HIV-1 Recruits UPF1 but Excludes UPF2 to Promote Nucleocytoplasmic Export of the Genomic RNA

Abstract

Unspliced, genomic HIV-1 RNA (vRNA) is a component of several ribonucleoprotein complexes (RNP) during the viral replication cycle. In earlier work, we demonstrated that the host upframeshift protein 1 (UPF1), a key factor in nonsense-mediated mRNA decay (NMD), colocalized and associated to the viral structural protein Gag during viral egress. In this work, we demonstrate a new function for UPF1 in the regulation of vRNA nuclear export. OPEN ACCESS Biomolecules 2015, 5 2809 We establish that the nucleocytoplasmic shuttling of UPF1 is required for this function and demonstrate that UPF1 exists in two essential viral RNPs during the late phase of HIV-1 replication: the first, in a nuclear export RNP that contains Rev, CRM1, DDX3 and the nucleoporin p62, and the second, which excludes these nuclear export markers but contains Gag in the cytoplasm. Interestingly, we observed that both UPF2 and the long isoform of UPF3a, UPF3aL, but not the shorter isoforms UPF3aS and UPF3b, are excluded from the UPF1-Rev-CRM1-DDX3 complex as they are negative regulators of vRNA nuclear export. In silico protein-protein docking analyses suggest that Rev binds UPF1 in a region that overlaps the UPF2 binding site, thus explaining the exclusion of this negative regulatory factor by HIV-1 that is necessary for vRNA trafficking. This work uncovers a novel and unique regulatory circuit involving several UPF proteins that ultimately regulate vRNA nuclear export and trafficking.

Keywords: HIV-1; UPF1; nonsense mediated decay; nuclear RNA export; ribonucleoprotein; viral evasion.

Figure 1

UPF1 nucleocytoplasmic shuttling promotes vRNA nucleocytoplasmic export (A); The UPF1 nuclear export and localization sequence (NES, NLS) domains are shaded in the depiction of UPF1. HeLa cells were co-transfected with wildtype proviral DNA (PNL4-3) alone (B); or a Rev-defective provirus pMRev(−) alone (C); or with UPF1^WT (D); UPF1^ΔNES (E); and UPF1^ΔNLS (F). vRNA shown in red and UPF1 proteins are shown in green (in all panels). White arrowheads identify HIV-1-transfected cells. Size bars indicate 10 µm. The corresponding Intensity plots for vRNA (red) and GFP-tagged UPF1 protein staining are shown from point A to B in the designate cells: dashed lines demarcate the nucleus (N) and cytoplasm (C) boundaries. Imaging results shown are representative of the phenotypes observed in >85% cells in each condition in 5 independent experiments. (G) Dunnett post-test analyses on vRNA localization results ±SEM values compared to the controls pNL4-3+GFP (pNL4-3) or GFP (Rev−); *** p < 0.001.

Figure 2

UPF1 depletion increases the abundance of vRNA in the nucleus while UPF1 Rescue enhances cyto-plasmic vRNA levels. (A) HeLa cells were mock transfected with pCI-FLAG and non-silencing siRNA (siNS) or co-transfected with HIV-1 proviral pNL4-3 DNA and pCI-FLAG and siNS. UPF1 was depleted by siUPF1 and UPF1 expression was rescued with FLAG-UPF1Rescue (UPF1R). At 30h post-transfection, cells were harvested and cytoplasmic-nuclear fractionation analysis was carried out as described in Materials and Methods. Western blot analysis was performed GAPDH (enriched in the cytoplasmic), Nucleolin (enriched in the nucleus), and UPF1 (identifying endogenous and overexpressed proteins). RNA was isolated from cell extracts and steady-state levels of vRNA and gapdh mRNA was measured by RT-PCR. vRNA levels were quantified by densitometry with ImageJ using gapdh mRNA signal intensity for normalization; pNL4-3 + siNS condition was set at a 100%. nd, not detectable. (B) Cells were mock-depleted with nonsilencing siRNA (siNS) or depleted using siUPF1 as described in Materials and Methods. At 40–48 h post-transfection, cells were harvested for western blotting for UPF1 (endogenous), Gag (identifying pr55Gag and p24) and GAPDH (loading control) (on left) and FISH/IF co-analyses for UPF1 (red), vRNA (green) and Gag (blue) (with merged renditions on right). UPF1 depletion led to decreased Gag expression as shown earlier (left panel; Ajamian et al., 2008) but also led to a blockade to vRNA nuclear export (white arrowhead < in lower row of right panel) in >80% cells examined (n = 48). See results for additional discussion of these findings.

