Role for a Filamentous Nuclear Assembly of IFI16, DNA, and Host Factors in Restriction of Herpesviral Infection

Abstract

Several host cell nuclear factors are known to restrict herpes simplex virus 1 (HSV-1) replication, but their mechanisms of action remain to be defined. Interferon-inducible protein 16 (IFI16) and the nuclear domain 10-associated proteins, such as promyelocytic leukemia (PML) protein, localize to input viral genomes, but they are also capable of restricting progeny viral transcription. In this study, we used structured illumination microscopy to show that after HSV DNA replication, IFI16 forms nuclear filamentous structures on DNA within a subset of nuclear replication compartments in HSV-1 ICP0-null mutant virus-infected human cells. The ability to form filaments in different cell types correlates with the efficiency of restriction, and the kinetics of filament formation and epigenetic changes are similar. Thus, both are consistent with the filamentous structures being involved in epigenetic silencing of viral progeny DNA. IFI16 filaments recruit other restriction factors, including PML, Sp100, and ATRX, to aid in the restriction. Although the filaments are only in a subset of the replication compartments, IFI16 reduces the levels of elongation-competent RNA polymerase II (Pol II) in all replication compartments. Therefore, we propose that IFI16 filaments with associated restriction factors that form in replication compartments constitute a "restrictosome" structure that signals in cis and trans to silence the progeny viral DNA throughout the infected cell nucleus. The IFI16 filamentous structure may constitute the first known nuclear supramolecular organizing center for signaling in the cell nucleus.IMPORTANCE Mammalian cells exhibit numerous strategies to recognize and contain viral infections. The best-characterized antiviral responses are those that are induced within the cytosol by receptors that activate interferon responses or shut down translation. Antiviral responses also occur in the nucleus, yet these intranuclear innate immune responses are poorly defined at the receptor-proximal level. In this study, we explored the ability of cells to restrict infection by assembling viral DNA into transcriptionally silent heterochromatin within the nucleus. We found that the IFI16 restriction factor forms filaments on DNA within infected cells. These filaments recruit antiviral restriction factors to prevent viral replication in various cell types. Mechanistically, IFI16 filaments inhibit the recruitment of RNA polymerase II to viral genes. We propose that IFI16 filaments with associated restriction factors constitute a "restrictosome" structure that can signal to other parts of the nucleus where foreign DNA is located that it should be silenced.

Keywords: DNA virus; chromatin; epigenetics; signaling; supramolecular organizing center.

FIG 1

IFI16 forms filamentous structures in replication compartments in cells infected with an HSV-1 ICP0-null virus. HFF cells were infected with 7134 virus at an MOI of 5. (A) Cells were fixed at 4, 6, 8, and 10 hpi and immunostained for IFI16 (green) and ICP8 (magenta). Images show nuclei of respective cells at indicated times postinfection. The scale bar represents 10 µm. n ≥ 3 experiments. (B) Enlarged section of the 6-hpi time point, indicated by the red quadrangle. (C) Frequency distributions of total RCs and RCs with filaments in a population of cells (≥50 cells per sample). (D) Single IFI16 filament (green) in an RC in a nucleus of a cell at the edge of a plaque. The scale bar length is 1 µm.

FIG 2

Role of viral DNA synthesis and human cell type in IFI16 filament formation. (A) HFF cells were infected with 7134 virus at an MOI of 5 in the presence or absence of PAA. Cells were fixed at 6 hpi, and immunostaining for IFI16 and ICP8 was performed. The scale bar length is 10 µm. (B) HFF cells were infected with 7134 virus at an MOI of 5. Mild fixation and DNase I treatment were performed at 6 hpi as outlined in Materials and Methods. Cells were then fixed and immunostained for IFI16 (green) and ICP8 (magenta). The enlarged section is denoted by a red quadrangle. Scale bar lengths in montages are 10 µm and 1 µm in the enlarged section. (C) As in panel B, but with RNase A treatment. (D) HFF, NOK, U2OS, and HeLa cells were infected with 7134 virus at an MOI of 5. Cells were fixed 6 hpi and immunostained for IFI16/ICP8. Panels A to D represent results from 2 experiments. (E) Cells were counted after immunostaining, and the percentages of cells with IFI16 filaments are shown for each cell type. (F) HFF, NOK, U2OS, and HeLa cells were treated with nontargeting or IFI16-specific siRNAs and infected with 7134 virus at an MOI of 0.1. Progeny viruses were harvested at 48 hpi, titrated on U2OS cells, and plotted as PFU/ml. Statistical analysis between columns was done via t test. (G) Fold restriction by IFI16 was calculated by dividing viral yields in the absence of IFI16 by viral yields in the presence of IFI16 and plotted for each cell type. Statistical analysis was done by t test. (H) Values obtained in panels F and G were plotted against each other. Linear regression analysis was performed with Prism.

