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. 2013 Nov 19;110(47):E4492-501.
doi: 10.1073/pnas.1316194110. Epub 2013 Nov 6.

Nuclear interferon-inducible protein 16 promotes silencing of herpesviral and transfected DNA

Megan H Orzalli  1 , Sara E Conwell, Christian Berrios, James A DeCaprio, David M Knipe
Affiliations

Nuclear interferon-inducible protein 16 promotes silencing of herpesviral and transfected DNA

Megan H Orzalli et al. Proc Natl Acad Sci U S A. .

Abstract

Mammalian cells have evolved mechanisms to silence foreign DNA introduced by viruses or by transfection. Upon herpesviral infection of cells, the viral genome is chromatinized in an attempt by the host cell to restrict expression of the viral genome. HSV ICP0 acts to counter host-intrinsic and innate responses to viral infection. We have found that nuclear interferon (IFN)-inducible protein 16 (IFI16) acts as a restriction factor against ICP0-null herpes simplex virus 1 (HSV-1) to limit viral replication and immediate-early gene expression. IFI16 promoted the addition of heterochromatin marks and the reduction of euchromatin marks on viral chromatin. IFI16 also restricted the expression of plasmid DNAs introduced by transfection but did not restrict SV40 DNA introduced into the cellular nucleus in the form of nucleosomal chromatin by viral infection. These results argue that IFI16 restricts unchromatinized DNA when it enters the cell nucleus by promoting the loading of nucleosomes and the addition of heterochromatin marks. Furthermore, these results indicate that IFI16 provides a broad surveillance role against viral and transfected DNA by promoting restriction of gene expression from the exogenous DNA and inducing innate immune responses.

