The PKR activator, PACT, becomes a PKR inhibitor during HIV-1 replication

Abstract

BackgroundHIV-1 translation is modulated by the activation of the interferon (IFN)-inducible Protein Kinase RNA-activated (PKR). PKR phosphorylates its downstream targets, including the alpha subunit of the eukaryotic translation Initiation Factor 2 (eIF2α), which decreases viral replication. The PKR Activator (PACT) is known to activate PKR after a cellular stress. In lymphocytic cell lines, HIV-1 activates PKR only transiently and not when cells replicate the virus at high levels. The regulation of this activation is due to a combination of viral and cellular factors that have been only partially identified.ResultsPKR is transiently induced and activated in peripheral blood mononuclear cells after HIV-1 infection. The addition of IFN reduces viral replication, and induces both the production and phosphorylation of PKR. In lymphocytic Jurkat cells infected by HIV-1, a multiprotein complex around PKR contains the double-stranded RNA binding proteins (dsRBPs), adenosine deaminase acting on RNA (ADAR)1 and PACT. In HEK 293T cells transfected with an HIV-1 molecular clone, PACT unexpectedly inhibited PKR and eIF2α phosphorylation and increased HIV-1 protein expression and virion production in the presence of either endogenous PKR alone or overexpressed PKR. The comparison between different dsRBPs showed that ADAR1, TAR RNA Binding Protein (TRBP) and PACT inhibit PKR and eIF2α phosphorylation in HIV-infected cells, whereas Staufen1 did not. Individual or a combination of short hairpin RNAs against PACT or ADAR1 decreased HIV-1 protein expression. In the astrocytic cell line U251MG, which weakly expresses TRBP, PACT mediated an increased HIV-1 protein expression and a decreased PKR phosphorylation. In these cells, a truncated PACT, which constitutively activates PKR in non-infected cells showed no activity on either PKR or HIV-1 protein expression. Finally, PACT and ADAR1 interact with each other in the absence of RNAs.ConclusionIn contrast to its previously described activity, PACT contributes to PKR dephosphorylation during HIV-1 replication. This activity is in addition to its heterodimer formation with TRBP and could be due to its binding to ADAR1. HIV-1 has evolved to replicate in cells with high levels of TRBP, to induce the expression of ADAR1 and to change the function of PACT for PKR inhibition and increased replication.

Effective Inhibition of HIV-1 Production by Short Hairpin RNAs and Small Interfering RNAs Targeting a Highly Conserved Site in HIV-1 Gag RNA Is Optimized by Evaluating Alternative Length Formats

“…Cell lysates were obtained 48 h after cotransfection in lysis buffer containing antiproteases and antiphosphatases (Roche, Basel, Switzerland). Seventy-five micrograms of total protein was resolved on a 10% denaturing polyacrylamide gel and transferred to a Hybond ECL nitrocellulose membrane (GE Healthcare, Little Chalfont, United Kingdom) as previously described (43). Membranes were incubated first with anti-protein kinase R (PKR) (phospho-T446) E120 (Abcam, Cambridge, United Kingdom), anti-IRF3 (phospho S396) 4D4G (Cell Signaling, Danvers, MA), and anti-ADAR1 (from B.L.…”

HIV-1 RRE RNA acts as an RNA silencing suppressor by competing with TRBP-bound siRNAs

“…34,83,84 For virus production, HEK 293T cells were transfected with HIV-1 provirus pNL4-3 and supernatants were harvested 48 h post-transfection. Viral content of supernatant was evaluated by performing a reverse-transcriptase assay as described.…”

“…Cell lysates were prepared, separated and transferred for immunoblotting as previously described. 34,83,84 Membranes were blocked for 1 h in 5% nonfat milk and 0.05% Tris-buffered saline (TBS)-Tween and incubated overnight at 4 C with anti-EGFP (Santa Cruz) antibody at a 1/1000 dilution, anti-Myc (Santa Cruz) antibody at a 1/1000 dilution, anti-actin (Chemicon) antibody at a 1/ 10,000 dilution, anti-Dicer349 5 or anti-Dicer (Medimabs, Montreal, QC, CA) at a 1/5000 dilution, anti-TRBPjbx 36 at a 1/500 dilution, anti-Ago2 7C6 (from Dr. T. Hobman) at a 1/5000 dilution, anti-HIV-1 p24 183-H12-5C 83,84 at a 1/1000 dilution, anti-Ad2/5 E1A (Santa Cruz) at a 1/1000 dilution, or anti-GAPDH (Santa Cruz) at a 1/1000 dilution in 5% milk/TBST. After 5 washes in TBST, membranes were incubated with peroxidase-conjugated secondary donkey anti-rabbit antibody (Amersham) for TRBP, Ago2, Dicer and E1A, and sheep antimouse (Amersham) for EGFP, Myc, Actin, p24 and GAPDH at a 1/5000 dilution.…”

HIV-1 translation and its regulation by cellular factors PKR and PACT

“…It is possible that other dsRNAs structures from the HIV-1 RNA activate PKR, but those have not been studied. PKR is extremely effective in restricting HIV-1 translation and replication in cell culture suggesting that it is effectively activated upon viral expression Benkirane et al, 1997;Clerzius et al, 2009Clerzius et al, , 2013Dimitrova et al, 2005;Muto et al, 1999;Ong et al, 2005). Furthermore, knocking down PKR by small interfering (si) RNAs or expressing a transdominant mutant of PKR increases HIV-1 production (Ong et al, 2005).…”

“…Infection of the lymphocytic cell line Jurkat by HIV-1 shows an activation of the already expressed PKR at the beginning of the infection (Clerzius et al, 2009), whereas during the infection of peripheral blood mononuclear cells (PBMCs) by HIV-1, PKR is induced and activated within the first days of the infection (Clerzius et al, 2013). In each case, this activation is not sustained throughout HIV replication and PKR is deactivated when the virus replicates at high levels (Clerzius et al, 2009(Clerzius et al, , 2013. Cumulative data show that viral and cellular factors contribute to PKR deactivation in HIV-1 expressing cells 4.3.2.…”