The viral ubiquitin ligase ICP0 is neither sufficient nor necessary for degradation of the cellular DNA sensor IFI16 during herpes simplex virus 1 infection

Abstract

The cellular protein IFI16 colocalizes with the herpes simplex virus 1 (HSV-1) ubiquitin ligase ICP0 at early times of infection and is degraded as infection progresses. Here, we report that the factors governing the degradation of IFI16 and its colocalization with ICP0 are distinct from those of promyelocytic leukemia protein (PML), a well-characterized ICP0 substrate. Unlike PML, IFI16 colocalization with ICP0 was dependent on the ICP0 RING finger and did not occur when proteasome activity was inhibited. Expression of ICP0 in the absence of infection did not destabilize IFI16, the degradation occurred efficiently in the absence of ICP0 if infection was progressing efficiently, and IFI16 was relatively stable in wild-type (wt) HSV-1-infected U2OS cells. Therefore, IFI16 stability appears to be regulated by cellular factors in response to active HSV-1 infection rather than directly by ICP0. Because IFI16 is a DNA sensor that becomes associated with viral genomes during the early stages of infection, we investigated its role in the recruitment of PML nuclear body (PML NB) components to viral genomes. Recruitment of PML and hDaxx was less efficient in a proportion of IFI16-depleted cells, and this correlated with improved replication efficiency of ICP0-null mutant HSV-1. Because the absence of interferon regulatory factor 3 (IRF3) does not increase the plaque formation efficiency of ICP0-null mutant HSV-1, we speculate that IFI16 contributes to cell-mediated restriction of HSV-1 in a manner that is separable from its roles in IRF3-mediated interferon induction, but that may be linked to the PML NB response to viral infection.

Fig 1

Localization of IFI16 in uninfected and infected HFs. (A) Upper row, Uninfected HFs were stained for IFI16 and DNA (Topro-3); lower row, HFs were infected with wt HSV-1 at an MOI of 4 and stained 2 h later for IFI16 (Abcam ab55328) and ICP0. (B) HFs were infected with HSV-1 expressing a RING finger deletion mutant ICP0 at an MOI of 4 and stained 4 h later for IFI16 and ICP0. (C) HFs were infected with wt HSV-1 at an MOI of 4 in the presence of 3 μM MG132 and stained 4 h and 6 h later for IFI16 and ICP0.

Fig 2

IFI16 is degraded more slowly than PML in wt HSV-1-infected HFs and HepaRG cells. HFs (A) and HepaRG cells (B) were mock infected (m) or infected with wt HSV-1 (MOI, 10) in the absence or presence of MG132 (3 μM) and harvested at 2, 4, 6, 8, and 10 h later. Whole-cell extracts were analyzed for IFI16, PML, ICP0, and tubulin. (C) The C-terminal region of ICP0 is required for maximal rates of degradation of PML but not IFI16. HepaRG cells were mock infected (m) or infected with wt HSV-1 or ICP0 mutants Δ633-680 and Δ594-775 (all at an MOI of 5), and samples taken at 2, 4, 6, and 8 h postinfection (hpi) were analyzed for IFI16, PML, ICP0, and actin.

Fig 3

ICP0 expression in the absence of HSV-1 infection is insufficient to mediate IFI16 degradation. (A) Control HepaRG cells expressing the tetracycline repressor and derivates in which inducible expression of either wt or RING finger deletion mutant ICP0 (as indicated) were analyzed for IFI16, PML, ICP0, and tubulin at 24 h after induction of ICP0 expression with doxycycline (Dox; 100 ng/ml). (B) Control or wt ICP0-inducible cells were treated with doxycycline and infected with either wt or ICP0-null mutant HSV-1 (MOI, 10) as indicated. Samples were harvested at 2, 4, 6, and 8 h after infection and analyzed for IFI16, PML, ICP0, UL42, and actin. (C) Control or ICP0-inducible cells were treated with doxycycline (100 ng/ml) for 24 h, and the inducible cells were further incubated for 2, 4, 6, and 8 h. Samples were analyzed for IFI16, ICP0, and actin. (D) Control or ICP0-inducible cells were infected (MOI, 10) with wt or ICP0-null mutant HSV-1 in the absence of induction, and samples were harvested at the indicated time points after infection for analysis of IFI16, ICP0, UL42, and actin. infn, infection.

Fig 4

Investigation of the properties of ICP0 required for efficient degradation of IFI16. Expression of ICP0 was induced in cells expressing wt or mutant proteins as indicated and then infected with ICP0-null mutant HSV-1 (MOI, 10) as shown. Samples were harvested at 4 and 8 h after infection and compared with uninduced controls for levels of IFI16, ICP0, UL42, and actin (and VP5 in the experiment shown in panel B). For each protein in panel B, the wt and 1-396nls panels show the same exposure of the samples analyzed on the same gel.