Keywords: DNA sensing; cellular response; host restriction; intrinsic resistance.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
IFI16 negatively regulates the replication of an ICP0-null virus. (A) IFI16 and (B) STING transcript and protein levels were decreased following transfection of HFF with nontarget control, IFI16-specific, or STING-specific siRNAs. (C) IFI16 depletion resulted in an increase in ICP0-null virus replication relative to cells transfected with control siRNAs. siRNA-transfected cells were infected with HSV-1 ICP0-null (7134) or a rescued virus (7134R) at an MOI of 0.1 and harvested at 48 hpi, and virus yield was determined by plaque assay on U2OS cells. (D) IFI16 or STING depletion decreased IFN-β transcript levels in response to ICP0-null virus infection. siRNA-transfected cells were infected with HSV-1 ICP0-null virus at an MOI of 10, and RNA was harvested at 6 hpi. Results are an average of three (A and B), four (C), or two (D) independent experiments, and error bars represent the SE of means (*P < 0.05 and **P < 0.01, Student t test).
Fig. 2.
Fig. 2.
Reducing IFI16 protein levels increases the expression of a viral immediate–early protein. (A) Immunoblot examining the levels of the HSV-1 ICP4 immediate–early protein in HFF cells treated with nontargeting control, IFI16, or STING siRNA. Treated cells were mock-infected or infected with an ICP0-null (7134) or rescued virus (7134R) at an MOI of 1. Lysates were prepared at 6 hpi and subjected to Western blot analysis. The cellular tubulin gene was used as a recovery and loading control. (B) IFI16-depleted cells were infected with HSV-1 d109 virus at an MOI of 1, and cells were fractionated at 2 hpi. Total DNA was prepared from the nuclear fraction and quantified. The viral ICP8 gene was normalized to the cellular γ-actin control.
Fig. 3.
Fig. 3.
Overexpression of IFI16 reduces HSV-1 gene expression and replication. U2OS cells transfected with an empty vector control or an N-terminal Myc-tagged IFI16 construct were infected with ICP0-null (7134) or rescued virus (7134R) at an MOI of 0.1 (A, Upper) or an MOI of 10 (B). Cell lysates were harvested at 6 hpi and analyzed for ICP4, IFI16, and Tubulin protein levels. (A, Lower) ICP4 and Tubulin band intensities from A (Upper) were quantified by ImageJ analysis and compared with a standard curve.
Fig. 4.
Fig. 4.
Stable expression of IFI16 in HEK293 cells inhibits viral gene expression and replication independently of IRF-3 activation. (A) Control HEK293 cells (293 EV), HEK293 cells stably expressing IFI16 (293 IFI16), or HFFs were infected with 7134 or d109 viruses. Total cell-associated RNA was harvested at 6 hpi and prepared for qRT-PCR. ISG54 transcripts were normalized to 18S RNA and made relative to mock-infected cells. (B) Total RNA was harvested at 4 or 8 hpi and analyzed by qRT-PCR for ICP4 and ICP27 mRNA. Transcripts were normalized to 18S RNA. (C) Cell lysates were harvested at 6 hpi and analyzed by Western blot for ICP4, IFI16, and Tubulin levels. (D) Virus yields at 24 hpi were determined by plaque assay on U2OS cells. Cells were infected with an MOI of 10 for A and an MOI of 0.1 for B–D.
Fig. 5.
Fig. 5.
IFI16 relocalizes to replication compartments during HSV-1 infection. HFF were infected with ICP0-null (7134) or rescued virus (7134R) at an MOI of 10 and fixed at 3 and 6 hpi. Cells were simultaneously stained with mouse anti-IFI16 (Abcam) and rabbit anti-ICP8 antibodies followed by Alexa Fluor 488-conjugated goat anti-mouse and Alexa Fluor 594-conjugated goat-anti rabbit secondary antibodies. Representative immunofluorescence images are shown. (A–C) Mock, (D–F, J–O) 7134, (G–I, P–R) 7134R.
Fig. 6.
Fig. 6.
IFI16 promotes heterochromatin association with viral DNA. Control and IFI16 siRNA-treated HFFs were infected with 7134 and 7134R viruses at an MOI of 1. Cell extracts were prepared at 6 hpi and ChIP was carried out by using antibody specific to (A) histone H3, (B) H3K4me3, (C) and H3K9me3. Immunoprecipitated ICP4, ICP27, and ICP8 promoter sequences were measured by qPCR and viral DNA sequences were normalized to immunoprecipitated GAPDH DNA. Histone marks (B and C) are represented as proportion of the total DNA immunoprecipitated by the H3 antibody.
Fig. 7.
Fig. 7.
Effect of IFI16 on transfected and SV40 DNA. (A) Immunoblot examining GFP and IFI16 expression in pCMV GFP-transfected HFFs treated with nontargeting control or IFI16 siRNA. The cellular tubulin gene was used as a recovery and loading control. (B) Quantification of GFP+ cells in the presence or absence of IFI16 by flow cytometry at 48 hpt. HFFs were transfected with an empty vector plasmid or pCMV GFP or pEF1 GFP at 48 h after siRNA treatment. The results are represented as the fold increase in GFP+ cells compared with their empty vector control. (C) siRNA-transfected HFF were infected with WT SV40 (MOI of 0.1) or transfected with pSV40 (0.5 μg). Cell lysates were prepared at 48 h posttreatment and analyzed by Western blot for TAg protein levels. IFI16 depletion was confirmed by Western analysis, and the cellular GAPDH gene was used as a recovery and loading control (*P < 0.05 and **P < 0.01, Student t test).
Fig. 8.
Fig. 8.
Model for IFI16 restriction of HSV gene expression. IFI16 binds to nucleosome-free DNA that accumulates in the nucleus. DNA-bound IFI16 undergoes a conformational change releasing the pyrin domain (light green) from an autoinhibited state. Activated IFI16 can signal from the nucleus to the cytoplasm to activate innate immune signaling pathways and recruit chromatin modification complexes that promote H3K9me3 on viral genomes, resulting in gene silencing.
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