We show here that inhibition of Akt by either siRNA or drug treatment results in significant inhibition of PIV5 protein expression and replication. In addition, Akt inhibitor treatment appears to have a broad spectrum of antiviral activity, decreasing the replication of NNSV from the rubulavirus, morbillivirus, pneumovirus, and rhabdovirus families. To the best of our knowledge, ours is the first study to report the potential of Akt inhibition as an antiviral therapeutic intervention. Because of the interest in Akt as a target for cancer therapy, there are many good compounds targeting Akt with low toxicity and high efficacy, and some of them have advanced to clinical trials (13
). It is possible that some of these compounds will be effective against infections caused by NNSVs in vivo. The development of a broad-spectrum antiviral against NNSVs would allow for therapeutic intervention in a wide array of human diseases.
We have shown that Akt inhibitors can be effective against RSV, the most important etiologic agent of pediatric viral respiratory infection, which remains a major cause of morbidity and mortality among infants and among immunocompromised subjects and the elderly (reviewed in reference 14
). Importantly, there is no vaccine for RSV, nor are there effective curative treatments for severe RSV disease, although aerosolized ribavirin and prophylactic immunoglobulin therapy are used in the clinical setting. However, the high cost of palivizumab prophylaxis due to the need for monthly injections during RSV season raises the question of cost effectiveness relative to health benefits. Therefore, there is a pressing need for safe and effective therapeutic interventions for RSV infection. In addition, Akt inhibitors can block MuV and MeV replication. While MuV and MeV infections have been well controlled in vaccinated population, they still pose a serious health threat in developing counties, where vaccine coverage is poor. VSV, a livestock pathogen, is a rhabdovirus similar to rabies virus, which causes a lethal infection in humans and also lacks an effective antiviral drug. The fact that the Akt inhibitor effectively blocked VSV replication provides evidence that Akt inhibition may be a good strategy for developing anti-rabies virus drugs.
One potential drawback is that the effects of the Akt inhibitors were variable in different cells for different viruses. For instance, in MDBK cells, a bovine cell line that is optimal for PIV5 growth, the Akt inhibitor had a dramatic effect on virus growth, reducing virus titers by 2 to 3 logs, while the same inhibitor reduced the PIV5 growth by only 1 to 2 logs in HeLa cells. We speculate that the differences are a reflection of how effective the inhibitors are in various cell lines, based on their tissue of origin and level of Akt isoform expression. Further optimization of Akt inhibitor treatment in primary cell culture and animal models of viral disease will enhance their potency for use in humans for therapy of infectious diseases caused by NNSV. While we have not tested the effects of Akt inhibitors on DNA virus replication, we do not expect the inhibitors to be effective against DNA virus. The AktIV inhibitor was originally identified as an inhibitor of the FOXO protein nuclear translocation, in which FOXO was expressed from a recombinant adenovirus (29
). In the report, the inhibitor did not appear to inhibit adenovirus gene expression, suggesting that Akt activation is not necessary for adenovirus replication.
Akt, a serine/threonine kinase, is activated through phosphorylation at different sites within the protein (6
). Since some of the inhibitors we used (e.g., the AktIV inhibitor) are known to inhibit phosphorylation of Akt (29
), it is likely that phosphorylation of Akt plays a role in viral RNA synthesis. Which kinase activates Akt in virus-infected cells and how it is activated itself are not known. It is well established that Akt is one of the major downstream targets for PI3K (6
). However, two well-known PI3K inhibitors, wortmannin and LY29002, had no effect on PIV5 replication (Fig. ), indicating that PI3K activation is not required for Akt activation in virus-infected cells. Therefore, we propose that a kinase downstream of PI3K, or potentially a novel kinase, is responsible for activating Akt in virus-infected cells.
