The HIV-1 Nef protein interacts with two components of the 40S small ribosomal subunit, the RPS10 protein and the 18S rRNA

Abstract

Background: Human immunodeficiency virus type 1 (HIV-1) Nef-encoded protein plays key functions at almost all stages of the viral life cycle, but its role in translation is largely unknown.

Methods: To determine the effect of Nef on translation we used an in vitro translation assay. The detection of Nef/RPS10 complexes and the presence of 18S rRNA and tRNAs in the complexes were performed by coimmunoprecipitation and RT-PCR assay.

Results: We observed that the HIV-1 Nef protein specifically impaired translation in vitro. We observed the interaction of Nef with RPS10 by coimmunoprecipitation assay. In addition 18S rRNA and tRNAs were present in the Nef/RPS10 complexes.

Conclusions: Our results are consistent with a model in which the Nef protein by binding to two components of the 40S small ribosomal subunit, RPS10 and 18S rRNA, and to a lesser extent to tRNAs, could lead to decreased protein synthesis.

Figure 1

HIV-1 Nef inhibits translationin vitro. The level of translation of the luciferase mRNA has been measured in the presence of increasing concentrations of rNef, rVpr or recombinant ovalbumin in RRL. Recombinant HIV-1 Vpr and ovalbumin were used to test the specificity of Nef action on translation. In addition an inactive form of Nef was obtained by heating at 100°C for 5 min (HI).

Figure 2

Expression of RPS10/rNef complexes in primary PBMCs, PBLs and MDMs treated with rNef. (A) TotalTotal cellular extracts from PBMCs treated with rNef (100 ng/ml) for 2 hours were immunoprecipitated with an anti-RPS10 mAb or an anti-Nef mAb. Immunoprecipitated material was analyzed by western blotting with an anti-Nef mAb or an anti-RPS10 mAb. Results are representative of three independent experiments. (B) Cytoplasmic and nuclear extracts from several cell lines (Vero cells, U937 cells), PBLs and MDMs treated with rNef (100 ng/ml) for 2 hours were immunoprecipitated with an anti-RPS10 mAb. Immunoprecipitated material was analyzed by western blotting with an anti-Nef mAb. The controls are input controls from total cell lysates (for IP RPS10 and mouse IgG control). Results are representative of three independent experiments. (C) Cytoplasmic and nuclear extracts from U937 cells treated with increasing concentrations of rNef (100–1500 ng/ml) for 2 hours or mock-treated were immunoprecipitated with an anti-RPS10 mAb. Immunoprecipitated material was analyzed by western blotting with an anti-Nef mAb. Results are representative of three independent experiments. β-actin and TBP (TATA binding protein) detection represents input loading controls of the lysates which were used in binding reactions.

Figure 3

Time course of RPS10/rNef complexes in cells treated with rNef. Cytoplasmic and nuclear extracts from U937 cells and PBLs treated with rNef (100 ng/ml) for up to 5 hours were immunoprecipitated with an anti-RPS10 mAb. Immunoprecipitated material was analyzed by western blotting with an anti-Nef mAb. Results are representative of three independent experiments

Figure 4

Presence of 18s rRNAs and tRNAs in immunoprecipitated complexes of Nef and RPS10. 18s rRNA and tRNAs are present in Nef (A) and RPS10 (B) co-immunoprecipitated complexes. MDMs were treated with rNef (100 ng/ml) for 2 h or left untreated (mock). Total cellular extracts were prepared and the detection of tRNAMet, tRNATryp, tRNAPhe, tRNALys3, tRNAyeast (as a negative control) and 18S rRNA was performed in lysates immunoprecipitated with Nef and RPS10 antibodies respectively, followed by RNA extraction and qRT-PCR amplification as previously described [28]. Results are representative of two independent experiments.

Figure 5

This scheme presents our hypothesis concerning the putative impact of Nef on ribosome biogenesis and on the translational process. The HIV-1 Nef protein binds to RPS10 and to 18S rRNA that could result in at least two distinct features: inhibition of ribosome biogenesis and direct inhibition of the translational process. ETS, external transcribed spacer; ITS, internal transcribed spacers 1 and 2; translation initiation factor eIF3.