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. 2016 Aug;97(8):1955-1967.
doi: 10.1099/jgv.0.000510. Epub 2016 May 23.

Promyelocytic leukemia protein isoform II inhibits infection by human adenovirus type 5 through effects on HSP70 and the interferon response

Zeenah Atwan  1 , Jordan Wright  1 , Andrew Woodman  1 , Keith N Leppard  1
Affiliations

Promyelocytic leukemia protein isoform II inhibits infection by human adenovirus type 5 through effects on HSP70 and the interferon response

Zeenah Atwan et al. J Gen Virol. 2016 Aug.

Abstract

Promyelocytic leukemia (PML) proteins have been implicated in antiviral responses but PML and associated proteins are also suggested to support virus replication. One isoform, PML-II, is required for efficient transcription of interferon and interferon-responsive genes. We therefore investigated the PML-II contribution to human adenovirus 5 (Ad5) infection, using shRNA-mediated knockdown. HelaΔII cells showed a 2-3-fold elevation in Ad5 yield, reflecting an increase in late gene expression. This increase was found to be due in part to the reduced innate immune response consequent upon PML-II depletion. However, the effect was minor because the viral E4 Orf3 protein targets and inactivates this PML-II function. The major benefit to Ad5 in HelaΔII cells was exerted via an increase in HSP70; depletion of HSP70 completely reversed this replicative advantage. Increased Ad5 late gene expression was not due either to the previously described inhibition of inflammatory responses by HSP70 or to effects of HSP70 on major late promoter or L4 promoter activity, but might be linked to an observed increase in E1B 55K, as this protein is known to be required for efficient late gene expression. The induction of HSP70 by PML-II removal was specific for the HSPA1B gene among the HSP70 gene family and thus was not the consequence of a general stress response. Taken together, these data show that PML-II, through its various actions, has an overall negative effect on the Ad5 lifecycle.

