The presence of the TAR RNA structure alters the programmed -1 ribosomal frameshift efficiency of the human immunodeficiency virus type 1 (HIV-1) by modifying the rate of translation initiation

Abstract

HIV-1 uses a programmed -1 ribosomal frameshift to synthesize the precursor of its enzymes, Gag-Pol. The frameshift efficiency that is critical for the virus replication, is controlled by an interaction between the ribosome and a specific structure on the viral mRNA, the frameshift stimulatory signal. The rate of cap-dependent translation initiation is known to be altered by the TAR RNA structure, present at the 5' and 3' end of all HIV-1 mRNAs. Depending upon its concentration, TAR activates or inhibits the double-stranded RNA-dependent protein kinase (PKR). We investigated here whether changes in translation initiation caused by TAR affect HIV-1 frameshift efficiency. CD4+ T cells and 293T cells were transfected with a dual-luciferase construct where the firefly luciferase expression depends upon the HIV-1 frameshift. Translation initiation was altered by adding TAR in cis or trans of the reporter mRNA. We show that HIV-1 frameshift efficiency correlates negatively with changes in the rate of translation initiation caused by TAR and mediated by PKR. A model is presented where changes in the rate of initiation affect the probability of frameshifting by altering the distance between elongating ribosomes on the mRNA, which influences the frequency of encounter between these ribosomes and the frameshift stimulatory signal.

Figure 1.

HIV-1 frameshift efficiency increases in the presence of inhibitors of cap-dependent translation initiation. (A) Major control steps of cap-dependent translation initiation in eukaryotes (15). The figure is adapted from Gebauer and Hentze (14). Only the factors we refer to in the text are named. The 40S ribosomal subunit associates with the ternary complex [initiation factor 2 (eIF2) plus GTP plus the initiator tRNA, Met-tRNA_i^Met] and with other factors, and binds to the 5′ cap structure of the mRNA. This binding requires the eIF4F complex formed by three initiation factors: eIF4E, the cap-binding protein, eIF4G, a scaffold protein and eIF4A, a RNA helicase that unfolds secondary structures. After each round of initiation, eIF2 is released from the ribosome in association with GDP. Phosphorylation of the α subunit of eIF2 (eIF2-α) prevents the recycling of eIF2-GDP in eIF2-GTP, blocking translation initiation. Thapsigargin induces endoplasmic reticulum stress, which stimulates the PERK kinase that phosphorylates eIF2-α, reducing the level of functional eIF2 (55–57). Rapamycin shuts down the mammalian target of rapamycin (mTOR) pathway, which blocks the phosphorylation of the translation repressor 4E-BP, and hypophosphorylated 4E-BP sequesters the initiation factor eIF4E (58,59). Hippuristanol is a selective inhibitor of eIF4A (60), which interferes with the binding of the 40S subunit to the mRNA. (B) Plasmid pDual-HIV contains the Rluc and the Fluc coding sequences under the control of a CMV promoter and separated by the HIV-1 frameshift region. (C) The frameshift efficiency was assessed in lysates from Jurkat cells transfected with 2 μg of pDual-HIV(-1) or (0) and, subsequently, treated with thapsigargin, rapamycin or hippuristanol or left untreated (see ‘Materials and Methods’ section for details). The frameshift efficiency with untreated cells transfected with pDual-HIV was arbitrarily set at 100%. Results are the means ± SD of at least four independent experiments.

Figure 2.

