TNPO3-Mediated Nuclear Entry of the Rous Sarcoma Virus Gag Protein Is Independent of the Cargo-Binding Domain

Abstract

Retroviral Gag polyproteins orchestrate the assembly and release of nascent virus particles from the plasma membranes of infected cells. Although it was traditionally thought that Gag proteins trafficked directly from the cytosol to the plasma membrane, we discovered that the oncogenic avian alpharetrovirus Rous sarcoma virus (RSV) Gag protein undergoes transient nucleocytoplasmic transport as an intrinsic step in virus assembly. Using a genetic approach in yeast, we identified three karyopherins that engage the two independent nuclear localization signals (NLSs) in Gag. The primary NLS is in the nucleocapsid (NC) domain of Gag and binds directly to importin-α, which recruits importin-β to mediate nuclear entry. The second NLS (TNPO3), which resides in the matrix (MA) domain, is dependent on importin-11 and transportin-3 (TNPO3), which are known as MTR10p and Kap120p in yeast, although it is not clear whether these import factors are independent or additive. The functions of importin-α/importin-β and importin-11 have been verified in avian cells, whereas the role of TNPO3 has not been studied. In this report, we demonstrate that TNPO3 directly binds to Gag and mediates its nuclear entry. To our surprise, this interaction did not require the cargo-binding domain (CBD) of TNPO3, which typically mediates nuclear entry for other binding partners of TNPO3, including SR domain-containing splicing factors and tRNAs that reenter the nucleus. These results suggest that RSV hijacks this host nuclear import pathway using a unique mechanism, potentially allowing other cargo to simultaneously bind TNPO3.IMPORTANCE RSV Gag nuclear entry is facilitated using three distinct host import factors that interact with nuclear localization signals in the Gag MA and NC domains. Here, we show that the MA region is required for nuclear import of Gag through the TNPO3 pathway. Gag nuclear entry does not require the CBD of TNPO3. Understanding the molecular basis for TNPO3-mediated nuclear trafficking of the RSV Gag protein may lead to a deeper appreciation for whether different import factors play distinct roles in retrovirus replication.

Keywords: Gag; TNPO3; nuclear import/export; retrovirus assembly.

FIG 1

Effects of TNPO3 expression on nuclear accumulation of Gag. (A) Schematic representation of RSV Gag constructs used, including the Gag domains matrix (MA), p2, p10, capsid (CA), spacer (SP), and nucleocapsid (NC) with an in-frame fusion of green fluorescent protein (GFP). Gag deletion mutants have yellow fluorescent protein (YFP) or GFP fusions, as indicated. TNPO3 consists of a three-domain structure that includes an N-terminal RanGTP-binding domain (RBD), a nuclear pore complex (NPC)-binding domain, and a C-terminal cargo-binding domain (CBD). (B) The localization of wild-type Gag.GFP (a) Gag.ΔNC.YFP (b), Gag.ΔMA_5–86.GFP (c) and Gag.ΔMA_5–148.GFP (d) expressed in QT6 cells are shown with DAPI (4′,6-diamidino-2-phenylindole)-stained images included to show the nucleus of the cell. (e) Localization of mCherry.TNPO3. (C) Cells coexpressing the Gag wild-type or mutant proteins with mCherry.TNPO3 are shown. (D) A scatterplot showing the percentage of Gag localized to the nucleus (% nuclear compared to the signal in the entire cell) for each cell analyzed, with or without coexpression of mCherry.TNPO3. At least 60 cells were analyzed from three independent experiments for each condition, with the standard error of the mean represented by the error bars. Statistical analysis was performed using the unpaired Student's t test, and significance (P < 0.0001) is indicted by an asterisk. In cases where mCherry.TNPO3 and Gag constructs were cotransfected, only cells expressing both Gag and mCherry.TNPO3 were analyzed quantitatively and shown in the graph. Images were chosen for display that are representative of the mean fluorescence intensity of the population analyzed. (E) Western blotting of cell lysates transfected with the indicated plasmids was performed using anti-GFP or α-mCherry antibodies, as appropriate, to show that full-length proteins were expressed at the expected sizes with minimal proteolysis for Gag.GFP, Gag variants, and mCherry.TNPO3. The vertical line between Gag.GFP and Gag. ΔMA_5–86.GFP represents the removal of an irrelevant lane in the gel. Gag.ΔNC.YFP was expressed on a separate blot, as was mCherry.TNPO3 (mCh.TPO3).

