The results that we present in this study address the issue of whether the NSS RNA virus VSV requires PI3k/Akt activity for efficient replication. Our results demonstrate that neither the inhibition of PI3k activity nor the inhibition of Akt activity decreases VSV gene expression or virus progeny production. This observation suggests that the activity of this pathway plays a minimal role in VSV replication. This finding is consistent with a recent report showing that in invertebrates, VSV infection results in the inhibition of the PI3k/Akt signaling pathway (31
Surprisingly, we also found contrasting actions when we examined how Akt inhibitors impacted virus replication. Treatment of cells with Akt inhibitors Akt-V and Akt-VIII did not alter VSV replication but did block the kinase-activating phophorylation events at Thr308 and Ser473 (Fig. ). In contrast, Akt inhibitor Akt-IV promoted Akt phosphorylation at residues Thr308 and Ser473 and showed strong inhibition of virus replication, which is in keeping with the data in an earlier report showing that this compound blocks RNA virus replication (35
). These findings suggest that the action by which Akt-IV inhibits virus replication is not a result of its targeting Akt kinase activity.
Our data suggest that a revision of the proposed mechanism of action for Akt-IV is in order. Based on results of drug treatments at 10 μM, previous reports postulated that Akt-IV was acting to block phosphorylation and, thereby, activation of Akt (19
). We find that at lower concentrations, Akt-IV increases the phosphorylation of Akt in multiple cell types. This increase in phosphorylation is PI3k dependent (Fig. ). Interestingly, our in vitro kinase assay data suggest that Akt-IV may directly activate PDK1, which phosphorylates Akt on Thr308. This potential increase in PDK1 activity may also account for the difference in the levels of Akt phosphorylation at residues Thr308 and Ser473 found in cells treated with Akt-IV (Fig. ).
Our observation that the Akt-IV inhibitor increases the levels of phospho-Akt suggests that the ascribed actions of this compound may be peripheral to the direct inhibition of Akt activity. The structure of the compound is consistent with the idea that Akt-IV may act as an ATP analog to block the active site of a kinase, but our screening assays did not identify Akt or any other kinase among the 80-plus kinases tested as a target. This result is consistent with findings described in other reports suggesting that Akt-IV does not alter the in vitro activity of Akt (40
). The addition of Akt-IV to cells did decrease the phosphorylation of downstream Akt substrates such as 4E-BP1 (Fig. and ). The dephosphorylation of 4E-BP1 is consistent with Akt-IV's targeting signaling downstream of Akt kinase activity, perhaps at the level of mTOR.
This observation of increased phosphorylation of Akt following drug treatment is not unique to Akt-IV, as the stimulation of Akt phosphorylation has been seen previously in response to several kinase inhibitors, such as rapamycin (36
) and the recently characterized Akt inhibitor Abbot compound A-443654 (24
). The difference in the actions of Akt-IV and A-443654 are highlighted by the results of our in vitro kinase-profiling assays; these show that Akt-IV does not directly inhibit Akt kinase activity in vitro (Table ), while A-443654 in an identical screen does (5
). Akt-IV and A-443654 both cause an increase in Akt phosphorylation and lead to the dephosphorylation of downstream effectors (17
), but their mechanisms of action must be distinct, as Akt-IV does not inhibit Akt in vitro. This pattern argues that Akt-IV has a unique mechanism of action, perhaps blocking the recruitment of a currently unidentified cofactor required for downstream signaling of Akt or inhibiting some other host cell process that is essential for viral replication (see Fig. ).
FIG. 6. Schematic of the PI3k/Akt signaling pathway depicting the possible sites of action by the PI3k inhibitors LY294002 and wortmannin and the Akt inhibitors Akt-VIII, Akt-V, and Akt-IV. PIP2, phosphatidylinositol 4,5-biphosphate; PIP3, phosphatidylinositol (more ...)
Depicted in Fig. is a simplified diagram of the PI3k/Akt signaling pathway highlighting the points at which inhibitors utilized in these experiments would exert their effects and inhibit Akt phosphorylation. The PI3k inhibitors LY294002 and wortmannin both inhibit the synthesis of PIP3, which is required for PDK1 activation of Akt. The Akt inhibitors Akt-V and Akt-VIII directly prevent phosphorylation and thus activation of Akt. Since Akt-IV does not prevent phosphorylation on Akt's activation sites or directly block kinase activity in vitro (Table ), we propose that Akt-IV acts downstream of Akt activation and possibly at the point of substrate recognition. We also propose that the antiviral activity associated with this compound is independent of the PI3k/Akt signaling pathway and occurs by a mechanism yet to be determined.
Our results show that Akt inhibitor Akt-IV is the only Akt inhibitor we tested that blocked early replication events in VSV, RSV, and VACV infection. The simplest explanation of this activity is a non-Akt-pathway target. The compound was isolated in a high-throughput screen in vivo that was not designed to uncover compounds that specifically target Akt (19
). Akt-IV, like the Akt inhibitor A-443654, may have multiple targets within the AGC kinase family (6
), although data from our kinase assay screen shows no obvious candidates. Alternatively, Akt-IV may target other aspects of normal cellular function. This implication may be important for the understanding of findings from studies that have used this compound as a specific Akt inhibitor (23
), particularly those which have found Akt-IV to be less effective than other Akt inhibitors such as Akt-V (10
). Speculatively, the mechanism of antiviral action could be attributed to a block of viral entry or perhaps to inhibition either of viral RNA transcription or the translation of viral mRNAs. Further studies to determine the level of viral RNAs in the cell will help determine which stage in the viral replication cycle is affected. Notably, all three of the viruses tested here replicate in the cytoplasm. Therefore, Akt-IV may potentially block the function of a host kinase (protein) in the cytoplasm, resulting in an effect similar to one of the host antiviral responses.
Because our results and those of other researchers have established that this compound effectively inhibits the replication of multiple negative-strand RNA viruses, it would be of significant interest to determine any additional targets of this compound. It may be possible to identify the antiviral target of Akt-IV in vitro simply by increasing the number of kinase targets in the kinase-profiling assay or in vivo by using an analytical approach that combines a drug affinity pull-down assay with mass spectrometry to identify proteins associated with Akt-IV as new targets. Both approaches have been used successfully in studies to assess off-target effects of several clinical drugs that have broad-spectrum antikinase activities (7
In conclusion, we demonstrate that the PI3k/Akt pathway does not appear to be necessary for VSV replication. This finding supports the conclusions of other groups that have determined that this pathway has minimal impact on negative-strand RNA virus replication (4
). Our studies do show that the inhibitor Akt-IV displays a mechanism of action that is different from what has been described previously and suggest that this compound deserves further study as a broad-spectrum antiviral agent. Our results show that the antiviral action of this drug is potent and sustained and blocks an early stage of viral replication. These results suggest the possibility that this compound may show a broader spectrum of antiviral activity than has been described to date. Therefore, based on our data, we propose that the Akt inhibitor Akt-IV has two distinct actions (or activities), the first being the inhibition of Akt by a unique mechanism and the second being the targeting of another, currently unknown kinase that is necessary for VSV to establish a productive replication cycle.