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Journal of Virology, July 2008, p. 6324-6336, Vol. 82, No. 13
0022-538X/08/$08.00+0     doi:10.1128/JVI.00455-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Oligomerization of ICP4 and Rearrangement of Heat Shock Proteins May Be Important for Herpes Simplex Virus Type 1 Prereplicative Site Formation{triangledown}

Christine M. Livingston,1 Neal A. DeLuca,2 Dianna E. Wilkinson,3 and Sandra K. Weller1*

Department of Molecular, Microbial and Structural Biology and The Molecular Biology and Biochemistry Graduate Program, The University of Connecticut Health Center, Farmington, Connecticut,1 Department of Molecular Genetics and Biochemistry, University of Pittsburgh, Pittsburgh, Pennsylvania,2 Division of Virology, National Institute for Biological Standards and Control, Hertsfordshire, United Kingdom3

Received 1 March 2008/ Accepted 14 April 2008


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ABSTRACT
 
Herpes simplex virus type 1 (HSV-1) DNA replication occurs in replication compartments that form in the nucleus by an ordered process involving a series of protein scaffold intermediates. Following entry of viral genomes into the nucleus, nucleoprotein complexes containing ICP4 can be detected at a position adjacent to nuclear domain 10 (ND10)-like bodies. ND10s are then disrupted by the viral E3 ubiquitin ligase ICP0. We have previously reported that after the dissociation of ND10-like bodies, ICP8 could be observed in a diffuse staining pattern; however, using more sensitive staining methods, we now report that in addition to diffuse staining, ICP8 can be detected in tiny foci adjacent to ICP4 foci. ICP8 microfoci contain UL9 and components of the helicase-primase complex. HSV infection also results in the reorganization of the heat shock cognate protein 70 (Hsc70) and the 20S proteasome into virus-induced chaperone-enriched (VICE) domains. In this report we show that VICE domains are distinct but adjacent to the ICP4 nucleoprotein complexes and the ICP8 microfoci. In cells infected with an ICP4 mutant virus encoding a mutant protein that cannot oligomerize on DNA, ICP8 microfoci are not detected; however, VICE domains could still be formed. These results suggest that oligomerization of ICP4 on viral DNA may be essential for the formation of ICP8 microfoci but not for the reorganization of host cell chaperones into VICE domains.


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INTRODUCTION
 
Productive herpes simplex virus type 1 (HSV-1) infection results in the formation of globular domains in the nucleus, called replication compartments, in which viral DNA replication and cleavage and packaging of the viral genome occur (40, 54). The HSV-1 genome encodes seven essential replication proteins, all of which are localized to replication compartments in productively infected cells: the single-stranded DNA binding protein ICP8, the heterotrimeric helicase-primase complex UL5/UL8/UL52, the origin-binding protein UL9, and the heterodimeric polymerase complex UL30/UL42 (53, 66). Replication compartments form via a series of protein scaffold intermediate structures called prereplicative sites that contain HSV-1 replication proteins and cellular proteins (7, 13, 37, 42, 45, 54, 68, 69). In this paper we confirm a previous report (8) that functional ICP8 protein is required for the formation of prereplicative sites.

In order to identify subassemblies or stages in the formation of replication compartments, it has been convenient to freeze the progression either by infection with viruses bearing mutations in replication proteins or by the use of pharmacological agents that inhibit viral DNA synthesis (see Fig. 1) (7, 68). Stage I is defined by the presence of nuclear domains (NDs) that resemble ND10 (promyelocytic leukemia protein [PML] bodies or PML oncogenic domains) and the absence of ICP8 as detected by immunofluorescence microscopy. Work from Everett and colleagues suggests that ND10s that are detected during stage I of infection represent reformed ND10-like foci that have been recruited to de novo HSV-1 nucleoprotein complexes (23, 27). These newly recruited PML-containing foci are reminiscent of ND10 in terms of protein composition. The immediate-early viral protein ICP0 localizes completely with the ND10-like foci and subsequently induces their disruption through the proteasome-dependent degradation of SUMOylated PML (where SUMO is the small ubiquitin-like modifier) and other SUMO-modified ND10 proteins, presumably through its E3 ubiquitin ligase activity (12, 22, 48, 49). The disruption of ND10-like foci corresponds to the transition from stage I to stage II in which the early protein ICP8 can be detected in a diffuse staining pattern. Cells in stage III contain discrete ICP8 foci that depend on the presence of UL5, UL8, UL9, and UL52 as cells infected with null mutants lacking these proteins appear to be blocked in stage II (7). If all three components of the helicase-primase complex are present including a functional UL52 protein, additional proteins can be recruited, resulting in the formation of stage IIIb foci which also contain the HSV-1 polymerase ([Pol] UL30), PML, and other cellular proteins (7, 10, 68). If replication is allowed to proceed, replication compartments are observed (stage IV).


Figure 1
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  		FIG. 1. Stages of replication compartment formation in HSV-1 infected cells. The stages of infection are depicted according to a previously described model (6, 7, 10, 44, 68). During stage I, ND10-like domains are detected. Cells that are in stage II were originally characterized by the disruption of ND10 and the detection of ICP8 in a diffuse pattern. Cells in stage IIIa display foci of ICP8 that contain the helicase-primase complex UL5/UL8/UL52 and the viral origin binding protein UL9. Stage IIIb foci contain the heterodimeric Pol complex UL30/UL42 and cellular proteins in addition to the five viral proteins detected in stage IIIb. Cells in stage IV form replication compartments when DNA replication is allowed to proceed.