FIG 2

Gag nuclear accumulation with TNPO3 mutants. (A) Schematic representation of the constructs used, including Gag containing a C-terminal YFP fusion and TNPO3.ΔCargo, in which the entire cargo domain and a small portion of the NPC domain was deleted. (B) The top row shows Gag.YFP expressed alone in QT6 cells. The bottom row displays the accumulation of Gag.YFP with coexpression of mCherry.TNPO3.ΔCargo. The image chosen is representative of the mean nuclear fluorescence intensity of the population examined. (C) Scatter diagram plotting the percentage of nuclear Gag in either cells expressing Gag alone or cells coexpressing Gag with TNPO3.ΔCargo indicates a significant increase in the nuclear population of Gag. At least 43 cells were analyzed from at least 3 independent experiments for each condition. The mean and standard error of the mean are shown, with analysis performed using an unpaired Student's t test. The asterisk signifies a P value of <0.0001. Only cells expressing both Gag and mCherry.TNPO3.ΔCargo were analyzed. (D) Western blot analysis was performed and RSV Gag.YFP was detected using an α-RSV antibody, and mCherry.TNPO3.ΔCargo, which appears as a doublet, was detected using an α-mCherry antibody. In both cases, proteins of the expected size were expressed.

FIG 3

In vitro affinity-tagged purifications of Gag and TNPO3 protein complexes. (A) Schematic representation of constructs used (see Materials and Methods for details). CA-NTD contains the N-terminal domain of CA. Glutathione S-transferase (GST) was fused to the N terminus of TNPO3, with the ΔCargo mutant having the entire CBD and a small portion of the NPC domain deleted. The GST-TNPO3.NPC construct contains most of the NPC domain and a portion of the RanGTP-binding domain. (B) In the affinity purifications, various recombinant Gag proteins were incubated with GST-TNPO3, GST-TNPO3.ΔCargo, or GST alone. In the left panel, one-tenth of the volume of the assay mixture was removed before the incubation step, separated by SDS-PAGE gel, and stained with AcquaStain to directly visualize the Gag proteins put into the binding assay (input). In the right panel, the bound protein elutes were separated by SDS-PAGE and analyzed by Western blotting using α-RSV antibodies. (C and D) The same assay was used as in panel B. Gag was detected in the linear range using a short exposure, whereas detection of MA required a longer exposure. At least 3 independent GST-tagged purifications were performed for each condition, and a representative image is shown. (E) Western blot analysis detecting purified Gag proteins using an α-RSV antibody to show full-length proteins of the expected molecular weights.

FIG 4

Effects of multiple import factors on Gag nuclear accumulation. (A) Visualization of subcellular localization of Gag.YFP (green) alone (first column) and with coexpression of individual import factors, mCherry.TNPO3 (red, second column), HA-importin-11 (magenta, third column), and HA-importin-β (magenta, fourth column). (B) Visualization of subcellular distribution of Gag.YFP with coexpression of importin-11 and TNPO3 (left column) and importin-β and TNPO3 (right column). (C) Scatterplot displaying the percentage of nuclear Gag alone, coexpressed with TNPO3 and importin-11 or importin-β individually, then coexpressed with the combination of TNPO3 plus importin-11 or TNPO3 plus importin-β. At least 27 cells were analyzed from two independent experiments for each condition. Group comparisons were analyzed using unpaired Student’s t tests. The mean and the standard error of the mean are represented, and an asterisk signifies a statistically significant difference (P < 0.0001) between each compared group. For conditions of Gag coexpressed with an import factor, only cells expressing both tagged proteins (Gag and the import factors) were analyzed, and the images shown are representative of the mean fluorescence intensity of the population.