Fig 5

Effect of inhibition of DNA replication on the rate of IFI16 degradation. (A) HFs were infected at an MOI of 5 in the presence and absence of ACG (50 μM). Samples were harvested at 2, 4, 6, and 8 h after infection and analyzed for IFI16, VP5, UL42 and actin in comparison with the mock-infected control (m). (B) An experiment similar to that shown in panel A, except that infection was at an MOI of 2 and ACG was used at 100 μM.

Fig 6

ICP0 is not essential for degradation of IFI16 during HSV-1 infection. (A) HepaRG cells were infected with wt HSV-1 (MOI, 2) or ICP0-null mutant dl1403 (ΔICP0) (MOI, 50), and samples were harvested at 4, 8, and 12 h after infection and analyzed for IFI16, PML, ICP0, VP5, UL42, and actin in comparison to mock-infected controls (m). (B) HepaRG cells that express HCMV protein IE1 in an inducible manner were left untreated (left most lane) or treated with 100 nM doxycycline (Dox) for 24 h. The induced cells were either mock infected (m) or infected with wt or ICP0-null mutant HSV-1 (ΔICP0) at an MOI of 10, and samples were harvested at 2, 4, 6, 8, and 10 h after infection, as indicated. Whole-cell extracts were analyzed by Western blotting for IFI16, IE1, VP5, ICP0, UL42, and actin. (C) U2OS cells were infected with wt HSV-1 (MOI, 10) or ICP0-null mutant dl1403 (ΔICP0) (MOI 20), and samples were harvested at 4, 8, and 12 h after infection and analyzed for IFI16, ICP0, VP5, UL42, and actin in comparison to mock-infected controls (m).

Fig 7

Recruitment of IFI16 to sites associated with HSV-1 genomes in the presence and absence of ICP0. (A) HFs were infected with dl0Y4 (ICP0-null mutant HSV-1 expressing EYFP-linked ICP4) at a low MOI, and viral plaques were imaged the following day. ICP4 was detected by EYFP autofluorescence, and IFI16 by staining with MAb ab55328 (Abcam). PML was detected using rAb ABD-030. Secondary antibodies were Alexa 663-linked anti-mouse and Alexa 555-linked anti-rabbit IgG. Top row, a view of the edge of a plaque, illustrating cells in which PML and IFI16 have been recruited to ICP4 foci and others that have asymmetric accumulations of IFI16 and PML prior to the accumulation of detectable levels of EYFP-ICP4; middle row, a high magnification of a typical cell exhibiting recruitment of IFI16 and PML into ICP4-associated foci; bottom row, a detail of highly infected cells, showing an example in which IFI16 and PML form thread-like structures. (B) An experiment similar to that shown in the top two rows of part A, but using vEYFP-ICP4 (wt HSV-1 expressing EYFP-linked ICP4).

Fig 8

Depletion of IFI16 increases the plaque formation efficiency of ICP0-null mutant but not wt HSV-1. (A) Western blot analysis of nontransduced HFs and HFs expressing control (shLuci) or IFI16-specific (shIFI16) shRNAs. (B and C) Relative plaque formation of wt (B) and ICP0-null mutant (C) HSV-1 in nontransduced, shLuci (control), or shIFI16 HFs, as indicated. The data from several independent experiments were averaged and then plotted ± standard deviations. Calculations were as described in reference .

Fig 9

Recruitment of PML and hDaxx to HSV-1 genomes is diminished in a proportion of IFI16-depleted cells. HF-shNeg and HF-shIFI16 were infected with dl0Y4 at a low MOI, and plaques were examined by ICP4 autofluorescence and staining for either PML (A) or hDaxx (B), using an Alexa 633-linked anti-rabbit secondary antibody. Typical cells with asymmetric ICP4 foci are shown for the control and shIFI16 cells, as indicated (top two rows of each panel). Examples of shIFI16 cells in which recruitment of the PML NB proteins is poor are shown in the bottom row of each panel.

Fig 10

Evidence for a reduced rate of response of hDaxx to the entry of HSV-1 genomes into the nucleus in IFI16-depleted cells. Control cells (left panels) and IFI16-depleted HFT cells (right panels) were infected with dl0Y4 at a low MOI, and plaques were examined by ICP4 autofluorescence and staining for hDaxx using an Alexa 633-linked anti-rabbit secondary antibody. The color images are a composite of arrays of 9 (control sample) or 16 (shIFI16 sample) images taken using a ×40 objective lens. Individual cells surrounding the core of the plaques and displaying clear asymmetric patterns of hDaxx staining were examined under zoom and scored for the presence of detectable ICP4 by switching off the red channel. The greyscale images below the color ones show the hDaxx and ICP4 channels of selected regions of the plaques. Other details and explanations are given in the text.