Although it is clear that viral proteins assemble in an ordered manner to initiate viral DNA replication, several questions remain about how the recruitment of viral and cellular proteins into prereplicative sites is orchestrated. Although we previously reported that ICP8 was localized in a diffuse pattern during stage II (44), we now report that by using more sensitive detection methods, such as in situ detergent extraction, very small microfoci can be detected at this stage. In addition, we now show that the signal that triggers the formation of ICP8 into microfoci is related to the recently reported ability of ICP4 to oligomerize on DNA (39).

We have previously shown that specific cellular chaperone proteins such as Hsp/Hsc70, Hsp40, and Hsp90 are reorganized during infection into foci localized adjacent to replication compartments (4, 5). In addition to chaperone proteins, virus-induced chaperone-enriched (VICE) domains contain ubiquitinated proteins, the 20S proteasomal subunit, and the stress response protein ATR-interacting protein (5, 67). VICE domains are reminiscent of structures previously reported by Everett that contain conjugated ubiquitin in nuclear foci in infected cells (20). The relationship between prereplicative site formation and the reorganization of host cell chaperone proteins in infected cells is addressed in this paper. We show that VICE domains can be detected after ND10-like bodies have been disrupted. VICE domains can be detected in PML-depleted cells, suggesting that PML itself is not required for VICE domain formation. We also show that the reorganization of Hsc70 into VICE domains can occur prior to the recruitment of ICP8 into microfoci, indicating that reorganization of host chaperone machinery can occur during the earliest stages of HSV-1 infection. These results suggest that the host chaperone machinery may play a role in the formation of replication compartments during the earliest stages of infection.


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MATERIALS AND METHODS
 
Cells and reagents. Vero cells (African green monkey kidney fibroblasts) were maintained at 37°C in the presence of 5% CO2 and were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 5% fetal bovine serum and 0.1% penicillin-streptomycin. Primary human foreskin fibroblast (HFF-1) Lk Luci cells constitutively express a short hairpin RNA against the luciferase gene and PML knockdown HFF-1 shPML1 cells constitutively express a short hairpin RNA directed against all major PML isoforms. Both PML cell lines were obtained from R. D. Everett and were maintained as described previously (26). BAY 57-1293 (N-[5-(aminosulfonyl)-4-methyl-1,3-thiazol-2-yl]-N-methyl-2-[4-(2-pyridinyl)phenyl]acetamide) was obtained from Gerald Kleymann (Bayer Pharmaceuticals; Wuppertal, Germany) (36). Phosphonoacetic acid (PAA) was purchased from Sigma Chemical Co.

Viruses and viral infections. Strain KOS served as wild-type HSV-1. The Hr114 mutant virus (32) contains an ICP6::lacZ insertion mutation in the UL52 (HSV-1 primase) gene in a KOS background. The Hp66 mutant virus (47) contains a LacZ insertion mutation in the UL30 (HSV-1 Pol) gene in a KOS background. Hr114 and Hp66 viruses were propagated on complementing BL-1 (32) and DTMA (43) cells, respectively. The n208 virus was described previously (16, 39). Vero cells were seeded onto round glass coverslips in 35-mm dishes and infected on the following day in 200 µl of serum-free medium containing virus inoculum from a frozen stock. Where appropriate, 400 µg/ml PAA or 100 µM BAY 57-1293 was added to the viral inoculum. Viral adsorption to the cell monolayer occurred for 1 h at 37°C, during which the culture dishes were rocked every 15 min. At the end of the hour, each cell monolayer was washed once with 1x phosphate-buffered saline (PBS) and Dulbecco's modified Eagle's medium containing 5% fetal bovine serum, and 0.1% penicillin-streptomycin was added to each dish. BAY 57-1293 or PAA was added to the culture medium (where appropriate) at the same concentrations as described above for the duration of infection. Infected cells were harvested and prepared for immunofluorescence microscopy between 5 and 6 h postinfection.

In situ extraction for removal of nucleosolic and cytosolic proteins. To remove non-matrix-bound nucleosolic and cytosolic proteins, infected or mock-infected cells were detergent extracted prior to fixation. Cell monolayers were washed once in ice-cold 1x PBS and then treated for 2 min on ice with cytoskeletal extraction buffer containing 0.5% Triton X-100 and a protease inhibitor cocktail tablet. The cytoskeletal extraction buffer was described previously (17). Nonextracted cells were examined in parallel with extracted cells. Extracted and nonextracted Vero cells were immediately fixed and permeabilized for immunofluorescence microscopy as described below.

Immunofluorescence analysis. Cells attached to glass coverslips were washed three times with 1x PBS, followed by fixation for 10 min in 4% paraformaldehyde, subsequent washing with 1x PBS, and permeabilization for an additional 10 min in 1% Triton X-100; this step was followed by washes with 1x PBS. Cells were incubated in 3 to 5% normal goat serum for 1 h at room temperature or overnight at 4°C. Primary antibodies were diluted in 3 to 5% normal goat serum. Coverslips were inverted onto 100-µl drops of primary antibodies on Parafilm for an incubation of 1 h at room temperature, followed by extensive washing with 1x PBS. Coverslips were again inverted onto 100-µl drops of secondary antibodies diluted in 3 to 5% normal goat serum for an incubation of 30 min at room temperature, followed by extensive washing. Coverslips were mounted to slides using a gelatin mounting medium containing 0.25% diazibicyclooctane (Sigma) to retard photobleaching.

Primary antibodies included monoclonal rat anti-Hsc70 (1:200; Stressgen), monoclonal mouse anti-ICP4 (1:200; US Biological), polyclonal rabbit anti-ICP8 clone 367 (1:400; a gift from William Ruyechan) (58), monoclonal mouse anti-ICP8 (1:200; Abcam), monoclonal mouse anti-UL30 1051a (0.1 mg/ml; a gift from Charles Knopf) (61), rabbit anti-UL9 R249 (1:200), rabbit anti-UL5 R220 (1:400), and rabbit anti-UL52 2403 (1:100). AlexaFluor secondary antibodies (Molecular Probes) conjugated with fluorophores excitable at wavelengths of 594, 488, or 647 nm were each diluted 1:200.