FIG 3

Formation of IFI16 filaments is accompanied by epigenetic silencing of progeny vDNA. (A) Western blot confirmation of lack of expression of IFI16 in Cas9 control or IFI16ko Tert-HF cells. (B to D) Chromatin immunoprecipitation after 7134 virus infection of Cas9 or IFI16ko cells was performed with antibodies specific for the H3K9me3. Results were plotted as percentage of immunoprecipitated protein normalized to the 2-h Cas9 control value for each experiment (n = 3). (B) ICP4 gene. (C) ICP8 gene. (D) ICP27 gene. *, P < 0.5, by unpaired two-tailed t test. Error bars represent standard deviation (SD).

FIG 4

Levels of elongation-competent and initiation-competent RNA polymerase II are enhanced in replication compartments in the absence of IFI16. (A and B) Tert-HF, Cas9, and IFI16ko cells were infected with 7134 virus and fixed at 6 hpi. Immunostaining was done for ICP8/Pol II or ICP8/Pol II S2P. n = 3 experiments. (C) Pol II S2P or Pol II signal intensities per area in individual replication compartments were determined and compared for different cell lines. Statistical analysis was done with a t test. (D) Pol II S2P signal intensities per area in individual replication compartments of individual cells were determined, averaged, and plotted against the total relative replication compartment area of the respective cell. Statistical analysis was done with a t test.

FIG 5

Other host restriction factors colocalize with IFI16 filaments. (A) HFF cells were infected with 7134 virus at an MOI of 5 and fixed at 6 hpi. Immunostaining was performed for all dual combinations of IFI16, PML, Sp100, and ATRX antibodies. The scale bar length is 1 µm. n = 3 experiments. (B) Colocalization analysis for all double combinations of antibodies specific for IFI16, PML, Sp100, ATRX, and ICP8 was performed with Fiji as described in Materials and Methods.

FIG 6

IFI16 organizes filamentous aggregation of ND-10 proteins. (A) Cas9 control and IFI16ko cells were infected with 7134 virus at an MOI of 5 and fixed at 6 hpi. Immunostaining was performed for PML (green) and ICP8 (magenta). The scale bar length is 10 µm. Images were taken with a Zeiss Axioplan microscope. (B) Tert-HF and Tert-HF-derived Cas9 and IFI16ko cells were infected with 7134 virus and processed as in panel A. Immunostaining was done for Sp100 (green) and ICP8 (magenta). (C) HFF cells stably expressing nontargeting or PML-specific shRNAs were infected with 7134 virus and processed as in panel A. Immunostaining was performed for IFI16/ICP8, PML/ICP8, or Sp100/ICP8. Scale bars represent 1 µm. (D) HFF cells were treated with nontargeting or PML-specific siRNAs, infected with 7134 virus, and processed as in panel A. Immunostaining was performed for IFI16/ICP8 or IFI16/PML. Scale bars represent 1 µm. n = 2 experiments.

FIG 7

Restrictosome working model. IFI16 assembles on progeny viral DNA molecules and recruits other restriction factors, including PML, Sp100, and ATRX. These factors together exert a silencing effect in cis on the viral genome. In addition, a trans-acting silencing appears to occur on other viral genomes in the same and different replication compartments. ATRX is only partially localized to IFI16 filaments, so it may be part of the trans-acting silencing mechanism.