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Figures

Fig. 1.
Fig. 1.
HelaΔII cells show physical and functional knockdown of PML-II. (a) Phase-contrast microscopic images of control Hela and shRNA Hela cells; bar, 100 µm. (b, c, d) HelaEV and HelaΔII cells were plated for 24 h, then RNA or protein samples were harvested. (b) PML-II mRNA was detected by RT-qPCR; results are normalized to the level detected in HelaEV cells and are the means and standard deviation of three technical replicates. (c) PML-II protein was detected by Western blotting. (d) IL-6 and ISG56 mRNAs were analysed as in (b).
Fig. 2.
Fig. 2.
Removal of PML-II increases Ad5 protein expression and virus yield. (a) HelaEV and HelaΔII cells were infected with wild-type Ad5 at m.o.i. of 5, and total protein extracts at various times post-infection were analysed by Western blotting. Upper panel: Ad5 late protein; middle panels: Ad5 E1B 55K and E2A DNA binding protein (DBP); lower panel: GAPDH. The 24 h samples were loaded at a 1 : 100 dilution compared to the earlier time points. (b) Total protein extracts of HelaEV and standard Hela cells, infected for 20 h as in (a), were analysed for hexon expression. (c) Adenovirus gene expression by FACS analysis. Upper panel: late gene expression; lower panel: DBP expression; grey curves are the background (mock-infected cells) while the black curves represent infected cells; the % of fluorescence-positive cells and their mean fluorescence intensity (MFI) are indicated on each panel. (d) Total virus in infected culture lysates was determined by fluorescent focus assay. Error bars show the standard deviation of replicates within an experiment; the experiment shown is representative of multiple experiments.
Fig. 3.
Fig. 3.
PML-II inhibits Ad5 infection by both IFN-dependent and -independent mechanisms. HelaEV and HelaΔII cells were plated for 24 h, transfected with 62.5 pmol ml−1 siRNA as indicated for 48 h and then either stimulated with poly I : C for 16 h (a, b) or infected with Ad5 wt300 at m.o.i. of 5 for 20 h (c), after which samples were prepared for analysis. (a) IRF3 protein or GAPDH (loading control) was detected by Western blot. (b) RT-qPCR analysis detecting ISG56 mRNA; results are the means and standard deviation of three technical replicates. (c) As panel (a), but detecting hexon protein using anti-late protein polyclonal antibodies.
Fig. 4.
Fig. 4.
Adenovirus E4 Orf3 inhibits IFN production and IFNβ promoter activation. (a) HEK293 cells were infected at a multiplicity of 10 p.f.u. cell−1 with wild-type Ad5 (wt300) or mutant inOrf3, or were mock-infected. Media was harvested at 8 h post-infection and IFN activity measured by plaque-reduction assay. (b) As panel (a) but using media from Ad5-infected MRC5 fibroblasts harvested at 16 h post-infection. (c) Media from HEK293 cell cultures infected as in (a) was harvested at 6 h post-infection and IFN activity measured using an ISRE-luciferase reporter construct in HEK293 cells. Known amounts of recombinant IFNα were analysed in parallel to provide a standard curve. (d) HEK293 cells were transfected with IFNβ promoter luciferase reporter and β-galactosidase control plasmids together with PML-IIΔRBCC (125 ng) and from 125–625 ng E4 Orf3 plasmid as appropriate and then stimulated with poly(I : C) and reporter activities assayed 8 h later. (e–i) HEK293 cells were transfected with reporter plasmids as in (d) plus 150–600 ng (150–750 ng, panels e, g) of either wild-type E4 Orf3 plasmid (e), or mutant E4 Orf3 R100A (f), N82A (g), L103A (h) or D105A-L106A (i), and then stimulated or not with poly(I : C) as indicated and assayed as in panel (d). Data are the means and standard deviation of three biological replicates.
Fig. 5.
Fig. 5.
Role of E4 Orf3 in the response of Ad5 to PML-II depletion and IFN-α. HelaEV and HelaΔII cells were either mock-treated (lanes 1, 2, 4, 6, 7 and 9) or treated with 1000 U ml−1 of IFN-α for 24 h (+I; lanes 3, 5, 8 and 10), then mock-infected or infected with Ad5 wt300 or inOrf3 as indicated for 20 h. Total protein lysates were collected and analysed by Western blotting for Ad5 late proteins (upper) or GAPDH (lower). Hexon protein bands were quantified using QuantityOne software, normalized to GAPDH and expressed relative to the value for wt300 in HelaEV cells.
Fig. 6.
Fig. 6.
Elevated HSP70 enhances the expression of Ad5 proteins when PML-II is reduced. (a, e) Samples were harvested from HelaEV and HelaΔII cells and analysed for HSP70 mRNA (a) or a selection of HSP mRNAs (e) by RT-qPCR, or (a, lower) for HSP70 protein by Western blot. (b) Hela cells were transfected or not with 125 pmol ml−1 siRNA as indicated and RNA was harvested after 48 h for analysis of HSP70 mRNA by RT-qPCR. (c) HelaEV and HelaΔII cells were transfected with 125 pmol ml−1 HSP70 or control siRNA for 48 h, then infected with Ad5 wt300 for 20 h before RNA was harvested and analysed for hexon mRNA by RT-qPCR. (d) HelaΔII cells were treated with siRNA and infected as in (c), then lysed and analysed by Western blotting as in Fig. 5. In (a–c), data were standardized to an internal control and then normalized to values from: (a) HelaEV; (b) siControl-treated Hela; (c) siControl-treated HelaEV. Panel (e) shows mRNA amounts measured separately for each amplicon, standardized in each case to an internal control. Graphs show the means and standard deviation of three technical replicates.
Fig. 7.
Fig. 7.
Effects of elevated HSP70 on NF-kB signalling do not cause enhanced Ad5 gene expression. (a) HelaEV cells were treated with HSP70 or control siRNA as in Fig. 6(c), then infected with Ad5 wt300 at m.o.i. of 5 for 20 h and ISG56 mRNA was quantified by RT-qPCR. (b, d) Cells as indicated were treated or not with 50 ng TNFα for 1 h (b) or with 100 nM of the NF-κB inhibitor QNZ for 45 min (d), then infected with Ad5 wt300 at m.o.i. of 5 for 20 h. Protein samples were harvested and analysed for late protein expression by Western blot. (c) HelaEV cells, treated with siRNA as in (a) were treated with TNFα as in (b), infected with Ad5 wt300 as in (a) and hexon mRNA quantified by RT-qPCR. Results in panels (a) and (c) are the means and standard deviation of three technical replicates.
Fig. 8.
Fig. 8.
HSP70 promotes nuclear accumulation of Ad5 hexon but has no effect on late promoter activity. (a) HelaΔII cells were treated with HSP70 or control siRNA as in Fig. 6(c), then infected with Ad5 wt300 at m.o.i. of 5 for 16 h. Cytoplasmic and nuclear fractions were analysed by SDS-PAGE and Western blotting. Replicate blots were probed with antibodies to the proteins indicated and bands quantified as in Fig 5, normalized to GAPDH (cytoplasmic) or SP1 (nuclear). (b) Hela cells were treated with siRNA as in (a) and then transfected with MLP or L4P luciferase reporter. Luciferase activity was measured after 20 h and normalized to a β-galactosidase transfection control as described by Wright et al. (2015). (c) HelaEV and HelaΔII cells were infected with wild-type Ad5 at m.o.i. of 5, and total protein extracts analysed by Western blotting at 20 h post-infection for the proteins indicated.
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References

    1. Ackrill A. M., Foster G. R., Laxton C. D., Flavell D. M., Stark G. R., Kerr I. M.(1991). Inhibition of the cellular response to interferons by products of the adenovirus type 5 E1A oncogene. Nucleic Acids Res 194387–4393.10.1093/nar/19.16.4387 - DOI - PMC - PubMed
    1. Agoff S. N., Hou J., Linzer D., Wu B.(1993). Regulation of the human hsp70 promoter by p53. Science 25984–87.10.1126/science.8418500 - DOI - PubMed
    1. Au W. C., Moore P. A., Lowther W., Juang Y. T., Pitha P. M.(1995). Identification of a member of the interferon regulatory factor family that binds to the interferon-stimulated response element and activates expression of interferon-induced genes. Proc Natl Acad Sci U S A 9211657–11661. - PMC - PubMed
    1. Bernardi R., Pandolfi P. P.(2007). Structure, dynamics and functions of promyelocytic leukaemia nuclear bodies. Nat Revs Mol Cell Biol 81006–1016. - PubMed
    1. Berscheminski J., Groitl P., Dobner T., Wimmer P., Schreiner S.(2013). The adenoviral oncogene E1A-13S interacts with a specific isoform of the tumor suppressor PML to enhance viral transcription. J Virol 87965–977.10.1128/JVI.02023-12 - DOI - PMC - PubMed

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