Imaging. All images were captured using a Zeiss LSM 510 Meta confocal microscope with the exception of panels B, D, E, and F of Fig. 2, which were imaged on an LSM 410 confocal microscope. The LSM 510 Meta confocal microscope is equipped with argon and HeNe lasers, and the LSM 410 confocal microscope is equipped with an argon-krypton laser to excite AlexaFluor 488, 594, and 647 fluorophores. All imaging was performed using a Zeiss 63x objective lens (numerical aperture, 1.4). Images were arranged using Adobe Photoshop, version 7.0, Adobe Illustrator, and Zeiss LSM 510 image viewer software. Cells were counted using either of the confocal microscopes; at least 100 ICP4-positive cells were assessed for the presence of Hsc70 foci (VICE domains) and/or PML foci (ND10).


Figure 2
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  		FIG. 2. ICP8 microfoci are detected during stage II of infection. Vero cells were infected at an MOI of 2 for 6 h with the primase mutant Hr114 (A, B, and D), with KOS in the presence of the helicase-primase inhibitor BAY 57-1293 (C), with the Pol UL30 mutant Hp66 (E), or with KOS in the presence of the Pol UL30 inhibitor PAA (F). The two nuclei in panel B are shown at x4 digital zoom and were detergent extracted prior to fixation, as described in Materials and Methods for removal of nucleosolic and cytosolic proteins. All other samples are shown at x2 digital zoom and were not detergent extracted. All samples were probed with polyclonal anti-ICP8 367 antibody. Images A and C were captured on the Zeiss LSM 510 Meta confocal microscope, and images B, D, E, and F were captured using the Zeiss LSM 410 confocal microscope.


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RESULTS
 
ICP8 microfoci are detected during stage II of HSV-1 infection. Our laboratory has defined four stages in the assembly of replication compartments by following the localization of ICP8 (Fig. 1) (6, 7, 10, 43, 68). Although each of the stages (I through IV) can be observed during wild-type infection, the formation of replication compartments occurs rapidly, and in order to clearly identify subassemblies at various stages, it has been necessary to interrupt the progression of infection either with mutant viruses or by the addition of drugs that inhibit viral DNA synthesis (7, 68). One of the hallmarks of the transition from stage I to II is the disruption of the ND10-like foci and the detection of ICP8. Although cells in stage II were initially characterized as exhibiting a diffuse ICP8 staining pattern, we now report that microfoci of ICP8 can be detected in cells infected with Hr114 (Fig. 2A) lacking the UL52 subunit of the helicase-primase complex. Actually, small ICP8 foci were previously seen in Hr114-infected cells (44); however, they were often obscured by a brighter diffuse ICP8 staining pattern. Microfoci are much more readily apparent in cells that have been extracted with detergent to remove nucleosolic and cytosolic proteins (Fig. 2B). Thus, both diffuse and microfocal staining patterns are observed in unextracted stage II cells (Fig. 2A); however, detergent-extracted cells lack the diffuse nucleosolic ICP8, allowing the microfoci to stand out much more prominently (Fig. 2B).

Since ICP8 microfoci appear to represent the earliest stage in the assembly of replication compartments, we next asked what conditions were required for their formation. In the experiment shown in Fig. 2A and B, cells were infected with the UL52 mutant Hr114; however, stage II microfoci were also observed in cells infected with viruses lacking the other members of the helicase-primase complex: UL8 and UL5 (data not shown). Furthermore, microfoci can be detected in cells infected with wild-type HSV-1 in the presence of the viral helicase-primase inhibitor BAY 57-1293 (Fig. 2C). BAY 57-1293 has been shown to inhibit DNA-dependent ATPase, helicase, and primase activities of the helicase-primase complex (36). These results suggest that stage II ICP8 microfoci are detected under conditions in which ND10 structures have been disrupted and the helicase-primase complex is inactive.

We next asked whether ICP8 microfoci seen in stage II are similar in morphology to the ICP8 foci seen in cells at the later stages of infection. Stage IIIa foci were previously defined as those detected in cells infected with the Pol mutant, Hp66, or with missense mutants in UL52 lacking primase activity. Stage IIIa foci contain ICP8, UL9, and all three subunits of the helicase-primase complex (6, 10). Stage IIIb foci on the other hand are seen in cells infected in the presence of PAA and contain all seven viral replication proteins as well as several cellular proteins (7, 68). As shown in Fig. 2E, Hp66-infected cells fall into two populations, those with numerous ICP8 foci, which are believed to represent cells in S phase, and those with fewer foci, which we have previously designated stage IIIa foci. The stage IIIa foci are believed to represent true intermediates in the formation of replication compartments and are larger and more robust than the microfoci seen in stage II cells (compare Fig. 2E to 2D). Figure 2F shows cells infected with KOS in the presence of PAA (stage IIIb), and as previously reported these foci resemble stage IIIa foci. In summary, stage II foci appear to be smaller than foci seen in cells in stages IIIa and IIIb.

