Mechanisms of Host IFI16, PML, and Daxx Protein Restriction of Herpes Simplex Virus 1 Replication

Abstract

The initial events after DNA virus infection involve a race between epigenetic silencing of the incoming viral DNA by host cell factors and expression of viral genes. Several host gene products, including the nuclear domain 10 (ND10) components PML (promyelocytic leukemia) and Daxx (death domain-associated protein 6), as well as IFI16 (interferon-inducible protein 16), have been shown to restrict herpes simplex virus 1 (HSV-1) replication. Whether IFI16 and ND10 components work together or separately to restrict HSV-1 replication is not known. To determine the combinatorial effects of IFI16 and ND10 proteins on viral infection, we depleted Daxx or PML in primary human foreskin fibroblasts (HFFs) in the presence or absence of IFI16. Daxx or IFI16 depletion resulted in higher ICP0 mutant viral yields, and the effects were additive. Surprisingly, small interfering RNA (siRNA) depletion of PML in the HFF cells led to decreased ICP0-null virus replication, while short hairpin RNA (shRNA) depletion led to increased ICP0-null virus replication, arguing that different PML isoforms or PML-related proteins may have restrictive or proviral functions. In normal human cells, viral DNA replication increases expression of all classes of HSV-1 genes. We observed that IFI16 repressed transcription from both parental and progeny DNA genomes. Taken together, our results show that the mechanisms of action of IFI16 and ND10 proteins are independent, at least in part, and that IFI16 exerts restrictive effects on both input and replicated viral genomes. These results raise the potential for distinct mechanisms of action of IFI16 on parental and progeny viral DNA molecules.IMPORTANCE Many human DNA viruses transcribe their genomes and replicate in the nucleus of a host cell, where they exploit the host cell nuclear machinery for their own replication. Host factors attempt to restrict viral replication by blocking such events, and viruses have evolved mechanisms to neutralize the host restriction factors. In this study, we provide information about the mechanisms of action of three host cell factors that restrict replication of herpes simplex virus (HSV). We found that these factors function independently and that one acts to restrict viral transcription from parental and progeny viral DNA genomes. These results provide new information about how cells counter DNA virus replication in the nucleus and provide possible approaches to enhance the ability of human cells to resist HSV infection.

Keywords: DNA virus; IFI16; ND10; nuclear replication; restriction factor.

FIG 1

IFI16 knockout via CRISPR/Cas in HFF cells leads to a defect in restriction of an HSV-1 ICP0-null virus. (A) Immunoblot of whole-cell lysates probed with antibodies specific for IFI16 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in HFF, Cas9-expressing, and IFI16 knockout cells. (B) Immunofluorescence. HFF, Cas9-expressing, and IFI16 knockout (with gRNA 1, 2, or 4) cells were fixed, permeabilized, and incubated with DAPI (4′,6-diamidino-2-phenylindole; blue) and an antibody specific to IFI16 (green). Total magnification, ×400. (C) Wild-type HFF cells (HFF), HFF cells expressing Cas9 (Cas9), or HFF cells expressing Cas9 and one of three IFI16-specific synthetic guide RNAs (gRNA 1, 2, or 4) were infected with the HSV-1 ICP0-null virus 7134 or the ICP0⁺ virus 7134R at an MOI of 0.1. Viral yields were collected at 48 hpi and titrated on U2OS cells. Results were plotted as numbers of PFU per milliliter or normalized to the Cas9 control level. For statistical analysis, t tests were performed. (D) HFF, Cas9-expressing, or IFI16 knockout cells (with gRNA 1, 2, or 4) were infected with 7134 at an MOI of 0.1. Viral yields were harvested at 24 hpi or 48 hpi and titrated on U2OS cells. Results were plotted as numbers of PFU per milliliter or normalized to the Cas9 control level. Statistical analysis was performed as described above.

FIG 2

Expression of IE, E, and L genes is earlier and increased in IFI16 knockout HFF cells. HFF, Cas9-expressing, and IFI16 knockout cells were infected with the 7134 and 7134R viruses at an MOI of 5 or mock infected. Cells were harvested for RNA and protein analysis at 4, 6, 8, 10, and 24 hpi. (A) ICP4, ICP27, ICP8, and gB transcript levels in HFF (black), Cas9-expressing (blue), and IFI16 knockout (red) cells were analyzed by qRT-PCR, and values were plotted relative to the 18S rRNA level. For statistical analysis, 2-way ANOVA including multiple comparisons was performed. Asterisks indicate significance compared to the values for HFF and Cas9-expressing cells. (B) Immunoblot showing ICP4, ICP27, ICP8, gB, cGAS, and GAPDH from 7134- or 7134R-infected cells.

FIG 3

IFI16 acts on gene expression from input and progeny genomes. HFF cells were treated with siRNAs specific for IFI16 or with nontargeting siRNA and infected with 7134 virus at an MOI of 5 in the presence or absence of PAA. Samples were harvested at 6, 8, and 10 hpi. (A to C) IFI16, ICP27, and ICP8 transcript levels at 10 hpi were analyzed by qRT-PCR and plotted relative to the 18S rRNA level. Numbers denote fold changes between the indicated columns. (D) Immunoblot probed with antibodies specific for ICP27, ICP8, IFI16, and GAPDH. (E) ICP8 protein levels at 10 hpi were quantified from the immunoblot, normalized to GAPDH, and plotted. Numbers indicate fold changes between columns. The diagram represents results for two biological replicates. (F) Total DNA was harvested at 1, 6, 8, and 10 hpi. Samples were analyzed via qPCR and plotted normalized to input DNA (1 hpi). For statistical analysis for panels B, C, and F, 2-way ANOVA including multiple comparisons was performed.

