Dynamic Response of IFI16 and Promyelocytic Leukemia Nuclear Body Components to Herpes Simplex Virus 1 Infection

Abstract

Intrinsic immunity is an aspect of antiviral defense that operates through diverse mechanisms at the intracellular level through a wide range of constitutively expressed cellular proteins. In the case of herpesviruses, intrinsic resistance involves the repression of viral gene expression during the very early stages of infection, a process that is normally overcome by viral tegument and/or immediate-early proteins. Thus, the balance between cellular repressors and virus-counteracting proteins determines whether or not a cell becomes productively infected. One aspect of intrinsic resistance to herpes simplex virus 1 (HSV-1) is conferred by components of promyelocytic leukemia nuclear bodies (PML NBs), which respond to infection by accumulating at sites that are closely associated with the incoming parental HSV-1 genomes. Other cellular proteins, including IFI16, which has been implicated in sensing pathogen DNA and initiating signaling pathways that lead to an interferon response, also respond to viral genomes in this manner. Here, studies of the dynamics of the response of PML NB components and IFI16 to invading HSV-1 genomes demonstrated that this response is extremely rapid, occurring within the first hour after addition of the virus, and that human Daxx (hDaxx) and IFI16 respond more rapidly than PML. In the absence of HSV-1 regulatory protein ICP0, which counteracts the recruitment process, the newly formed, viral-genome-induced PML NB-like foci can fuse with existing PML NBs. These data are consistent with a model involving viral genome sequestration into such structures, thereby contributing to the low probability of initiation of lytic infection in the absence of ICP0.

Importance: Herpesviruses have intimate interactions with their hosts, with infection leading either to the productive lytic cycle or to a quiescent infection in which viral gene expression is suppressed while the viral genome is maintained in the host cell nucleus. Whether a cell becomes lytically or quiescently infected can be determined through the competing activities of cellular repressors and viral activators, some of which counteract cell-mediated repression. Therefore, the events that occur within the earliest stages of infection can be of crucial importance. This paper describes the extremely rapid response to herpes simplex virus 1 infection of cellular protein IFI16, a sensor of pathogen DNA, and also of the PML nuclear body proteins PML and hDaxx, as revealed by live-cell microscopy. The data imply that these proteins can accumulate on or close to the viral genomes in a sequential manner which may lead to their sequestration and repression.

FIG 1

Examples of recruitment of PML NB proteins to sites associated with HSV-1 genomes, as detected by staining for ICP4. The panels show views of cells close to developing ICP0-null mutant HSV-1 plaques in HFs, stained for ICP4 (green) and PML (red) (A) or ICP4 (green) and hDaxx (red) (B). Cells indicated by “a” show the pattern of PML or hDaxx expected of uninfected cells. Cells labeled “b” show various typical asymmetric patterns of ICP4 and PML NB protein staining, showing recruitment of PML or hDaxx to sites associated with HSV-1 DNA. The cell labeled “c” in panel B exhibits some faint foci of ICP4, each of which is associated with hDaxx staining. The cell labeled “d” in panel B shows a less common phenotype, in which hDaxx foci are distributed in a highly asymmetric pattern, very likely associated with parental HSV-1 genomes, but before ICP4 expression has reached a detectable level.

FIG 2

The PYD domain is required for the recruitment of IFI16 to HSV-1 genomes. (A) A map of a lentivirus vector that expresses EYFP-linked IFI16. (B) Western blot analysis of cell extracts from a cell line transduced with the vector, analyzed for IFI16 (left) and EGFP (right). There are three endogenous IFI16 isoforms, which differ in the number of “S” regions in the hinge region between Hin-200A and Hin-200B. Only a single EYFP-linked isoform is expressed in these cells. (C) A map of the coding sequence of IFI16, indicating the PYD and the two HIN domains, with two linker sequences (S1 and S2). Also marked are the locations of the triple point mutations in mutant m3 and the region deleted in the mutant ΔHIN2. (D) The nuclear distribution of the wild-type (wt) and mutant m3 and ΔHIN2 mutant forms of IFI16, detected by autofluorescence in transduced cell lines. (E) The distributions of wt and mutant m3 and ΔHIN2 mutant forms of IFI16 in cells at the edge of developing ICP0-null mutant plaques, indicating prominent recruitment of the wt and ΔHIN2 IFI16 strains, but not the m3 mutant, to sites that are closely associated with HSV-1 genomes (detected by staining for ICP4). (F) Depletion of PML does not compromise efficient recruitment of IFI16 to HSV-1 genomes. The panels show either uninfected HFs or examples of cells at the edge of ICP0-null mutant HSV-1 plaques stained for hDaxx and IFI16, in control and PML-depleted cells (left and right pairs of images, respectively).

