Here we demonstrate that VSV causes the dephosphorylation and subsequent inactivation of Akt and its signaling pathway at an early stage of infection and that dephosphorylation is found to be dependent on virus replication. This finding is in agreement with previous observations that VSV replication induces the dephosphorylation of 4EB-P1 (21
) and downstream effectors of Akt (42
) and that VSV replication is not dependent on an active PI3k/Akt signaling pathway (24
). This runs counter to what has been seen for other viruses and even other negative-strand RNA viruses, such as influenza A virus and RSV, which are known to activate Akt (26
). VSV's inactivation of Akt is reminiscent of the Akt inhibition seen during measles infection (6
). Measles virus is thought to inactivate Akt in a replication-independent manner through the induction of a cellular lipid phosphatase that alters the concentration of PIP3 at the membrane (7
), while we find that VSV blocks in a replication-dependent manner that is independent of PIP3 and involves the viral matrix protein.
VSV was able to interrupt normal receptor tyrosine kinase-driven Akt activation. Insulin and EGF stimulation was markedly blunted in infected cells, and this dominance of signaling was present throughout the course of the infection. This appears to be due to the effect of virus infection on Akt specifically and not due to the inactivation of tyrosine kinase signaling, as signaling to PI3k to synthesize PIP3 and activate the mitogen-activated protein (MAP) kinase extracellular signal-regulated kinases 1/2 (ERK1/2) (data not shown) was still intact. Thus, virus infection effectively decouples Akt activation from growth factor-mediated stimulation.
This decoupling/inactivation of Akt highlights a novel mechanism of interacting with this signaling pathway. Infection of cells with virus did decrease phosphorylation of Akt but did not alter total cellular levels or the activity of PDK1 (Fig. ), PDK1's subcellular localization (Fig. ), or the levels of phosphorylation of other PDK1 substrates (Fig. ). Analysis of subcellular fractions determined that VSV did not keep Akt from translocating to the membrane. Akt levels at the membrane were in fact found to be approximately 3-fold higher than found in mock-infected cells. This observation is consistent with the significant increase in PIP3 levels detected during VSV replication. Thus, VSV must block the activation of Akt after membrane localization by either disrupting the interaction of PDK1 and Akt at the membrane during infection or blocking access to the phosphorylation site(s) on Akt.
Our data are consistent with the model in which VSV replication blocks the phosphorylation of Akt, and this block is dominant over the external stimuli of growth factors to phosphorylate and activate Akt. The rapid decrease in the level of phosphorylated Akt detected during VSV replication is likely due to constitutive cellular phosphatase activity leading to “run-down.”
This block/disruption of Akt phosphorylation appears to be mediated at least in part by the viral matrix protein (M). M, a peripheral membrane protein, was sufficient to induce the dephosphorylation of Akt (Fig. ) in transfection experiments. This control of Akt is due at least in part to the protein's ability to block transcription and nuclear/cytoplasmic transport, as a mutant of M that is defective in blocking nuclear/cytoplasmic transport and host transcription was defective in forcing Akt inactivation (Fig. ). The generation and characterization of new M protein mutants may help further identify which amino acids are important for M-induced Akt dephosphorylation and whether a specific cellular localization of M is necessary for this phenotype.
A moderate reduction in Akt phosphorylation (~20% with significant variation) was also found in cells transiently expressing either the VSV P, G, or L protein (Fig. ). This effect was not as dramatic as with the M protein, but it is possible that during a virus infection there could be an additive effect of the combination of these single factors that leads to the greatly reduced levels of Akt phosphorylation that we observe. During the course of our studies, we also noticed that increasing the incubation time of VSV G transient expression (>20 h) resulted in a significant drop in the level of Akt phosphorylation (data not shown). We did not pursue this finding further, as this time point also correlated with complete syncytia of the cell monolayer, a phenotype not observed during a VSV infection and therefore one that we presumed to be an artifact of transfecting cells in tissue culture.
What gain does the virus derive from Akt inactivation? Earlier publications have suggested that active Akt signaling can decrease VSV replication (42
). In addition, Akt signaling has recently been shown to be essential for generating the interferon (IFN)-dependent antiviral response and to complement the function of IFN-activated JAK-STAT pathways in cells (33
). Thus, the inactivation of Akt by VSV may serve to blunt the IFN response in productively infected cells.
One aspect of interest from these findings pertains to VSV's potential as an oncolytic agent. VSV has previously been shown to be an effective oncolytic agent in a variety of tumor models (8
), both on its own and in combination with other therapies (2
). While there have been several studies analyzing why cancer cells are susceptible to infection (11
), the primary signaling pathway by which the virus induces apoptosis in these cells has not been elucidated, though both the Bcl-2 pathway and ASK1/DAXX pathways have been implicated (27
). Inactivation of Akt/PKB can stimulate both of these pathways (12
), suggesting that this action is a key regulator of VSV-mediated cell killing and may explain how cells can be directed into different apoptotic pathways (28
Our findings could help guide the future development of new oncolytic VSV strains. The natural ability of VSV to block oncogenic signaling through Akt can be useful in identifying potential synergistic effects of combination therapies. As an example, Alain et al. (2
) recently reported that pretreatment of a malignant glioma with the mTORC1 inhibitor rapamycin potentiated the oncolytic effect of VSV in vivo
and ex vivo
. Based on our findings, the combination of VSV and the mTOR inhibitor is predicted to have delivered a “double hit” to the Akt signaling axis (2
) making it a highly potent antiproliferative combination.