransferase domain of NS5 can be phosphorylated intracellularly, is conserved in all flaviviruses, and is a critical residue in the catalytic mechanism. A negative charge at this residue inactivates the 2'-0 methyltransferase activity necessary to form a 5' cap structure of the viral RNA. Here we show pharmacologic inhibition of Casein Kinase 1 (CK1) suppresses yellow fever virus (YFV) production. We also demonstrate the alpha isoform of Casein Kinase 1 (CK1alpha), a kinase previously identified as phosphorylating Hepatitis C Virus NS5A protein, also phosphorylates serine 56 of YFV methyltransferase. Overall these results suggest CK1 activity can influence flaviviral replication.