Figure 3

UPF1 exports vRNA via CRM1 and not the NXF1 export pathway. HeLa cells were transfected with pNL4-3 alone and treated with LMB (A); co-transfected with Flag-UPF1 and treated with LMB (B); co-transfected with HA-TapA17 (C); or co-transfected with HA-TapA17 and FLAG-UPF1 (D). FISH/IF co-analyses to identify the localization of vRNA (green in all rows), either endogenous UPF1 (red, A) or FLAG-UPF1 expressed in trans (red, B and D) or HA-TapA17 (blue in C and D). Merged renditions are shown on right-most panels. White arrowheads identify cells exhibiting LMB-induced block to nuclear vRNA export in (in A and B) and cells expressing HIV-1 (in C and D). Imaging results are representative of the phenotypes observed in 78%–85% cells in each condition in 3 experiments. Size bars, 10 µm; (E) Western blot analysis for Gag, FLAG-UPF1, HA-TapA17 and GAPDH (loading control). ImageJ was used to quantify the relative expression levels of Gag (normalized to GAPDH levels). Results shown represent averages from 2 independent experiments with <10% deviation between experiments. nd: not detectable.

Figure 4

UPF1 is found in complex with Rev-CRM1-DDX3-Nup62. (A) HeLa cells were mock transfected with pCI-FLAG or transfected with FLAG-UPF1 alone or with either pMRev− or Rev+ pNL4-3 DNAs. FLAG-UPF1 was immunoprecipitated as described in Materials and Methods. Input lysates and bound complexes were analyzed by western blotting for CRM1, FLAG-UPF1, Gag, DDX3 and GAPDH (loading and output control). HC: Heavy Chain IgG. Results shown are representative of three independent experiments; (B) HeLa cells were mock transfected with pCI-FLAG or FLAG-UPF1 or co-transfected with FLAG-UPF1 and Rev-R-YC. FLAG-UPF1 was immunoprecipitated and western blotting of input lysates and bound complexes for Rev, FLAG-UPF1, DDX3 and CRM1. HC: Heavy Chain IgG; (C) HeLa cells were mock transfected with pCI-FLAG or transfected with FLAG-UPF1. Cells were harvested and FLAG-UPF1 was immunoprecipitated as described in Materials and Methods. Western blotting analysis for FLAG-UPF1 and Nup62. IgG HC: IgG heavy Chain; (D) Glycerol gradient analyses of UPF1 complexes in HIV-1-expressing cells. HIV-1/FLAG-UPF1-expressing cells were immunoprecipitated with anti-FLAG serum. FLAG-UPF1 complexes were eluted and fractionated on a glycerol gradient. Fractions were collected and FLAG-UPF1, pr55^Gag, CRM1 and UPF3b were probed in each fraction by Western blotting. vRNA abundance is shown on the bottom panel, as determined by slot blot analyses; (E) HeLa cells were mock transfected with pCI-FLAG or co-transfected with HIV-1 and FLAG-UPF1. At 30 h post--transfection, Gag was immunoprecipitated using monoclonal anti-p24 antisera. Western for UPF1 (FLAG), Gag, CRM1, Nup62 and DDX3 were performed. Light Chain IgG.

