Characterization of staufen1 ribonucleoproteins by mass spectrometry and biochemical analyses reveal the presence of diverse host proteins associated with human immunodeficiency virus type 1

Abstract

The human immunodeficiency virus type 1 (HIV-1) unspliced, 9 kb genomic RNA (vRNA) is exported from the nucleus for the synthesis of viral structural proteins and enzymes (Gag and Gag/Pol) and is then transported to sites of virus assembly where it is packaged into progeny virions. vRNA co-exists in the cytoplasm in the context of the HIV-1 ribonucleoprotein (RNP) that is currently defined by the presence of Gag and several host proteins including the double-stranded RNA-binding protein, Staufen1. In this study we isolated Staufen1 RNP complexes derived from HIV-1-expressing cells using tandem affinity purification and have identified multiple host protein components by mass spectrometry. Four viral proteins, including Gag, Gag/Pol, Env and Nef as well as >200 host proteins were identified in these RNPs. Moreover, HIV-1 induces both qualitative and quantitative differences in host protein content in these RNPs. 22% of Staufen1-associated factors are virion-associated suggesting that the RNP could be a vehicle to achieve this. In addition, we provide evidence on how HIV-1 modulates the composition of cytoplasmic Staufen1 RNPs. Biochemical fractionation by density gradient analyses revealed new facets on the assembly of Staufen1 RNPs. The assembly of dense Staufen1 RNPs that contain Gag and several host proteins were found to be entirely RNA-dependent but their assembly appeared to be independent of Gag expression. Gag-containing complexes fractionated into a lighter and another, more dense pool. Lastly, Staufen1 depletion studies demonstrated that the previously characterized Staufen1 HIV-1-dependent RNPs are most likely aggregates of smaller RNPs that accumulate at juxtanuclear domains. The molecular characterization of Staufen1 HIV-1 RNPs will offer important information on virus-host cell interactions and on the elucidation of the function of these RNPs for the transport of Gag and the fate of the unspliced vRNA in HIV-1-producing cells.

Keywords: Gag; HIV-1; Staufen1; genomic RNA; gradient centrifugation; mass spectrometry; ribonucleoprotein; virus-host interactions.

Figure 1

Generation and isolation of stable neomycin-resistant cell lines expressing Staufen1-TAP and TAP proteins. (A) Structures of the Tandem Affinity Purification (TAP) tag cassette and Staufen1-TAP fusion protein. TAP tag consists of two sequences responsible for the affinity purification – IgG binding domain of Protein A and Calmodulin-binding peptide separated by a unique cleavage site for Tobacco Etch Virus (TEV) protease. (B,C) Expression levels of St-TAP in stable 293T and Jurkat T cell clones detected by western blotting analysis using a monoclonal mouse anti-Staufen1 antibody that recognizes both Staufen1⁵⁵ (55 kDa), Staufen1⁶³ (63 kDa), the endogenous isoforms and exogenous St-TAP (∼75 kDa) fusion protein, respectively. GAPDH is the loading control. 293T cell clone #11 and Jurkat T cell clone #13 were used in experiments. (D) Expression levels of TAP protein in 7 (#1–#7) control 293T clones and verified by western blotting. Clone #6 was used in control experiments. (E) Stable cell lines TAP#6 and St-TAP#11 were stained with mouse monoclonal anti-protein A antibody. The primary antibody was detected by Alexa Fluor 488 goat anti–mouse IgG antibody and the stained cells were visualized by epifluorescence microscopy.

Figure 2

Characterization of the tandem affinity-isolated Staufen1^55 kDa RNPs by western blot and mass spectrometry analysis. (A) Separation of proteins from cell lysates (Inputs) before (left panels) and after (right panels) tandem affinity purification (Staufen1 complexes) 0.25 mg of total protein obtained from extracts of TAP (control) or Staufen1-TAP expressing cell lines in the absence or presence of HIV-1 were used for affinity purification. Initially, the membrane was blotted with mouse anti-Staufen1 monoclonal antibody shown on the top left and right panels. The star (*) indicates the position of the purified St-CBP protein. (B) The proteins from the eluted Staufen1^55 kDa complexes and those from TAP alone (control) were purified from 75 mg of total protein, separated on SDS-PAGE and subsequently stained with Bio-Safe Coomassie Blue. Four bands were excised from gel lanes (II) and (III) for the identification of potential contaminating proteins. 23 bands from St-TAP and St-TAP HIV-1 (lanes IV and V) were excised and analyzed with mass spectrometry. Lane I represents the molecular weights in kDa of the protein standards. (C) Dual affinity purification of Staufen1 complexes derived from Jurkat T St-TAP stable cell lines in the absence or in the presence of HIV-1.

