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. 2013 Mar 1;190(5):2311-9.
doi: 10.4049/jimmunol.1202749. Epub 2013 Jan 23.

Proteasomal degradation of herpes simplex virus capsids in macrophages releases DNA to the cytosol for recognition by DNA sensors

Kristy A Horan  1 , Kathrine Hansen, Martin R Jakobsen, Christian K Holm, Stine Søby, Leonie Unterholzner, Mikayla Thompson, John A West, Marie B Iversen, Simon B Rasmussen, Svend Ellermann-Eriksen, Evelyn Kurt-Jones, Santo Landolfo, Blossom Damania, Jesper Melchjorsen, Andrew G Bowie, Katherine A Fitzgerald, Søren R Paludan
Affiliations

Proteasomal degradation of herpes simplex virus capsids in macrophages releases DNA to the cytosol for recognition by DNA sensors

Kristy A Horan et al. J Immunol. .

Abstract

The innate immune system is important for control of infections, including herpesvirus infections. Intracellular DNA potently stimulates antiviral IFN responses. It is known that plasmacytoid dendritic cells sense herpesvirus DNA in endosomes via TLR9 and that nonimmune tissue cells can sense herpesvirus DNA in the nucleus. However, it remains unknown how and where myeloid cells, such as macrophages and conventional dendritic cells, detect infections with herpesviruses. In this study, we demonstrate that the HSV-1 capsid was ubiquitinated in the cytosol and degraded by the proteasome, hence releasing genomic DNA into the cytoplasm for detection by DNA sensors. In this context, the DNA sensor IFN-γ-inducible 16 is important for induction of IFN-β in human macrophages postinfection with HSV-1 and CMV. Viral DNA localized to the same cytoplasmic regions as did IFN-γ-inducible 16, with DNA sensing being independent of viral nuclear entry. Thus, proteasomal degradation of herpesvirus capsids releases DNA to the cytoplasm for recognition by DNA sensors.