Figure 4

UPF1 is found in complex with Rev-CRM1-DDX3-Nup62. (A) HeLa cells were mock transfected with pCI-FLAG or transfected with FLAG-UPF1 alone or with either pMRev− or Rev+ pNL4-3 DNAs. FLAG-UPF1 was immunoprecipitated as described in Materials and Methods. Input lysates and bound complexes were analyzed by western blotting for CRM1, FLAG-UPF1, Gag, DDX3 and GAPDH (loading and output control). HC: Heavy Chain IgG. Results shown are representative of three independent experiments; (B) HeLa cells were mock transfected with pCI-FLAG or FLAG-UPF1 or co-transfected with FLAG-UPF1 and Rev-R-YC. FLAG-UPF1 was immunoprecipitated and western blotting of input lysates and bound complexes for Rev, FLAG-UPF1, DDX3 and CRM1. HC: Heavy Chain IgG; (C) HeLa cells were mock transfected with pCI-FLAG or transfected with FLAG-UPF1. Cells were harvested and FLAG-UPF1 was immunoprecipitated as described in Materials and Methods. Western blotting analysis for FLAG-UPF1 and Nup62. IgG HC: IgG heavy Chain; (D) Glycerol gradient analyses of UPF1 complexes in HIV-1-expressing cells. HIV-1/FLAG-UPF1-expressing cells were immunoprecipitated with anti-FLAG serum. FLAG-UPF1 complexes were eluted and fractionated on a glycerol gradient. Fractions were collected and FLAG-UPF1, pr55^Gag, CRM1 and UPF3b were probed in each fraction by Western blotting. vRNA abundance is shown on the bottom panel, as determined by slot blot analyses; (E) HeLa cells were mock transfected with pCI-FLAG or co-transfected with HIV-1 and FLAG-UPF1. At 30 h post--transfection, Gag was immunoprecipitated using monoclonal anti-p24 antisera. Western for UPF1 (FLAG), Gag, CRM1, Nup62 and DDX3 were performed. Light Chain IgG.

Figure 5

Interaction models and a mechanism for UPF2 exclusion. (A) Structural model for the interaction between UPF1 (cyan) and DDX3 (yellow) and structural alignment with the UPF1-UPF2 complex. UPF2 is represented as a gray surface (upper). An amplified image of the model for the UPF1-DDX3 complex is presented (bottom). Relevant residues obtained for UPF1 (orange) and DDX3 (pink) through alanine scanning or described in previous work as relevant for interaction with UPF2 (green) are shown as sticks. (B) Structural model for the interaction between UPF1 (cyan) and Rev (magenta) and structural alignment with the UPF1-UPF2 complex. UPF2 is represented as a gray surface (upper). An amplified image of the model for the UPF1-Rev complex is presented (bottom). Relevant residues obtained for UPF1 (orange) and Rev (pink) through alanine scanning or described in previous work as relevant for interaction with UPF2 (green) are shown as sticks. Drawing was performed with the Pymol software as described in materials and methods.

Figure 5

Interaction models and a mechanism for UPF2 exclusion. (A) Structural model for the interaction between UPF1 (cyan) and DDX3 (yellow) and structural alignment with the UPF1-UPF2 complex. UPF2 is represented as a gray surface (upper). An amplified image of the model for the UPF1-DDX3 complex is presented (bottom). Relevant residues obtained for UPF1 (orange) and DDX3 (pink) through alanine scanning or described in previous work as relevant for interaction with UPF2 (green) are shown as sticks. (B) Structural model for the interaction between UPF1 (cyan) and Rev (magenta) and structural alignment with the UPF1-UPF2 complex. UPF2 is represented as a gray surface (upper). An amplified image of the model for the UPF1-Rev complex is presented (bottom). Relevant residues obtained for UPF1 (orange) and Rev (pink) through alanine scanning or described in previous work as relevant for interaction with UPF2 (green) are shown as sticks. Drawing was performed with the Pymol software as described in materials and methods.

Figure 6

UPF2 is absent from the HIV-1 Gag RNP and UPF3b co-immunoprecipitates with UPF1 under HIV-1 conditions. (A) HeLa cells were mock transfected with pCI-FLAG or co-transfected with proviral pNL4-3 DNA and FLAG-UPF1^WT. At 30 h post-transfection, cells were harvested and Gag was immunoprecipitated using a monoclonal anti-p24 antiserum as described in Materials and Methods. Western blot analysis for FLAG-UPF1, UPF3b, Gag (pr55^Gag), UPF2, and eF1α (positive control) was performed. HC: IgG Heavy Chain. LC: IgG Light Chain. Results are representative of two experiments. (B) HeLa cells were mock transfected with pCI-FLAG or FLAG-UPF1 or co-transfected with HIV-1 pNL4-3 DNA. Cells were harvested and FLAG-UPF1 was immunoprecipitated. Input lysates and bound complexes were analyzed by western blotting for UPF2, FLAG-UPF1, UPF3b, GAPDH (loading and output control) and Gag (pr55^Gag). Results shown are representative of three independent experiments. HC: IgG Heavy Chain, LC: IgG Light Chain.

