Tuning of AKT-pathway by Nef and its blockade by protease inhibitors results in limited recovery in latently HIV infected T-cell line.

Amit Kumar,Kashif Aziz Khan,Georges Herbein,Alexandra Borch,Audrey Varin,Christine Rouzioux,Nathan Clumeck,Sébastien Pasquereau,Renate König,Olivier Rohr,Jean-Stéphane Gatot,Caroline Vanhulle,Wasim Abbas,Sophie Bouchat,Kabamba Kabeya,Anis Larbi,Laurence Colin,Carine Van Lint,Laurie Coquard,Tamás Fülöp ,Nadège Delacourt ,Stéphane De Wit

doi:10.1038/srep24090

Abstract

Akt signaling plays a central role in many biological processes, which are key players in human immunodeficiency virus 1 (HIV-1) pathogenesis. We found that Akt interacts with HIV-1 Nef protein. In primary T cells treated with exogenous Nef or acutely infected with Nef-expressing HIV-1 in vitro, Akt became phosphorylated on serine473 and threonine308. In vitro, Akt activation mediated by Nef in T-cells was blocked by HIV protease inhibitors (PI), but not by reverse transcriptase inhibitors (RTI). Ex vivo, we found that the Akt pathway is hyperactivated in peripheral blood lymphocytes (PBLs) from cART naïve HIV-1-infected patients. PBLs isolated from PI-treated patients, but not from RTI-treated patients, exhibited decreased Akt activation, T-cell proliferation and IL-2 production. We found that PI but not RTI can block HIV-1 reactivation in latently infected J-Lat lymphoid cells stimulated with various stimuli. Using luciferase measurement, we further confirmed that Nef-mediated reactivation of HIV-1 from latency in 1G5 cells was blocked by PI parallel to decreased Akt activation. Our results indicate that PI-mediated blockade of Akt activation could impact the HIV-1 reservoir and support the need to further assess the therapeutic use of HIV-1 PI in order to curtail latently infected cells in HIV-1-infected patients.

Highlights

(1 to 10 ng/ml) and a dose response of recombinant Nef (rNef) treatment on Akt activation (D) in peripheral blood lymphocytes (PBLs) isolated from healthy donors. (E), Internalization of rNef by CD4+ T cells determined by flow cytometry
Western blot detection of activated pAkt (Ser[473], Thr308) in the lysates derived from 5 × 106 PBLs treated with 100 ng/ml of Nef with or without Akt (Akt inhibitor VIII) and phosphoinositide 3-kinase (PI3K) inhibitors (LY294002 and Wortmannin) (n = 3). (G), Akt activation in PBLs by wild-type HIV-1, but not by isogenic HIV-1∆ Nef
We observed that treatment of PBLs with rNef led to Akt phosphorylation in a dose dependent (Fig. 1A) and time dependent manner as determined (Fig. 1B) by western blotting and confocal microscopy (Fig. 1C,D)

Summary

Results

Exogenous Nef enters into PBLs and increases Akt phosphorylation. Nef on Akt activity, which is activated by its phosphorylation on Ser[473] and Thr[308] residues[20,21]. Our results indicate that rNef, alone or in conjunction with TCR stimulation is able to significantly activate Akt in PBLs from healthy donors Since both proteins have been reported to translocate to lipid rafts[1,31,32], we questioned whether the HIV-1 Nef/Akt interaction involves lipid rafts. We assessed the respective role of PI or RTI treatment on HIV-1 reactivation from latency by measuring viral production in TCR-stimulated latently-infected J-Lat 8.4 cells treated with rNef. J-Lat 8.4 cells have full length HIV-1 minus env and nef genes. We found rNef treatment resulted in almost two-fold increase in the expression of luciferase activity as compared to mock treated cells which was subsequently blocked by protease inhibitor (Fig. 8 upper panel). Our results indicate that PI-mediated blockade of Akt activation could impact the HIV-1 reservoir especially when driven by viral proteins such as Nef

Discussion

Conclusion

Materials and Methods