Vif increases phosphorylation of AKT at Ser 473
To investigate the effect of Vif on AKT signaling pathway, HEK-293T cells were transfected with HA AKT, HA Myr AKT and HA KD AKT along with Myc Vif. After 24 hrs of transfection, cells were lysed and analyzed by western blotting. Vif was increasing the expression of Myr AKT while there was no effect of Vif on the expression of wild type HA AKT as well as the kinase deficient KD AKT. Vif was also increasing the levels of phospho-AKT Ser 473 in Myr AKT and Myc Vif co-transfection experiment (Fig. 1a).
We also checked the endogenous levels of phospho-AKT Ser 473 in the presence of Vif. HEK-293T cells were transfected with increasing amounts of Myc Vif encoding plasmid. After 24 hrs of transfection, cell lysates were analyzed by western blotting using anti-phospho-AKT Ser 473. It was observed that Vif was increasing the levels of phosphor-AKT Ser 473 in dose dependent manner while the expression of unmodified AKT was unaffected by Vif (Fig. 1b). This observation was also confirmed by using inhibitor of AKT activity or phosphorylation i.e. AKTi. HEK-293T cells were transfected with Myc Vif with or without AKTi treatment. After 24 hrs, cells were subjected to western analysis. AKTi was reducing the levels of phosho-AKT Ser 473 as expected. Vif was found to induce the phosphorylation of AKT at Ser 473 even in the presence of AKTi (Fig. 1c). These results indicate that HIV-1 Vif can increase the activity of AKT by inducing its phosphorylation at Ser 473.
Vif induces ubiquitin-mediated proteasomal degradation of Mdm2
Mdm2 is the downstream target of AKT. When PI3/AKT pathway is activated, AKT is phosphorylated and further it phosphorylates and stabilizes Mdm221. As, Vif was increasing the activity of AKT by inducing its phosphorylation, the effect of Vif on the levels of Mdm2 was also investigated. HEK-293T cells were co-transfected with HA Mdm2 and Myc Vif plasmids for 24 hrs. Western blotting analysis of these cells showed that Mdm2 is degraded in the presence of Vif (Fig. 1d). Vif was able to degrade Mdm2 at endogenous level also (Fig. 1e). The cycloheximide chase of Mdm2 alone and in the presence of Vif also showed that the stability of Mdm2 is reduced in the presence of Vif (Fig. 1f).
These results were confirmed using constitutively active form of AKT i.e. Myr AKT which stabilizes Mdm2 expression. HEK-293T cells were co-transfected with HA Mdm2, HA Myr AKT and Vif for 24 hrs and cells were subjected to western analysis. As expected, Myr AKT was increasing the expression of Mdm2. Vif was found to reduce the expression level of Mdm2 even in the presence of Myr AKT which is itself increased by Vif (Fig. 1g). This result was also validated using inhibitor of AKT activity i.e. AKTi. Vif was increasing the levels of phospho-AKT Ser 473 even in the presence of AKTi as described above but the expression level of Mdm2 was found to be reduced in the presence of Vif (Fig. 1h).
To find out the mechanism of Vif-mediated degradation of Mdm2, HEK-293T cells were co-transfected with HA Mdm2 and Myc Vif in presence or absence of MG132 (proteasomal inhibitor). Vif-mediated degradation of Mdm2 was found to be reversed by MG132 (Fig. 1i). This indicates the post-translational regulation of Mdm2 by Vif. The ubiquitination levels of Mdm2 in presence of Vif were also checked. HEK-293T cells were co-transfected with HA Mdm2, Myc Vif and His Ub plasmids. Cells were also treated with MG132 to accumulate ubiquitinated species. Cells were lysed and cell lysate was incubated with Ni-NTA beads which bind His tagged ubiquitinated proteins. The ubiquitination of Mdm2 was found to be increased in the presence of Vif (Fig. 1j). These results indicate that Vif induces ubiquitin-mediated proteasomal degradation of Mdm2.
Vif increases expression of Tat and exhibits direct interaction
The expression of Tat in the presence of Vif was investigated by co-transfecting HEK-293T cells with HA Tat and Myc Vif for 24hrs. Cell lysates were analyzed by western blotting to check the expression of Tat and Vif. Vif was found to stabilize the expression of Tat (Fig. 2a). Cycloheximide chase of Tat either alone or with Vif also showed that the stability of Tat is more in the presence of Vif (Fig.2b). As, Mdm2 is known to increase the LTR transcriptional activity of Tat15 and we are reporting the ubiquitin mediated proteasomal degradation of Mdm2 by Vif, the effect of Vif on LTR transcription mediated by Tat was also investigated by co-transfecting HEK-293T cells with B LTR luciferase reporter plasmid (pBlue3’LTR-luc) and Tat either alone or in combination with Vif encoding plasmid for 24 hrs. As expected, LTR activation was observed in the presence of Tat. Vif alone also showed little activation of LTR. Surprisingly, In the presence of both Vif and Tat proteins, LTR activation was observed to be much higher than that of Tat alone (Fig. 2c), indicating Vif mediated stabilization of Tat. Further, to find out the importance of these observations in context of HIV-1 virus, we co-transfected Vif deficient HIV-1 proviral DNA (pNL4-3ΔVif) into Tzm-Bl cells either alone or along with Myc Vif for 24 hours. It was observed that the activation of LTR in the presence of both pNL4-3ΔVif and Vif was higher than in the presence of pNL4-3ΔVif or Vif alone (Fig. 2d).