FIG 4

IFI16, PML, and Daxx exert their effects on replication of an ICP0-null virus in ways that are independent of each other. (A) HFF, Cas9-expressing, or IFI16 KO cells were treated with pooled siRNAs specific for PML or Daxx or with nontargeting siRNA and infected with 7134 virus at an MOI of 0.1. Viral yields were collected at 48 hpi and titrated on U2OS cells. (B) HFF cells were treated with pooled siRNAs specific for IFI16, PML, or Daxx or with nontargeting siRNA and infected with 7134 virus at an MOI of 0.1. Viral yields were collected at 48 hpi and titrated on U2OS cells. Viral yields were plotted as numbers of PFU per milliliter or normalized to nontargeting siRNA. For statistical analysis, t tests were performed. Asterisks located directly above columns indicate significance compared to the siNT value. (C) Relative IFI16, PML, and Daxx mRNA levels were analyzed via qRT-PCR prior to infection. (D) Immunoblot of protein samples harvested before infection and probed with antibodies specific for IFI16, PML, Daxx, and GAPDH. siRNA treatment is indicated by letters, as follows: I, IFI16; P, PML; and D, Daxx. (E) HFF cells were treated with siRNAs specific for PML or with nontargeting siRNA and infected with 7134 virus at an MOI of 0.1 or 10. Viral yields were collected at 48 or 24 hpi, respectively, and titrated on U2OS cells. The effectiveness of siRNA treatment was confirmed by immunoblot analysis of cell lysates prior to infection. Statistical analysis by the t test was performed as described above.

FIG 5

Different knockdown strategies affect HSV-1 replication differently. HFF cells expressing a PML-specific shRNA or control HFFs were treated with PML-specific siRNAs or nontargeting siRNA. Cells were infected with 7134 virus at an MOI of 0.1 or 5, and viral yields were harvested at 48 or 24 hpi, respectively, and titrated on U2OS cells. Total viral yields (A and B) or yields normalized to those for control HFFs treated with siNT (C and D) were plotted. (E) Immunoblot of lysates from mock- or 7134-infected cells at 6 hpi, probed with antibodies specific for PML, GAPDH, ICP8, and ICP27. (F) PML transcript levels at 6 hpi in mock- or 7134 virus-infected cells were assessed via qRT-PCR and plotted relative to 18S rRNA levels. (G and H) ICP8 and ICP27 transcript levels were measured by qRT-PCR and plotted relative to 18S rRNA levels. Numbers denote fold changes between columns. For all statistical analyses for this figure, t tests were performed.

FIG 6

A PML-related function promotes ICP0-null virus replication and viral IE and E gene expression. HFF cells were treated with siRNAs specific for PML, a single siRNA from that pool (siRNA 06), or nontargeting siRNA and infected with 7134 virus at an MOI of 5. Samples were harvested at 4 to 10 hpi, and transcript levels of PML (A), ICP8 (B), and ICP27 (C) were analyzed by qRT-PCR and plotted relative to 18S rRNA levels. (D) Immunoblots of cell lysates obtained at 4 to 10 hpi, probed with antibodies specific for ICP8, ICP27, PML, and GAPDH. For statistical analyses for panels B and C, 2-way ANOVA including multiple comparisons was performed.

FIG 7

A PML-related function acts at the stage of viral DNA synthesis. HFF cells were treated with siRNAs specific for PML or with nontargeting siRNA and infected with 7134 virus at an MOI of 5 in the presence or absence of PAA. Samples were harvested at 6, 8, and 10 hpi. (A to C) IFI16, ICP27, and ICP8 transcript levels at 10 hpi were analyzed by qRT-PCR and plotted relative to 18S rRNA levels. Numbers denote fold changes between the indicated columns. (D) Immunoblot probed with antibodies specific for ICP27, ICP8, IFI16, and GAPDH. (E) ICP8 protein levels at 10 hpi were quantified from the immunoblot, normalized to GAPDH, and plotted. Numbers indicate fold changes between the respective columns. The diagram represents data for two biological replicates. (F) Total DNA was harvested at 1, 6, 8, and 10 hpi. Samples were analyzed via qPCR and plotted with normalization to input DNA (1 hpi). For statistical analyses for panels B and F, 2-way ANOVA including multiple comparisons was performed.

FIG 8

Absence of PML reduces replication compartment formation. HFF cells were cultivated on coverslips and treated with siRNAs specific for PML or with nontargeting siRNA. Cells were infected with 7134 virus at an MOI of 5 or mock infected and then were fixed at 6 hpi. Immunofluorescence staining was performed with antibodies specific for PML and ICP8. (A) Images showing nuclei of representative mock-infected cells in the presence or absence of PML. Channels specifically detected PML (green) or ICP8 (red), and merged images are also shown. (B) Same as panel A, but with 7134 virus infection. (C) Cells showing ICP8 expression above the background were sorted into two groups according to the morphology of staining (diffuse versus replication compartment). Percentages of cells with diffuse or replication compartment-like ICP8 staining are depicted for both siNT and siPML conditions. (D) Percentages of cells showing ICP8 staining above the background for either the siNT or siPML background.