Figure 7

UPF3aL and UPF2 block vRNA export by associating to UPF1 as determined by FISH/IF and co-immunoprecipitation analyses. HeLa cells were co-transfected with pNL4-3 with pCI-FLAG (A); and FLAG-tagged UPF proteins: UPF3b (B); UPF3aL (C); UPF3aS (D); UPF2 (E); or UPF2 1-1096 (F); and processed for FISH/IF co-analyses to determine the localization of vRNA (green) and UPF proteins (B–F) (red); White arrowheads identify cells transfected with pNL4-3 alone. (G) Dunnett post-test analyses on vRNA localization results ±SEM values compared to the controls pNL4-3+pCI-FLAG: *** p < 0.001. Co-immunoprecipitation analyses confirm that UPF2 association blocks vRNA export. HeLa cells were mock transfected with pCI-FLAG or co-transfected with HIV-1 pNL4-3 proviral DNA and pCI-FLAG or FLAG-UPF3aL or FLAG-UPF3b (H). HeLa cells were transfected as in (H) but FLAG-UPF3aS was included instead of FLAG-UPF3b (I). HeLa cells were transfected as in (H) except that FLAG-tagged UPF2 proteins were co-transfected: UPF2WT or UPF21173 or UPF21-1096 (J). At 48 h post-transfection, cells were harvested and FLAG-tagged UPF proteins were individually immunoprecipitated as described in Materials and Methods. Input and bound (Output) complexes were analyzed by western blotting for UPF proteins as indicated. The abundance of vRNA and gapdh RNA was estimated by RT-PCR (in Input only). Results shown are representative of 2 independent experiments.

Figure 7

UPF3aL and UPF2 block vRNA export by associating to UPF1 as determined by FISH/IF and co-immunoprecipitation analyses. HeLa cells were co-transfected with pNL4-3 with pCI-FLAG (A); and FLAG-tagged UPF proteins: UPF3b (B); UPF3aL (C); UPF3aS (D); UPF2 (E); or UPF2 1-1096 (F); and processed for FISH/IF co-analyses to determine the localization of vRNA (green) and UPF proteins (B–F) (red); White arrowheads identify cells transfected with pNL4-3 alone. (G) Dunnett post-test analyses on vRNA localization results ±SEM values compared to the controls pNL4-3+pCI-FLAG: *** p < 0.001. Co-immunoprecipitation analyses confirm that UPF2 association blocks vRNA export. HeLa cells were mock transfected with pCI-FLAG or co-transfected with HIV-1 pNL4-3 proviral DNA and pCI-FLAG or FLAG-UPF3aL or FLAG-UPF3b (H). HeLa cells were transfected as in (H) but FLAG-UPF3aS was included instead of FLAG-UPF3b (I). HeLa cells were transfected as in (H) except that FLAG-tagged UPF2 proteins were co-transfected: UPF2WT or UPF21173 or UPF21-1096 (J). At 48 h post-transfection, cells were harvested and FLAG-tagged UPF proteins were individually immunoprecipitated as described in Materials and Methods. Input and bound (Output) complexes were analyzed by western blotting for UPF proteins as indicated. The abundance of vRNA and gapdh RNA was estimated by RT-PCR (in Input only). Results shown are representative of 2 independent experiments.

Figure 8

Proposed model for UPF1-mediated vRNA nucleocytoplasmic export. During the HIV-1 expression phase, the shuttling ability of UPF1 is important for vRNA nucleocytoplasmic export. In the nucleus, UPF1 can be recruited to the vRNA via its C-terminal domain (including the NLS domain, Figure 1A) and acts as an adaptor protein to bridge the nuclear HIV-1 RNP with export factors through, most likely, its NES region (i). However, UPF3aL can potentially block the recruitment of UPF1 to prevent the assembly of an export-competent nuclear RNP by recruiting UPF2 (ii). Once assembled, the UPF1 nuclear export complex, composed of the vRNA, UPF1, Rev, CRM1, DDX3 and DDX1 then associates with nucleoporins Nup98 and/or Nup214, as well as Nup62 to form a nuclear complex (N-UPF1-HIV-1 RNP, (iii)). This complex can then be efficiently exported (iv). Once in the cytoplasm, several factors including UPF1 recycle back to their original locations. UPF1 can either act in the context of a cytoplasmic RNP (C-UPF1-HIV-1 RNP) to promote vRNA stability and utilization (translation) to ensure Gag synthesis (v), or, according to the current results in this manuscript, escapes interaction with UPF2 (vi) within the cytoplasm or nucleus (“?” indicates open question) to control vRNA nucleocytoplasmic export.