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. 2012 Jan 20;422(2):317-25.
doi: 10.1016/j.virol.2011.10.033. Epub 2011 Nov 27.

Adenovirus E4-ORF3-dependent relocalization of TIF1α and TIF1γ relies on access to the Coiled-Coil motif

Elizabeth I Vink  1 , Mark A Yondola, Kai Wu, Patrick Hearing
Affiliations

Adenovirus E4-ORF3-dependent relocalization of TIF1α and TIF1γ relies on access to the Coiled-Coil motif

Elizabeth I Vink et al. Virology. .

Abstract

The adenovirus E4-ORF3 protein promotes viral replication by relocalizing cellular proteins into nuclear track structures, interfering with potential anti-viral activities. E4-ORF3 targets transcriptional intermediary factor 1 alpha (TIF1α), but not homologous TIF1β. Here, we introduce TIF1γ as a novel E4-ORF3-interacting partner. E4-ORF3 relocalizes endogenous TIF1γ in virus-infected cells in vivo and binds to TIF1γ in vitro. We used the homologous nature, yet differing binding capabilities, of these proteins to study how E4-ORF3 targets proteins for track localization. We mapped the ability of E4-ORF3 to interact with specific TIF1 subdomains, demonstrating that E4-ORF3 interacts with the Coiled-Coil domains of TIF1α, TIF1β, and TIF1γ, and that the C-terminal half of TIF1β interferes with this interaction. The results of E4-ORF3-directed TIF1 protein relocalization assays performed in vivo were verified using coimmunoprecipitation assays in vitro. These results suggest that E4-ORF3 targets proteins for relocalization through a loosely homologous sequence dependent on accessibility.

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Figures

Fig. 1
Fig. 1
E4-ORF3 alters the localization of endogenous TIF1γ in vivo. A. Endogenous TIF1γ localization in mock-infected Hela cells was analyzed by indirect immunofluorescence using anti-TIF1γ primary antibody and FITC-conjugated secondary antibody. B. PML localization in the same cell using anti-PML primary antibody and TRITC-conjugated secondary antibody. C. Merge of panels A and B; arrows indicate colocalization of TIF1γ and PML nuclear bodies. D–F. TIF1γ localization at early times after infection with wild-type Ad5 at a multiplicity of infection of 200 virus particles/cell. D. TIF1γ localization performed described as in A. E. E4-ORF3 localization using an anti-E4-ORF3 primary antibody and TRITC-conjugated secondary antibody. F. Merge of panels D and E. G. HeLa cells were infected with wild type Ad5 at a multiplicity of infection of 1000 virus particles/cell and analyzed as in A. H–J. TIF1γ localization at early times after infection with an E4-ORF3 mutant virus at 200 virus particles/cell. H. TIF1γ localization performed as described in A. I. Ad DBP localization using an anti-DBP primary antibody and TRITC-conjugated secondary antibody. J. Merge of panels H and I. K–M. TIF1γ localization at late times after infection with wild-type Ad5 at 200 virus particles/cells performed as described in D–F.
Fig. 2
Fig. 2
The wild-type, but not the N82A mutant, E4-ORF3 protein coimmunoprecipitates with endogenous TIF1γ in vitro. HeLa cells were mock-infected (M) or infected with Ad vectors that expressed either the wild-type (WT) or the N82A (N82A) mutant HA-tagged E4-ORF3 proteins. Immunoprecipitations from whole cell extracts (WCE) were performed using anti-TIF1γ antibody, and Western blots were probed using antibodies against TIF1γ (top panel) and HA (bottom panel). Lanes 1–3 show proteins present in the starting WCE. Lanes 4–6 show proteins immunoprecipitated with the anti-TIF1γ antibody.
Fig. 3
Fig. 3
Diagram of the EYFP-TIF1 fusion proteins described in the text. Abbreviations are: R, Ring finger domain; BB, B Box domain; CC, Coiled-Coil domain; CTH, C-terminal half; α, TIF1α coding sequences; β, TIF1β coding sequences; γ, TIF1γ coding sequences.
Fig. 4
Fig. 4
E4-ORF3 rearrangement of the Coiled-Coil domains isolated from TRIM family proteins. HeLa cells were transfected with expression vectors encoding EYFP-tagged Coiled-Coil domains from TIF1α (A–D), TIF1β (E–H), TIF1γ (I–L), or PML (M–P), then mock-infected (A, E, I, and M) or infected with wild-type Ad5 (F–H, J–L, N–P). At 18 hours post-infection (A–L) or 8 hours post-infection (M–P), cells were fixed and stained with an antibody directed against E4-ORF3 followed by a TRITC labeled secondary antibody (C, G, K, O). EYFP fusion protein localization is shown in B, F, J, and N. EYFP:E4-ORF3 merged images are shown in D, H, L, and P.
Fig. 5
Fig. 5
E4-ORF3 interaction with the TIF1 Coiled-Coil domain in different protein contexts. HeLa cells were transfected with expression vectors containing EYFP fused to TIF1α containing the Coiled-Coil domain of TIF1β (A–D), or TIF1β containing the Coiled-Coil domain of TIF1α (E–H) or TIF1γ (I–L). Cells were mock-infected (A, E, I) or infected with wild-type Ad5 (B–D, F-H, J–L). Eighteen hours later, the cells were fixed and stained with an antibody directed against E4-ORF3 followed by a TRITC labeled secondary antibody (C, G, K). EYFP fusion protein localization is shown in A–B, E–F, I–J. EYFP:E4-ORF3 merged images are shown in D, H, and L.
Fig. 6
Fig. 6
The C-terminal half of TIF1β interferes with E4-ORF3-directed TIF1 relocalization. Hela cells were transfected with expression vectors containing EYFP-TIF1β (A–D), EYFP-βRBCC (E-H), or EYFP-TIF1α-βCTH (I–J). Cells were mock-infected (A, E, I) or infected with wild-type Ad5 (B–D, F–G, J–L). Eighteen hours later, the cells were fixed and stained with an antibody directed against E4-ORF3 followed by a TRITC labeled secondary antibody (C, G, K). EYFP fusion protein localization is shown in A–B, E–F, I–J. EYFP:E4-ORF3 merged images are shown in D, H, and L.
Fig. 7
Fig. 7
Interaction EYFP-TIF1 fusion proteins with wild-type E4-ORF3 in vitro. HeLa cells were transfected with expression vectors containing EYFP-TIF1 fusion proteins indicated across the top (see Fig. 3 for depictions) and subsequently mock-infected (Mock, bottom) or infected with an Ad vector expressing HA-tagged wild-type E4-ORF3 protein (+HA-ORF3-WT, bottom). Proteins in cell extracts were immunoprecipitated using an EGFP antibody that cross-reacted with the EYFP and then subject to Western blot analysis using anti-HA (top panel) and anti-GFP (bottom panel) antibodies.
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