ICP8 microfoci contain UL9 and the helicase-primase complex but not UL30. Next, we asked whether the protein composition of stage II ICP8 microfoci was similar to stage IIIa foci, which were previously reported to contain UL9 and all three components of the helicase-primase complex (UL5, UL8, and UL52) but not Pol UL30 (44). Figure 3 shows that UL9, UL5, and UL52 can be detected in ICP8 microfoci of KOS-infected cells treated with the helicase-primase inhibitor BAY 57-1293 (Fig. 3, top three rows). The bottom panel of Fig. 3A, on the other hand, shows that the vast majority of UL30 is diffusely localized in stage II cells. Although we cannot rule out that a small amount of UL30 is localized to ICP8 microfoci, it is clear that the localization of UL30 is quite different from that of UL9, UL5, and UL5. Thus, it appears that stage II foci seen in cells treated with BAY 57-1293 are similar in composition to stage IIIa foci. Interestingly, however, in a majority of cells infected with Hr114 (lacking the UL52 protein), the ICP8 microfoci do not contain UL5 (data not shown). This suggests that in the absence of UL52 of the helicase-primase complex, the UL5 subunit is not recruited to ICP8 microfoci. This is consistent with previous reports suggesting that UL5 and UL52 need to be expressed together and that in the absence of UL52, UL5 may be unstable (9, 46). Thus, if UL5 and UL52 are present but inactive (in the presence of the BAY inhibitor), they are recruited to ICP8 microfoci. UL9 appears to localize to ICP8 microfoci even in the absence of an intact helicase-primase complex such as in Hr114-infected cells (Fig. 3B). These results suggest that UL9 may localize to microfoci before the helicase-primase complex or that UL9 can localize to ICP8 microfoci independently of the helicase-primase complex.


Figure 3
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  		FIG. 3. ICP8 microfoci contain UL9 and members of the viral helicase-primase complex but not UL30. Vero cells were infected with 2 PFU/cell KOS in the presence of the helicase-primase inhibitor BAY 57-1293 (A) or 2 PFU/cell Hr114 (B) and were fixed for immunofluorescence analysis 6 h postinfection. Cells were double labeled with mouse anti-ICP8 plus either rabbit anti-UL9 R249, rabbit anti-UL5 R220, or rabbit anti-UL52 2403. In the bottom row of panel A, cells were doubly labeled with rabbit anti-ICP8 367 and mouse anti-UL30 1051b (a gift from Charles Knopf). All imaging was performed on the Zeiss LSM 510 Meta confocal microscope.

ICP8 microfoci are detected adjacent to foci of ICP4. In order to identify the events that trigger the formation of ICP8 microfoci, we looked at the relationship between these prereplicative sites and other viral protein structures that can be detected at the earliest times after infection. Everett and his collaborators have used live-cell imaging experiments to define the first nucleoprotein complexes which form on incoming viral genomes (27, 28). These investigators have described discrete foci containing immediate-early proteins ICP4 and ICP27, which are mostly localized to the inner rim of the nucleus, a pattern reminiscent of the localization of incoming viral genomes (27). In response to the formation of these nucleoprotein complexes or perhaps some other signal generated by the presence of incoming viral genomes, Everett et al. have suggested that PML-containing foci are recruited to viral genomes (27, 28). The earliest viral protein structures detected after infection are discrete foci containing ICP4 and ICP27, and it appears that the PML-containing ND10-like structures form at a position adjacent to ICP4 foci (27). According to our definition, these cells would correspond to stage I.

Next, we were interested in the relationship between the ICP4 nucleoprotein complexes observed during stage I and the formation of ICP8 microfoci during stage II. In the experiment shown in the top panel of Fig. 4, Vero cells were infected with KOS at a multiplicity of infection (MOI) of 10 for 30 min and double labeled for ICP4 and the ND10 marker PML. This experiment confirms the previous observation that ICP4 and PML foci are detected adjacent to one another very early in infection (Fig. 4, top row). In order to investigate the relationship between ICP4 and ICP8 at the earliest stages of infection, Vero cells were infected with Hr114 (stage II) and stained for ICP4 and ICP8 at 6 h postinfection. ICP4 and ICP8 are detected in adjacent foci in these cells (Fig. 4, middle row). Thus, during stage II, ICP8 microfoci are distinct from and adjacent to the ICP4 foci. This is intriguing, given that ICP8 and ICP4 have been reported to colocalize in fully formed replication compartments (37, 55). In the experiment shown in the bottom panel of Fig. 4, we have confirmed that ICP8 and ICP4 are colocalized in small replication compartments in cells infected with KOS for 3 h. In order to explain the observation that ICP8 and ICP4 are in distinct foci at early times but are colocalized in replication compartments, we observed cells at frequent intervals during the formation of replication compartments and found that some ICP8 appeared to be recruited to ICP4 foci. For instance, higher magnification of the image shown at the bottom of Fig. 4 (inset) reveals that tiny ICP8 foci appear to surround a larger ICP4-containing replication compartment. We did not observe cells in which ICP4 appeared to be recruited to ICP8 foci. This may indicate that ICP8 can be gradually recruited into the ICP4 foci, eventually creating the nascent replication compartments.


Figure 4
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  		FIG. 4. ICP8 microfoci are localized adjacent to ICP4 foci. Vero cells were infected with KOS at an MOI of 10 and were fixed for immunofluorescence analysis 30 min (top row) or 3 h (bottom row) postinfection. Double labeling using polyclonal anti-PML and monoclonal ICP4 antibodies or polyclonal anti-ICP8 367 and monoclonal ICP4 antibodies was performed. Vero cells were infected with 2 PFU/cell primase null Hr114 virus (middle row) and harvested for immunofluorescence analysis at 6 h postinfection. All images were captured using a Zeiss LSM 510 Meta confocal microscope.