HIV-1 frameshift efficiency decreases when a high amount of TAR is present. (A) Sequence and structure of wild-type TAR RNA. (B) Plasmids pDual-HIV-TAR and pDual-HIV-50TAR are derivatives of pDual-HIV with the TAR-coding sequence inserted, respectively, about 40 nt downstream from the CMV promoter or at an additional distance of 50 nt from this promoter. Plasmid pTAR generates the free TAR sequence in trans from the reporter mRNA expressed from pDual-HIV. The frameshift efficiency was assessed in lysates from Jurkat cells (C) and 293T cells (D) transfected with 2 μg of pDual-HIV or pDual-HIV-TAR or pDual-HIV-50TAR or co-transfected with 2 μg of pDual-HIV and 2 μg of pTAR. The frameshift efficiency with Jurkat cells and 293T cells transfected with pDual-HIV was arbitrarily set at 100% in (C) and (D), respectively. Results are the means ± SD of at least four independent experiments. The P-values, calculated according to the Student's t-test, are indicated.

Figure 3.

PKR is involved in the decrease in HIV-1 frameshift efficiency observed when a high amount of TAR is present. (A) Sequence and structure of TAR mutants that cannot bind PKR (31) used in this study. (B) The frameshift efficiency is not affected by the TAR mutants. The frameshift efficiency was assessed in lysates from Jurkat cells co-transfected with pDual-HIV and plasmids expressing wild-type TAR or mutants of TAR. The frameshift efficiency with pDual-HIV was arbitrarily set at 100%. Results are the means ± SD of at least five independent experiments. The P values are indicated. The values with pDual-HIV and pTARΔbulge* or pDual-HIV and pTARuucg* were not significantly different from the value with pDual-HIV, but were significantly higher than the value with pDual-HIV and pTAR. (C) The presence of a Tat mutant (Tat*) that inhibits PKR decreases the frameshift efficiency. The frameshift efficiency was assessed in lysates from Jurkat cells co-transfected with pDual-HIV, pDual-HIV-TAR or pDual-HIV-50TAR and a plasmid coding for Tat* or an empty vector in a 1:1 ratio. The frameshift efficiency with pDual-HIV without Tat* was arbitrarily set at 100%. Results are the means ± SD of at least four independent experiments. The P-values are indicated. The values with pDual-HIV-TAR and pDual-HIV-50TAR, with or without Tat*, were not significantly different from the value with pDual-HIV with Tat* but significantly lower than the value with pDual-HIV without Tat*.

Figure 4.

Wild-type TAR, but not the TAR mutants, increases or decreases HIV-1 frameshift efficiency in a dose-dependent manner. The frameshift efficiency was assessed in lysates from stable 293T transfectants expressing the (-1) or (0) dual-luciferase HIV reporter transfected with pTAR (A), pTAR▵bulge* (B) or pTARuucg* (B) in different amounts ranging from 0 to 2.3 μg. The asterisks indicate the frameshift efficiencies that significantly differ from the frameshift efficiency without pTAR (P < 0.0005). Results are the means ± SD of at least six independent experiments.

Figure 5.

The effect of TAR on HIV-1 frameshift efficiency disappears when PKR expression is silenced. (A) The frameshift efficiency was assessed in lysates from stable 293T transfectants expressing the (-1) or (0) dual-luciferase HIV reporter and transfected first with a eGFP siRNA mix (negative control) or a PKR siRNA mix, and, after 24 h, with pTAR in different amounts ranging from 0 to 2.3 μg. Results are the means ± SD of at least three independent experiments. (B) Control of the silencing of PKR expression. Equal amounts of proteins from lysates of stable 293T transfectants expressing a dual-luciferase HIV reporter and transfected with either the eGFP siRNA mix (lane 1) or PKR siRNA mix (lane 2) were separated by SDS–PAGE, transferred on a nitrocellulose membrane and immunoblotted with a mouse anti-PKR monoclonal antibody. Anti-α-tubulin blotting was used as an internal control for loading.

Figure 6.

Changes in the rate of translation initiation influence the frameshift efficiency by modifying the spacing between elongating ribosomes. This model shows elongating ribosomes that reach the frameshift region and explains how the rate of translation initiation, which determines the spacing between these ribosomes, affects the frameshift efficiency (see the text). Note that a ribosome must encounter a folded frameshift stimulatory signal to make a frameshift, but this encounter does not ensure that frameshifting will occur.