The interaction between Tat and Vif was also investigated. In vitro synthesized HIV-1 Vif protein and GST Tat fusion protein were used to study their interaction. For in vitro binding studies, GST Tat and GST alone (control) were allowed to bind to GST beads. Thereafter, in vitro translated Vif protein was added to the complex and incubated for 2 hours at 4°C. Vif protein was visualized on a nitro-cellulose membrane by western blot analysis using specific antiserum (obtained from NIH, MD, USA). GST Tat was able to pull down Vif specifically while GST alone failed to do so (Fig. 2e). This led to the conclusion that Vif specifically interacts with HIV-1 Tat protein. To further check the interaction between the three proteins viz. Tat, Vif and AKT, HEK-293T cells were transfected with HA Tat, Myc Vif and HA AKT. Cell lysates were subjected to immuno-precipitation with anti-Myc antibody tagged agarose beads and bound proteins were analyzed by western blotting using anti-HA antibody. Both proteins Tat and AKT were found to equally interact with Vif (Fig. 2f). These results indicate that Vif interacts with both Tat and AKT independently and can form triplet.
Role of AKT siganaling pathway in HIV-1 Vif mediated stabilization of Tat
As, Tat is known to play important role in activation of AKT signaling pathway and we are also reporting the up-regulation of phosphorylated AKT by Vif, so the role of AKT signaling pathway in Vif mediated stabilization of Tat was investigated using chemical inhibitor of AKT (AKTi). HEK-293T cells were transfected with HA Tat either alone or with Myc Vif and cells were treated with AKTi. Vif was observed to increase the expression of Tat protein as before but inhibition of AKT activity by AKTi was able to decrease the expression of Tat even in the presence of Vif (Fig. 3a). The effect of AKTi treatment on HIV-1 replication was also investigated in U1 cells (monocytes latently infected with HIV-1). HIV-1 replication was induced in U1 cells using PMA and cells were treated with AKTi. The inhibition of AKT activity by AKTi was found to reduce the HIV-1 replication (Fig. 3b). When Myr AKT, wild type AKT and KD AKT were co-expressed with HA Tat in HEK-293T cells, it was observed that the expression of Tat is downregulated in presence of both wild type AKT (Fig. 3c) and KD AKT (Fig. 3e) while there was no significant effect of Myr AKT on the Tat levels (Fig. 3d). This indicated the involvement of other host factor in stabilizing Tat which becomes functional upon activation of AKT signalling pathway and compensates for its downregulation by AKT.
Since Mdm2 is the immediate downstream target of AKT signalling pathway and also known to mediate the K63 ubiquitination of Tat by direct interaction resulting in the enhancement of LTR transcriptional activity of Tat and viral replication15, the expression of Tat in presence of Mdm2 was examined. HEK-293T cells were co-transfected with HA Tat and HA Mdm2 for 24hrs. The expression of Tat protein was found to be enhanced in the presence of Mdm2 (Fig. 3f). Cycloheximide chase of Tat in the presence or absence of Mdm2 also showed that stability of Tat was increased in the presence of Mdm2 (Fig. 3g). These results indicated that Vif stabilizes the expression of Tat via Mdm2.
Mdm2 increases the expression of NQO1 and vice versa
As, we have previously reported that NQO1 stabilizes the expression of HIV-1 Tat protein20, so the expression of NQO1 in the presence of Mdm2 was also examined to investigate the mechanism of Mdm2 mediated stabilization of Tat. HEK-293T cells were co-transfected with Flag NQO1 and HA Mdm2 for 24hrs. The expression of NQO1 was found to be increased in the presence of Mdm2 (Fig. 4a). Mdm2 was found to increase the expression of NQO1 at endogenous level also (Fig. 4b). Cycloheximide chase of NQO1 in presence or absence of Mdm2 also showed that stability of NQO1 was enhanced in the presence of Mdm2 (Fig. 4c). The ubiquitination of NQO1 in presence of Mdm2 was also investigated. HEK-293T cells were co-transfected with Flag NQO1, HA Mdm2, and His Ub plasmids. Cells were also treated with MG132 to accumulate ubiquitinated species. Cells were lysed and cell lysate was incubated with Ni-NTA beads which bind His tagged ubiquitinated proteins. Ubiquitination levels of NQO1 were found to be downregualted in the presence of Mdm2 (Fig. 4d). To check the interaction between Mdm2 and NQO1, HEK-293T cells were transfected with Flag NQO1 and HA Mdm2 for 24 hrs and cell lysates were subjected to immunoprecipitation using anti-NQO1 antibody bound agarose beads. The western blot analysis of pulled down proteins using anti-HA antibody indicated the interaction between NQO1 and Mdm2 (Fig. 4e).