Formation of ICP8 microfoci may require the ability of ICP4 to form oligomers on DNA. The observation that ICP8 microfoci can be detected adjacent to ICP4 foci suggests that the ability of ICP4 to form nucleoprotein complexes on viral DNA may be a critical step in the formation of viral prereplicative sites and replication compartments. To test this hypothesis, an ICP4 mutant virus (n208) that cannot oligomerize on DNA was tested. The n208 mutant, which expresses a C-terminal truncation of the ICP4 protein, was previously reported to transactivate some early proteins but exhibited a severe defect in viral DNA replication and viral growth (16). Interestingly, the DeLuca laboratory has recently demonstrated that the n208 mutant ICP4 protein cannot oligomerize on DNA, as assessed by an electrophoretic mobility shift assay (39). We next asked whether ICP8 microfoci could form in cells infected with n208 and compared the result to ICP8 microfocus formation in wild-type (KOS) infection in the presence of BAY 57-1293. Figure 5 shows results for three different MOIs: 2, 10, and 20. In cells infected with n208 at all MOIs, ICP8 was predominantly detected in a diffuse nuclear pattern, although at higher MOIs some cells exhibited a few microfoci. On the other hand, all KOS-infected cells exhibited efficient microfocus formation (Fig. 5, KOS/BAY). Thus, ICP8 microfoci can easily be detected in KOS-infected cells in the presence of BAY 57-1293 but not in cells infected with n208 (Fig. 5). To rule out the possibility that the inhibitor BAY 57-1293 was responsible for microfocus formation in these cells, Vero cells were infected with n208 in the presence of BAY 57-1293. We again found that ICP8 microfoci could not be detected (data not shown). These results suggest that ICP4 is required either directly or indirectly for the formation of ICP8 microfoci. It is possible that ICP4 oligomerization is directly responsible for nucleating ICP8 microfocus formation. Alternatively, it is possible that some other function of ICP4 that is lacking in n208 may contribute to inefficient ICP8 microfocus formation, such as the ability to recruit ICP27 or lowered levels of early proteins such as ICP8. Further experiments will be needed to distinguish between these possibilities.


Figure 5
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  		FIG. 5. Formation of ICP8 microfoci is impaired in cells infected with an ICP4 mutant that cannot oligomerize on DNA. Vero cells were infected with 2, 10, or 20 PFU/cell of n208 or KOS in the presence of BAY 57-1293 for 6 h. Cells were labeled with rabbit anti-ICP8 367 and imaged using a Zeiss LSM 510 Meta confocal microscope.

VICE domains are detected adjacent to ICP8 microfoci and ICP4 foci during stage II of infection. We previously reported the reorganization of cellular chaperone proteins, the 20S proteasome, and ubiquitinated proteins into VICE domains, which form adjacent to replication compartments (5). The formation of VICE domains does not require viral DNA replication since VICE domains can form in the presence of a Pol UL30 inhibitor, albeit to a reduced extent compared with KOS infection (5). We were next interested in the relationship between VICE domain formation and the other virus-induced foci that are detected during stages I and II. Figure 6 shows VICE domains that have formed adjacent to replication compartments 6 h following wild-type (KOS, top row) infection. The spatial relationship between VICE domains and the earliest prereplicative sites was examined in cells infected with Hr114. Figure 6 shows that VICE domains can be detected adjacent to both ICP4 and ICP8 foci in a three-lobed structure in stage II cells infected with Hr114 (Fig. 6, Hr114, insets). Thus, ICP4, ICP8, and Hsc70 foci are often detected together although pairwise combinations of ICP4 and ICP8, ICP8 and Hsc70, or ICP4 and Hsc70 can also be detected. In the case of the pairwise combinations, it is possible that a third focus may lie above or below the focal plane of the other two. These results indicate that VICE domains can form in cells that are in stage II and that there appears to be a spatial relationship between VICE domains and developing prereplicative site foci.


Figure 6
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  		FIG. 6. VICE domains form during stage II of infection at a position adjacent to ICP8 microfoci and ICP4. Vero cells were infected with 2 PFU/cell KOS, Hr114, or n208 and were harvested 6 h postinfection. Cells were labeled with rabbit anti-ICP8 367, mouse anti-ICP4, and rat anti-Hsc70 and imaged using a Zeiss LSM 510 Meta confocal microscope.

We next asked whether formation of ICP8 microfoci is required for formation of VICE domains. We turned again to the ICP4 n208 mutant protein that cannot oligomerize on DNA. Vero cells were infected with n208 for 6 h and stained for ICP4, ICP8, and Hsc70. As shown above, in cells infected with n208, ICP8 microfoci were not detected; however, foci of Hsc70 could be observed (Fig. 6, bottom row). It should be noted, however, that the formation of ICP4 foci is less efficient in cell infected with n208 than in Hr114-infected cells. These data indicate that VICE domains can form independently of ICP8 microfoci.

VICE domain formation correlates with the disruption of ND10-like foci. Since the formation of ICP8 microfoci in stage II cells was not necessary for VICE domain formation, we next asked whether the events that occur during the transition from stage I to stage II are necessary for the formation of VICE domains. Specifically, we asked if the disruption of ND10 is required for the formation of VICE domains. We performed a time course analysis of KOS infection to determine the relationship between ND10 disruption and the formation of VICE domains. Vero cells were infected with KOS at an MOI of 10, and cells were harvested every 30 min over a 3-h period, followed by staining with antibodies recognizing Hsc70, ICP4, and PML. A minimum of 100 cells at each time point were counted, and the percentage of ICP4-positive cells that contained intact ND10 and/or intact Hsc70-containing VICE domains was determined. Figure 7A shows that the percentage of cells containing ND10-like structures declines over the first 2 h of infection, followed by an increase in the percentage of cells containing VICE domains starting at 2.5 h postinfection. Thus, the formation of VICE domains correlates perfectly with the disruption of ND10-like structures. A similar pattern was observed when cells were infected with KOS at an MOI of 2 (data not shown). Thus, VICE domains appear to form after ND10-like structures have been disrupted. This conclusion is consistent with our previous report that VICE domain formation is impaired in cells infected with an ICP0 null mutant virus. This result may indicate that if ND10s are not disrupted, VICE domain formation is impaired. Further experiments will be necessary to establish whether the connection between ND10 disruption and VICE formation is direct or indirect.