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Figures

FIGURE 1
FIGURE 1
Herpesviruses induce IFN responses in macrophages - dependent on IFI16. PMA-differentiated THP1 macrophages were infected with (A,C) HSV-1; or (B,D) CMV. (A, B) Total RNA was harvested 6 h post infection, and IFNβ mRNA was determined by RT-PCR. (C, D) Supernatants from cells treated for 16 h as indicated were analyzed for CXCL10 protein levels by ELISA. (E) Whole cell extracts from PMA-differentiated THP1 macrophages, treated as indicated were isolated and phospho-IκBα was determined by Luminex. (F, G) PMA-differentiated THP1 macrophages stably transfected with control vector (EV) or IFI16 shRNA (sh-IFI16) or (H, I) MDMs transfected with si-CTRL or si-IFI16 RNA were infected with (F, H) HSV-1 or (G, I) CMV. Total RNA was harvested 6 h post infection, and IFNβ mRNA was determined by RT-PCR. Data represent mean +/- SD of duplicates. * p<0.05.
FIGURE 2
FIGURE 2
IFI16 and STING co-localize following infection with herpesviruses. MDMs were mock infected or infected with HSV-1 or CMV for 4 h. Subcellular distribution of IFI16 and STING was determined by confocal microscopy. White box indicates area displayed in zoom column, scale bar 10 μm.
FIGURE 3
FIGURE 3
Herpesvirus DNA is present in the cytosol of infected macrophages and co-localizes with IFI16. (A) PMA-differentiated THP1 cells were mock-infected or infected with HSV-1 at MOI 3 for 6 h. Cytosolic and nuclear extracts were isolated and analyzed for IFI16, DDX41, RCC1, and β-actin by Western blotting. (B, C) PMA-differentiated THP1 cells were infected with (B) HSV-1 (C) CMV, and (D) Vero cells were infected with HSV-1 for 4 h and viral DNA was visualized by virus specific FISH probes (green) and co-stained with anti-capsid specific antibodies. White box indicates area displayed in zoom column, scale bar 10 μm. (E, F) MDMs were infected with HSV-1 or CMV for 4 hr and viral DNA was visualized by virus specific FISH probes (green) and co-stained with anti-IFI16 specific antibodies. White box indicates area displayed in zoom column, scale bar 10 μm.
FIGURE 4
FIGURE 4
Induction of type I IFN response and IFI16 co-localisation is independent of nuclear delivery of the viral genome. (A). MDMs were treated with vehicle or the nuclear export inhibitor LMB and infected with HSV-1 for 4 h. Viral DNA was visualized by HSV-1 specific FISH probes (green) and co-stained with anti-IFI16 specific antibodies, scale bar 10 μm. PMA-differentiated THP1 macrophages were (B, C) pre-treated with LMB and infected with HSV-1 for 4 h. (B) HSV-1 DNA foci per 100 cells and (C) percentage DNA foci negative for Vp5 were determined, data represent mean +/- SD. (D) PMA-differentiated THP1 macrophages were infected with the HSV-1 mutant TsB7 at 33°C (permissive) or 39°C (non-permissive) for 4 h and the percentage of HSV-1 DNA foci negative for Vp5 determined, data represent mean +/- SD. (E-F) PMA-differentiated THP1 macrophages were infected with TsB7 and HSV-1 for 6 hr and total RNA and whole-cell extracts were isolated for measurement of IFNβ mRNA (RT-qPCR) and phospho-IκBα (Luminex), respectively, data represent mean +/- SD of duplicates. (G) PMA-differentiated THP1 macrophages and U20S cells were infected with TsB7 and HSV-1 for 6 hr at 33°C or 39°C. For THP1 cells, live virus was used, and for U2OS cells UV-inactivated virus was used. Total RNA was isolated for measurement of ISG56 mRNA (RT-qPCR) data represent mean +/- SD of triplicates. * p<0.05 (H) MDMs were infected with TsB7 at 33°C or 39°C for 4 hr and viral DNA visualized by HSV-1 specific FISH probes (green) and co-stained with anti-IFI16 specific antibodies, scale bar 10 μm.
FIGURE 5
FIGURE 5
Ubiquitination of the HSV-1 capsid. (A) BMMs were infected with HSV-1 (MOI 3) for 2 hr and co-stained with Anti-Vp5, anti-ubiquitin (Ub), and anti-proteasome (p20S). White box indicates area displayed in zoom row Arrows indicates the following co-localizations: 1, capsid and p20S; 2, caspid and Vp5; and 3, capsid p20S and Vp5 scalebar: 20 μm. (B) Lysates from PMA-differentiated THP1 macrophages infected for 30 or 90 min with 300 MOI of HSV-1 were subjected to IP using anti-Vp5-coupled beads. Total Ubiquitin and K48-coupled ubiquitin in the precipitate was detected using Western blotting. Levels of Vp5 and βactin in the input lysate used for IP was determined by Western blotting. (C) Lysates from PMA-differentiated THP1 macrophages infected for 90 min with 300 MOI of HSV-1 or the HSV-1 mutant TsB7 at either 33 °C or 39 °C were subjected to IP using anti-Vp5-couples beads. Total Ubiquitin in the precipitate was detected using Western blotting.
FIGURE 6
FIGURE 6
The HSV-1 capsid is degraded by the proteasome. (A, B) PMA-differentiated THP1 macrophages were un-treated or pre-treated with MG132 (10 μg/ml) and infected with HSV-1 or CMV (MOI 3). Total cell lysates were isolated at the indicated time points post infection and the capsid protein Vp5 (A) or CMV p28 (B) were determined by Western blot. (C) Lysates from PMA-differentiated THP1 macrophages infected for 90 min with HSV-1 (MOI 300) in the presence or absence of MG132 were subjected to IP using anti-Vp5-coupled beads. Total and K48 Ubiquitin in the precipitate was detected using Western blotting. (D, E) BMMs were pretreated with MG132 and infected with HSV-1 (MOI 3) for 4 h. The cells were fixed and probed with anti-Vp5 and a HSV-1-specific FISH probe. The data are presented as (D) number of HSV-1 DNA foci per100 cells and (E) percentage HSV-1 DNA foci negative for Vp5. Data represents mean +/- SD, * p<0.05. (F) PMA-differentiated THP1 macrophages or HFFs were treated with vehicle or pre-treated with MG132 and infected with live (THP1 cells) or UV-inactivated (HFFs) HSV-1 (MOI 3) for 6 h and total RNA was isolated for measurement of ISG56 mRNA (RT-qPCR) data represent mean +/- SD of triplicates. * p<0.05.
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References

    1. Kawai T, Akira S. The role of pattern-recognition receptors in innate immunity: update on Toll-like receptors. Nature Immunology. 2010;11:373–384. - PubMed
    1. Paludan SR, Bowie AG, Horan KA, Fitzgerald KA. Recognition of herpesviruses by the innate immune system. Nat.Rev.Immunol. 2011;11:143–154. - PMC - PubMed
    1. Janeway CA., Jr. Approaching the asymptote? Evolution and revolution in immunology. Cold Spring Harb.Symp.Quant.Biol. 1989;54(Pt 1):1–13. - PubMed
    1. Poltorak A, He X, Smirnova I, Liu M, van Huffel C, Du X, Birdwell D, Alejos E, Silva M, Galanos C, Freudenberg M, Ricciardi-Castagnoli P, Layton B, Beutler B. Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice: Mutations in Tlr4 gene. Science. 1999;282:2085–2088. - PubMed
    1. Imai Y, Kuba K, Neely GG, Yaghubian-Malhami R, Perkmann T, van Loo G, Ermolaeva M, Veldhuizen R, Leung YH, Wang H, Liu H, Sun Y, Pasparakis M, Kopf M, Mech C, Bavari S, Peiris JS, Slutsky AS, Akira S, Hultqvist M, Holmdahl R, Nicholls J, Jiang C, Binder CJ, Penninger JM. Identification of oxidative stress and Toll-like receptor 4 signaling as a key pathway of acute lung injury. Cell. 2008;133:235–249. - PMC - PubMed

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