While factors required for its activation remain unclear, our studies provide insight as to the mechanism by which Akt regulates PIV5 RNA synthesis. Chemical inhibition of Akt results in a significant decrease in phosphorylation of the viral P protein. In addition, Akt can phosphorylate P in vitro. The phosphorylation status of P is thought to be a key regulator of the switch by vRNAP from viral mRNA synthesis (transcription) to a viral RNA replication (12
). Previous studies have shown that the P proteins of various NNSV are phosphorylated by host kinases (33
). For VSV, there are two regions (N terminus region, domain I, and C terminus region, domain II) within the P protein that are phosphorylated. One of the host kinases known to phosphorylate the N-terminal region of VSV P is casein kinase II (CKII) (4
). While the host kinase for the C-terminal region has not been identified (16
), there is some circumstantial evidence that the host kinase may be Akt. Kim et al. first reported that K-252a, a broad nonspecific kinase inhibitor (isolated from Nocardiopsis
sp.), and its derivative molecule KT5926 have anti-VSV activities in BHK cells (31
). KT5926 does not inhibit CKII (30
) but has been shown to target a host kinase that matches the size of Akt (59
). In addition, staurosporine inhibits VSV RNA synthesis (53
) and has been shown to target Akt in addition to protein kinases A and C (27
Like other P proteins of paramyxoviruses, RSV P is a major cofactor for vRNAP and plays an essential role in viral RNA synthesis. Phosphorylation of P plays an essential role in its functions (20
). There are five major phosphorylation sites within the P protein (43
). Mutating these five phosphorylation sites resulted in a recombinant RSV that is severely attenuated in animals and is reduced in growth in some tissue culture cells (38
). Interestingly, this mutant P protein was still phosphorylated in infected cells, albeit at much lower levels than wild-type P (38
). The P protein purified from bacteria can be phosphorylated by casein kinase II, and this phosphorylated P is as active as the P protein treated with cell extract (presumably containing the host kinase that phosphorylates P) in an in vitro viral RNA synthesis assay, indicating that CKII can phosphorylate P (39
). However, CKII has not been shown to phosphorylate P in infected cells. We found that inhibition of Akt reduced the phosphorylation of the P protein, indicating that Akt plays a critical role in the phosphorylation of the P protein in RSV-infected cells.
Our data lead us to a specific model for the role of Akt in the replication of NNSV (Fig. ). Infection of cells by NNSV results in the activation of Akt. While the mechanism by which Akt is activated is unclear, PI3K is unlikely involved, as treatment with PI3K inhibitors has no effect on viral replication. Activated Akt then phosphorylates the viral P protein, which is an essential cofactor for vRNAP, regulating viral RNA synthesis. Since Akt inhibition causes a significant decrease in viral protein production, it is possible that phosphorylation of P by Akt is responsible for regulating the switch between transcription and RNA replication by the vRNAP in favor of transcription. Increased transcription would then allow for increased viral protein production and, subsequently, genome replication and virion morphogenesis. Inhibition of Akt thus blocks viral replication at an early stage postentry. In the paramyxoviruses which encode V proteins or other accessory proteins, such as W or C, we propose that these proteins regulate viral RNA synthesis by interacting with Akt and inhibiting its function, either by decreasing its kinase activity or by sequestering Akt itself. Thus, we propose that Akt plays a critical role in viral replication by regulating RNA synthesis through the phosphorylation of the P protein in infected cells.
FIG. 8. A model for the involvement of Akt in viral RNA synthesis. The replication of viral RNA and synthesis of viral mRNA require P and L complex. The phosphorylation status of the P protein plays a critical in viral RNA synthesis. Akt phosphorylates P, thus (more ...)
The studies presented in this paper arose from our hypothesis that the V protein of PIV5 exerts its numerous activities by interacting with host proteins. We initially used a computational method to predict potential V-interacting proteins and then determined empirically that V binds specifically to one of the predicted interactors, Akt1/PKB. This interaction has functional consequences in that Akt overexpression alleviates V-mediated inhibition of PIV5 RNA synthesis in a minigenome system. Beyond this effect on the viral polymerase activity, it is possible that the V-Akt interaction plays additional roles in viral infection. Akt plays an important role in many cellular signaling pathways, such as those for apoptosis and interferon signaling and production (23
). The V protein of PIV5 is known to inhibit apoptosis induced by viral infection (57
). Therefore, the interaction between V and Akt is consistent with the role of V in apoptosis. Furthermore, V has also been shown to block interferon production and interferon signaling as well as to block interleukin-6 expression (18
). It will be interesting to examine whether the interaction between V and Akt plays a role in these processes. Finally, PIV5 infection slows down the cell cycle, and the V protein of PIV5 is thought to play a critical in the process (35
). It is tempting to speculate that the interaction of V and Akt may contribute to the slowing down of the cell cycle by PIV5, since Akt signaling also affects cell cycle control (6
). Thus, Akt activation, and interaction with V, may play multiple roles in the replication and pathogenesis of NNSV.