Figure 7
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  		FIG. 7. VICE domain formation correlates with the disruption of ND10-like foci, and their formation occurs independently of PML protein. In panel A, Vero cells were infected with 10 PFU/cell KOS and were fixed for immunofluorescence analysis along half-hour increments until 3 h postinfection. Cells were stained with mouse anti-ICP4 and rat anti-Hsc70. Infected cells were assessed for presence of Hsc70 foci using a Zeiss 510 Meta confocal microscope. At least 100 cells of each sample were counted. In panel B, PML knockdown (shPML1) or control (Lk Luci) HFF-1 cells were infected with either 1 or 10 PFU/cell KOS and were harvested at 6 h postinfection. Cells were double labeled with ICP4 and Hsc70 antibodies as described above and assessed for formation of Hsc70 foci using a Zeiss LSM 410 confocal microscope. At least 100 cells were counted in each case, and the experiment was performed three times. Representative Lk Luci (control) and shPML1 (PML depleted) cells stained for all PML isoforms are shown in panel C.

PML is the organizing protein for the formation of ND10; therefore, ND10s do not form in PML–/– cell lines (35). To determine whether PML is required for the formation of VICE domains, we obtained an HFF-1 cell line depleted of PML protein from Everett et al. (26). shPML1 cells express a short hairpin RNA directed against all major isoforms of PML. An HFF-1 cell line (Lk Luci cells) expressing a short hairpin RNA directed against the luciferase gene was used as a control (26). PML protein is depleted in the majority of shPML1 but not Lk Luci HFF-1 cells as detected by immunofluorescence analysis and Western blotting (26). Representative Lk Luci cells (control) and typical shPML1 cells (PML knockdown) stained for PML are shown in Fig. 7C. Lk Luci cells exhibit PML staining in ND10s whereas ND10s were not detected in the majority of shPML1 cells. To test whether VICE domains can form in the absence of ND10s, we infected Lk Luci and shPML1 cells with KOS at MOIs of 1 and 10 for 6 h. At least 100 ICP4-positive cells in each case were assessed for whether they contained Hsc70 foci (VICE domains). We consistently found that there is little or no difference in the percentages of control and PML knockdown cells that contain VICE domains (Fig. 7B). Thus, we conclude that VICE domains can form even in the absence of PML protein and that VICE domains form during the initial stages of infection after ND10s have been disrupted.

We next asked whether PML plays a role in the formation of ICP8 microfoci during stage II of infection. We infected control Lk Luci cells and shPML1 cells with KOS or Hr114 at an MOI of 2 for 6 h. Replication compartments and VICE domains were detected in Lk Luci and shPML1 cells that were infected with wild-type HSV-1 (Fig. 8, KOS). Figure 8 shows that ICP8 microfoci can be detected in Hr114-infected Lk Luci and shPML1 cells. Additionally, the trimeric arrangement of ICP4, ICP8, and Hsc70 detected in Lk Luci and shPML1 cells resembled that seen in Hr114-infected Vero cells (compare insets of Fig. 8 with insets of Fig. 6). Thus, we conclude that ICP8 microfoci adjacent to foci of ICP4 and Hsc70 can form in the absence of PML.


Figure 8
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  		FIG. 8. ICP8 microfoci form in the absence of PML at a position adjacent to ICP4 and Hsc70. HFF-1 Lk Luci (control) and shPML1 (PML depleted) cells were infected with 2 PFU/cell KOS or Hr114 and harvested at 6 h postinfection. Cells were triple labeled with mouse anti-ICP4, rabbit anti-ICP8 367, and rat anti-Hsc70 antibodies. Images were captured using an LSM 510 Meta confocal microscope.


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DISCUSSION
 
We and others previously reported that the formation of replication compartments requires the ordered assembly of viral and cellular proteins. In this paper we have confirmed and extended previous observations relating to the earliest steps in this process. The following observations are reported in this paper. (i) Consistent with previous reports, we have observed the formation of ND10-like domains in infected cells at a position adjacent to ICP4-containing nucleoprotein complexes (ICP4 foci) and the subsequent disruption of these ND10-like domains. The disruption of ND10-like foci coincides with a stage in infection previously designated stage II (7). (ii) During stage II, we previously reported that ICP8 was diffusely localized; however, we now report that small microfoci of ICP8 can be detected which also contain UL9 and components of the helicase/primase complex. (iii) Microfoci of ICP8 localize adjacent to ICP4 foci; however, in cells infected with the ICP4 mutant n208, no ICP8 microfoci were detected. These results suggest that oligomerization of ICP4 is required for the formation of ICP8 microfoci. (iv) ICP8 microfoci which form during stage II are smaller and more numerous than ICP8 prereplicative sites that form during stage IIIa of infection; however, the protein composition is similar. (v) We previously reported the rearrangement of host cell chaperones such as Hsc70 into VICE domains (4, 5). VICE domains can be detected during stage II of infection in a trimeric arrangement with ICP4 foci and ICP8 microfoci. (vi) VICE domains can form even in cells infected with n208, suggesting that their formation does not depend on the ability of ICP4 to oligomerize on DNA. (vii) VICE domain formation occurs after ND10s are disrupted; however, they can form in cells depleted of PML, suggesting that the prior existence of PML-containing ND10s is not essential for their formation.