Figure 3

Staufen1 RNPs contain proteins involved in the localization, stabilization and trafficking of mRNAs and HIV-1 vRNA. Graphical representation and comparison of Staufen1-associated proteins isolated from 293T cells in the absence (top panel) and the presence of HIV-1 (middle panel). The newly identified proteins were grouped on the basis of their main function or the process in which they are involved in both control and HIV-1 conditions. The subcellular distribution of proteins found in Staufen1-TAP (blue bars) and Staufen1-TAP HIV-1 (orange bars) RNPs is depicted (bottom panel). The information for each protein was obtained from the UniProt database.

Figure 4

Confirmation of the in vivo association of Staufen1 with several of the host factors that were identified in Staufen1-contain RNP complexes. (A–E) CRM1, ABCE1, TDP-43, AUF1 and IMP1 (as IMP1-VenusC) were immunoprecipitated from 500 mg total protein lysate of HeLa cells. For the immunoprecipitation of IMP1 HeLa cells were transfected with IMP1-VenC or Venus-full length (as a control) and immunoprecipitated with a mouse anti-GFP antibody. The images to the immediate right of the IPs show the patterns of co-localization of CRM1, cytoplasmic RNAse L inhibitor (ABCE1), AUF1, TAR DNA-binding protein (TDP-43) and IMP1 with Staufen1-HA. The Manders’ coefficients (average from >10 cells per experiment derived from Staufen-HA expressing cells only, in %) are shown to provide an estimate of the co-localization. At the far right, the distribution of each host protein (in red) is shown in relation to Gag (blue) and the viral genomic RNA (vRNA, green) in HIV-1-expressing cells as determined by laser scanning confocal microscopy. Cells overexpressing IMP1-VenusC are indicated with white arrows in (E). Size bars are 10 μm.

Figure 5

Staufen1 co-fractionates with Gag and vRNA in gradient density fractionation analyses. (A) HeLa cells were either mock transfected with empty vector, pcDNA3, or with a plasmid that expresses Staufen1-HA. The transfected cells were collected 24 h later, lysed and were either mock-treated or treated with RNAse A. The lysates were then fractionated on 5–50% sucrose gradients and 20 fractions were collected for further analysis by western blotting for viral and host proteins, as indicated. HIV-1 viral genomic RNA (vRNA, 9 kb) was assessed in each fraction by slot blot analysis. TL represents the total lysates. (B) HeLa cells were co-transfected with pNL 4–3 and Staufen1-HA. The presence of Staufen1-HA, precursor Gag and p24 were assessed in each fraction by western blotting analysis. HIV-1 viral genomic RNA (9 kb, vRNA) was assessed in each fraction by slot blot analysis. Staufen1, Gag and vRNA were quantitated in each fraction by densitometry and relative levels are depicted for each fraction (Blue: Staufen1-HA, Red: Gag, Green-vRNA).

Figure 6

The sedimentation of Staufen1 and vRNA indensitygradientsdid not significantly change in the absence of Gag expression. (A) HeLa cells were co-transfected with pNL4-XX (harboring two mutations in the gag open reading frame to prevent Gag synthesis) with either empty vector control (A), Staufen1-HA (B), or Gag-RRE (C). The cells lysates were then fractionated on 5–50% sucrose gradients and 20 fractions were collected for further analysis by western blotting analyses, as indicated. TL represents the total lysates. Staufen1, Gag and vRNA were quantitated in each fraction by densitometry and relative levels are depicted for each fraction (Blue: Staufen1-HA, Red: Gag, Green: vRNA).

Figure 7

The relative levels of Gag in light and dense gradient fractions are modulatedin Staufen1-depleted cells. HeLa cells were mock transfected or co-transfected with pNL4-3 with either non-silencing control siRNA (siNS) or a siRNA to deplete endogenous Staufen1 (siStaufen1) and were harvested and lysed 36 h later and analyzed by western blotting. Corresponding expression levels for Staufen1, Gag, UPF1 and GAPDH proteins are shown in (A). The cell lysates from siNS-treated (B) and siStaufen1-treated (C) cells were fractionated on 5–50% sucrose gradients and 19–20 fractions were collected for further analysis by western blotting, as indicated. TL represents the total lysates. HIV-1 viral genomic RNA (9 kb, vRNA) was assessed in each fraction by slot blot analysis. Staufen1, Gag and vRNA were quantitated in each fraction by densitometry and relative levels are depicted for each fraction (Blue: Staufen1-HA, Red: Gag, Green: vRNA).