As NQO1 is known to stabilize the proteins by inhibiting their degradation by 20S proteasome22, so the reverse regulation of Mdm2 by NQO1 was also checked. HEK-293T cells were co-transfected with HA Mdm2 and Flag NQO1 for 24hrs. The expression of Mdm2 was found to be increased in the presence of NQO1 (Fig. 4f). NQO1 was also found to stabilize Mdm2 protein at endogenous level (Fig. 4g). This observation was further confirmed by using diminutol (chemical inhibitor of NQO1 activity). HEK-293T cells were treated with different doses of diminutol for 6hrs. The expression of Mdm2 was found to be down-regulated in the presence of diminutol (Fig. 4h). Cycloheximide chase of Mdm2 in presence of diminutol also confirmed that stability of Mdm2 is reduced in the presence of diminutol (Fig. 4i). These results indicated that NQO1 stabilizes the expression of Mdm2 by inhibiting its 20S proteasomal degradation.
The expression of NQO1 in the presence of AKTi was also investigated. Inhibition of AKT activity was found to reduce the expression level of NQO1 in HEK-293T cells. This result was further validated in ThP-1 cells (natural host of HIV-1). AKTi treatment was found to reduce the levels of NQO1 in ThP-1 cells also (Fig. 4j). These results indicated that activation of PI3/AKT signalling pathway up-regulates the expression of HIV-1 Tat protein via Mdm2 mediated stabilization of NQO1.
Regulation of HIV-1 Tat expression via USP17 in ubiquitin-dependent manner
To confirm the ubiquitin-independent up-regulation of Tat expression by Mdm2 via NQO1, the ubiquitination assay was performed. HEK-293T cells were transfected with Flag Tat, HA Mdm2 and His Ub plasmids for 24hrs. Cells were treated with MG132 for 8hrs followed by immunoprecipitation using Ni-NTA resin. Western blot analysis was done to detect ubiquitinated species of Tat using anti-Flag antibody. Surprisingly, ubiquitination of Tat was found to be reduced in the presence of Mdm2 (Fig. 5a). This indicated the involvement of ubiquitination in Mdm2 mediated stabilization of Tat.
As, we have previously reported the stabilization of HIV-1 Tat protein in ubiquitination dependent manner by USP7 which is a deubiquitinase23, so we explored the possibility of the involvement of a deubiquitinase in Mdm2 mediated stabilization of Tat. We found a deubiquitinase, USP17 which was stabilizing the expression of Tat when co-expressed in HEK-293T cells (Fig. 5b). Cycloheximide chase of Tat in presence of USP17 also indicated that stability of Tat is increased in the presence of USP17 (Fig. 5c). Ubiquitination of Tat was also found to be increased by USP17 (Fig. 5d). To check the interaction between Tat and USP17, HEK-293T cells were transfected with HA Tat and co-immunprecipitaion was performed using anti-USP17 antibody bound agarose beads. This showed that USP17 interacts with HIV-1 Tat protein specifically (Fig. 5e). The regulation of USP17 expression by HIV-1 was also investigated in U1 cells. U1 cells were treated with PMA to activate latent virus for different time intervals followed by western blotting. The expression of USP17 was found to be induced by HIV-1 replication (Fig. 5f). These results indicated that USP17 stabilizes the expression of Tat in ubiquitin-dependent manner and is induced by HIV-1 infection also.
Mdm2 increases the expression of USP17 via direct interaction
The effect of Mdm2 on the expression of USP17 was investigated by co-expressing them in HEK-293T cells. The levels of USP17 were found to be increased in the presence of Mdm2 (Fig. 6a). The dose-dependent effect of Mdm2 on the levels of USP17 was also observed (Fig. 6b). Mdm2 was stabilizing the expression of USP17 at endogenous level also (Fig. 6c). The ubiquitination of USP17 in presence of Mdm2 was also investigated. HEK-293T cells were co-transfected with His USP17, HA Mdm2, and His Ub plasmids. Cells were also treated with MG132 to accumulate ubiquitinated species. Cells were lysed and cell lysate was incubated with Ni-NTA beads which bind His tagged ubiquitinated proteins followed by western blotting using anti-USP17 antibody. Ubiquitination of USP17 was found to be reduced by Mdm2 indicating the ubiquitin-dependent regulation of USP17 expression by Mdm2 (Fig. 6d). To check the interaction between Mdm2 and USP17, cell lysates of HEK-293T cells were subjected to co-immunoprecipitation using anti-Mdm2 antibody bound agarose resin followed by western blotting. Mdm2 was found to interact with USP17 specifically (Fig. 6e). To further confirm Mdm2-mediated stabilization of USP17, Mdm2 was downregulated using specific siRNA in HEK-293T cells. The expression of USP17 was found to be reduced in the presence of Mdm2 siRNA (Fig. 6f). The expression of USP17 in the presence of AKTi was also investigated. Inhibition of AKT activity was found to reduce the expression level of USP17 in HEK-293T cells. This result was further validated in ThP-1 cells. AKTi treatment was found to reduce the levels of USP17 in ThP-1 cells also (Fig. 6g). These results indicated that Mdm2 stabilizes Tat protein via inducing the expression of USP17.