Revised model for the stages of replication compartment formation. Based on the observations made in this paper, a more detailed model for the formation of various subassemblies and the requirements for their formation is now emerging (Fig. 9). During stage I, ND10-like domains are detected adjacent to nucleoprotein complexes containing ICP4. ND10-like domains are subsequently disrupted, a process that is mediated by the E3 ubiquitin ligase activity of ICP0, which mediates the degradation of SUMOylated PML and Sp100 (19). The disruption of ND10-like domains during the transition from stage I to stage II appears to correlate with the formation of VICE domains, which are also detected adjacent to ICP4 foci. Stage II cells are also characterized by the detection of ICP8 microfoci, which form in a trimeric arrangement with ICP4 foci and VICE domains. Stage II foci contain ICP8, UL9, and components of the helicase-primase complex, and their formation can be blocked in cells infected with an ICP4 mutant that cannot oligomerize on DNA (Fig. 5) or with an ICP0 null mutant at low MOIs (C. Livingston and S. Weller, unpublished results). The transition from stage II to stage III can be blocked in cells infected with wild-type virus in the presence of the helicase-primase inhibitor BAY 57-1293 or with a null mutant in one of the helicase-primase components. Stage IIIa foci contain the same five viral proteins detected in stage II. The progression of stage IIIa to IIIb is prevented under conditions in which either the HSV Pol UL30 is absent or when the helicase-primase proteins are present but the primase is inactivated by a point mutation (10). We have previously suggested that the recruitment of HSV Pol UL30 to stage IIIa foci requires an active primase (10). It appears that the progression from stage II to IIIa requires the presence of an active helicase and all three helicase-primase subunits, implying that the helicase-primase subunits themselves may be required to form a scaffold which can recruit other replication proteins. The progression from stage IIIa to IIIb requires the presence of an active primase (10). When replication is allowed to proceed, replication compartments are detected (stage IV).


Figure 9
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  		FIG. 9. Revised model for the formation of replication compartments. (A) During stage I of infection, ND10-like domains are detected adjacent to ICP4-containing nucleoprotein complexes. The transition from stage I to stage II involves both the disruption of ND10-like foci and the formation of ICP8 microfoci. The data in this report suggest that when cells are infected with n208, the formation of ICP8 microfoci is impaired; however, it is expected that ND10-like foci would be disrupted since ICP0 expression is not impaired in this mutant. Stage II is characterized by ICP8 microfoci that form adjacent to ICP4 and Hsc70 foci. The transition from stage II to stage IIIa can be blocked in cells infected with the UL52 mutant virus Hr114 or in the presence of the helicase-primase inhibitor BAY 57-1293. The transition from stage IIIa to stage IIIb is blocked during infection with a Pol UL30 mutant, Hp66, or when the viral primase is inactive (10). The addition of Pol UL30 to prereplicative sites is the hallmark of stage IIIb foci, and inhibition of Pol UL30 with PAA blocks infection at this stage. Productive infection results in the formation of stage IV replication compartments. (B) A model of events that take place during stages I and II of infection is shown. Incoming viral DNA is bound by the immediate-early protein ICP4 to form a viral nucleoprotein complex. ND10s reform on ICP4-containing nucleoprotein complexes and are subsequently disrupted by the E3 ligase activity of ICP0. VICE domains, marked by Hsc70 staining, begin to form and are detected adjacent to ICP4 foci. ICP8 microfoci are detected as punctate foci, often in a trimeric assembly with ICP4 and Hsc70 foci.

Formation of ICP4-containing nucleoprotein complexes. The immediate-early protein ICP4 plays an essential role in repression of immediate-early gene expression and transactivation of early and late gene expression (15, 65). ICP4 is a sequence-specific DNA binding protein that forms dimers in solution (30, 50). Although it has been known for some time that repression of immediate-early proteins involves the tight binding of ICP4 to high-affinity binding sites in the promoters of immediate-early genes (34), the mechanism of gene activation is less clear. ICP4 has been shown to specifically interact with transcription factors such as TFIID and TAF250 (11, 33). This result implies that ICP4 may help recruit transcription factors to viral promoters; however, strong ICP4 binding sites are not present in many early and late promoters. Furthermore, although the DNA binding domain of ICP4 is essential for activation of target genes (1), mutation of putative binding sites in early and late promoters has little effect on gene activation (1, 33, 59). These apparent contradictions have now been reconciled by recent observations from the DeLuca laboratory. Kuddus and DeLuca have demonstrated that ICP4 can oligomerize on DNA and that cooperative binding requires the ICP4 C terminus, which is believed to contain sequences necessary for dimerization (39). A C-terminally truncated ICP4 mutant, n208, that fails to oligomerize on DNA can only weakly transactivate early gene expression (16). Thus, ICP4 may promote transcription by facilitating the formation of preinitiation complexes through recruitment of TFIID even on viral promoters containing relatively weak ICP4 binding sites. It has also been recently shown that the presence of TATA-binding protein and Pol II on the promoters of HSV early and late genes during infection requires the presence of ICP4 on the promoter sequences, whereas TATA-binding protein and Pol II binding to an immediate-early promoter was independent of the presence of ICP4 (57). The binding of ICP4 to the early and late promoters in this study was independent of the presence of strong ICP4 binding sites, again suggesting that while DNA binding activity is important for activation, the presence of strong specific binding sites is not.

The finding that ICP4 can oligomerize on DNA is consistent with the previous demonstration that ICP4 is recruited to viral genomes early in infection (28). In that report, Everett et al. concluded that the ability of ICP4 to form nucleoprotein complexes was dependent on the proper folding of the DNA binding domain of ICP4 since a temperature-sensitive ICP4 mutant with a defect in DNA binding failed to form foci at the nonpermissive temperature (28). In this paper, we confirm that early after infection, ICP4 foci can be observed in infected nuclei. In contrast, in cells infected with the ICP4 mutant that fails to oligomerize on DNA (n208), nucleoprotein complex formation is inefficient. Furthermore, we demonstrate that the recruitment of ICP8 to microfoci adjacent to ICP4 foci is severely compromised in n208-infected cells. As a result, infection is stalled, and replication compartments can only be observed in a minority of cells infected at a high MOI. Thus, we have demonstrated that an ICP4 mutant that cannot oligomerize on DNA is also defective in the formation of nucleoprotein complexes that are competent to recruit ICP8. The fact that ICP4 appears to be required for ICP8 recruitment to the earliest prereplicative sites is also consistent with previous reports suggesting that proper folding and localization of ICP4 are required for nuclear localization of ICP8 (38) and that ICP4 may interact with ICP8 (64). In summary, our results confirm and extend previous reports and indicate that ICP4 oligomerization on DNA is important not only for efficient transactivation but also for the formation of the earliest prereplicative sites.