FIG 3

Detection of the rapid response of IFI16 to HSV-1 infection. (A) HFs expressing EYFP-IFI16 were infected with ICP0-null mutant HSV-1 (MOI of 50), and then images were captured at the indicated times after addition of the virus. (B) A similar experiment was conducted using wt HSV-1 infection (MOI of 20). (C) Images from a sequence of a cell close to the edge of an ICP0-null mutant HSV-1 plaque. (D) HFs expressing EYFP-IFI16 were infected with virus dl0C4, and images from a sequence of a cell at the edge of a developing plaque, showing the IFI16, ICP4, and merged signals as indicated, are presented.

FIG 4

Dynamics of PML.I and hDaxx in uninfected cells and their rapid recruitment to sites associated with HSV-1 genomes. (A). Uninfected HepaRG cells transduced to express either G(PA)C-PML.I or G(PA)C-hDaxx were imaged before and after photoactivation of the PA-EGFP moiety within the red boxed area. Detection of PA-EGFP at intervals after photoactivation reveals migration of activated molecules from the activated region to the rest of the nucleoplasm. (B). Experiments were performed as described for panel A, but cells at the edge of developing ICP0 mutant HSV-1 plaques with characteristic asymmetrically distributed foci were examined under the same activation and time course conditions. Activated hDaxx was detectable in the asymmetric foci at the first time point after bleaching (about 2 s), becoming more prominent as time progressed. Activated PML accumulated in the foci at the nuclear periphery more slowly.

FIG 5

IFI16 and hDaxx respond to HSV-1 infection with similar kinetics. HFs expressing EYFP-IFI16 and ECFP-hDaxx were infected with ICP0-null mutant HSV-1 (MOI of 100). After a 15-min absorption period, the cells were examined by live-cell microscopy, with images captured every 90 s. A selection of images is shown, with the times after adding the virus indicated in the top row. The upper three rows show the IFI16, hDaxx, and merged channels, with the numbered arrows pointing to IFI16 foci that transiently appeared during the sequence. The corresponding foci are also indicated on the hDaxx and merged panels. The sets of smaller images show details from each time point of the boxed areas marked for the 48-min sample. The left and right columns of these images show the images corresponding to the lower and upper boxes, respectively, for each time point, using the same numbering system. In this sequence covering just 9 min, four IFI16 foci appeared simultaneously with an hDaxx signal, although, as shown at dot 3, the latter was weak at the 55.5-min time point. The presence of IFI16 was transient as shown at dots 1 and 2 in this sequence, while hDaxx was more stable. A longer sequence from the same time course is shown in Video S5 in the supplemental material, in which several other examples of the same phenomena can be seen.

FIG 6

IFI16 responds to HSV-1 infection more rapidly than PML. HFs expressing EYFP-IFI16 and ECFP-PML were infected with ICP0-null mutant HSV-1 (MOI of 25). After a 20-min absorption period, the cells were examined by live-cell microscopy, with images captured every minute, starting 35 min after addition of the virus. A selection of images from the time course is presented, showing a cell in which a novel focus of IFI16 appeared, which later became PML positive before merging with a preexisting PML NB structure. The lower set of three rows presents expansions for each time point of the boxes in the merged image corresponding to the 82-min time point. Further details are provided in the text.

FIG 7

hDaxx responds more rapidly than PML to HSV-1 infection. HFs expressing both ECFP-hDaxx and EYFP-PML were infected with ICP0-null mutant HSV-1, and cells at the edge of a developing plaque were examined. A selection of images from an image sequence are presented, indicating the time (arbitrary) after the first image shown (which is image 54 of the original sequence [frame 24 as presented in Video S7 in the supplemental material]). The lowermost row shows a magnified view of the region that is boxed in the merged image of the leftmost column. Further details are provided in the text.

FIG 8

A model for the dynamics of IFI16 and PML NB components with respect to HSV-1 genomes. A full explanation is provided in the text. The hexagon on the left represents a full capsid bound to the outer side of a nuclear pore through which the naked viral genome (tangled line) is transferred into the nucleoplasm.