In this paper we demonstrate that at very early times postinfection, ICP8 microfoci are distinct from and adjacent to ICP4 nucleoprotein complexes. On the other hand, we have observed that by about 3 h postinfection, ICP4 and ICP8 colocalize within replication compartments, consistent with previous observations (37, 55). Interestingly, in this study cells with small ICP4-containing replication compartments were observed which were surrounded by small ICP8 foci. This observation suggests that replication compartments form by recruitment of ICP8 into preexisting ICP4 foci. Live-cell imaging will be required to confirm this hypothesis.

ND10-like foci form adjacent to ICP4 nucleoprotein complexes and are subsequently disrupted. In uninfected cells, PML and other ND10 proteins are found in discrete nuclear foci called PML bodies or ND10s. ND10s are thought to represent nuclear depots of cellular proteins such as PML, Sp100, hDaxx, and SUMO that function in diverse cellular pathways (for a review, see reference 52). Other cellular proteins have also been reported in ND10s, including DNA repair factors, proteasomal subunits, chaperone proteins, and ubiquitin (2, 18, 29, 31, 56). Following exposure to various stressful stimuli such as DNA damage or heavy metal exposure, ND10s are disrupted, allowing components to be recruited where needed (51). For instance, after a DNA double strand break, some ND10 proteins relocalize to the site of the lesion (3, 14, 21). In response to viral infection, ND10 proteins are recruited to viral genomes, forming ND10-like foci adjacent to ICP4 nucleoprotein complexes (28). The signal for recruitment of PML and other ND10 proteins to viral genomes during infection has not been identified (24, 48, 62). Although active viral transcription has been shown to increase the association of ND10-like domains with viral genomes (60, 62), a recent report suggests that ND10 proteins can be recruited to viral genomes even during a quiescent infection in which ICP4 is not expressed (24). In any case, in this study we have confirmed that ND10-like foci can be detected adjacent to ICP4 foci during the earliest stages of infection. The reformation of ND10-like foci may reflect an attempt by the cell to silence incoming viral genomes (24, 25, 63).

VICE domains form after ND10-like domains have been disrupted. VICE domains contain cellular heat shock proteins such as Hsc70 and components of protein processing machinery such as ubiquitin and the 20S proteasome. VICE domain formation requires live virus (Livingston and Weller, unpublished data) and active protein synthesis but does not require productive viral DNA replication (5). Components of VICE domains appear to play important roles during infection. For instance, the Sandri-Goldin laboratory demonstrated that HSV-1 infection in the presence of a dominant-negative Hsc70 (K71M Hsc70) results in reduced viral titers and the prevention of replication compartment formation (41). We have confirmed that Hsc70 is important for replication compartment formation using both the dominant-negative Hsc70 and by depleting cells of Hsc70 with small interfering RNA (Livingston and Weller, unpublished results).

In this paper we address VICE domain formation in the context of the formation of prereplicative sites and replication compartments. We have shown that VICE domains are detected only after the disruption of ND10-like foci. This intriguing result may indicate a relationship between ND10-like foci and the newly formed VICE domains. It may be relevant that some components of ND10 (PML, the 20S proteasome, and ubiquitin) are also detected in VICE domains (D. Wilkinson and S. Weller, unpublished results) (5, 20). These observations may suggest that VICE domains are formed from components of ND10-like domains after they have been disrupted. Although PML has been detected in VICE domains (Wilkinson and Weller, unpublished results), we report in this paper that VICE domains form with equal propensity in the presence and absence of PML. Thus, unlike ND10 formation, which has been shown to require PML, VICE domain formation does not. It is possible, however, that other ND10 components may play a role in VICE domain formation in the absence of PML. This may be reminiscent of the recent finding that even in cells which lack PML, other components of ND10-like foci appear to localize adjacent to ICP4-containing nucleoprotein complexes (26). Thus, in PML knockdown cells infected with an ICP0 null virus, ND10 components Sp100 and hDaxx localize adjacent to ICP4-containing nucleoprotein complexes. It is possible that Sp100 and hDaxx play a role in the formation of VICE domains adjacent to ICP4-containing nucleoprotein complexes. Taken together, these results suggest that there may be a relationship between ND10-like foci and VICE domains, and it will be of considerable interest to use live-cell imaging to test this relationship.

In summary, we describe a novel ICP8-containing prereplicative site that forms following the disruption of ND10 at very early times postinfection. The oligomerization of ICP4 on DNA is important not only for efficient viral transactivation (39) but also for the formation of the earliest ICP8 prereplicative sites. On the other hand, oligomerization of ICP4 is not essential for the reorganization of host cell chaperones into VICE domains. We are intrigued by the possibility that a heterotrimeric arrangement of ICP4 foci, ICP8 microfoci, and VICE domains may be important for the progression of viral infection.


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ACKNOWLEDGMENTS
 
We acknowledge members of the Weller laboratory for helpful comments and discussions. We also thank the Center for Cell Analysis and Modeling for assistance with the confocal microscopy.

This work was supported by Public Health Service grant AI21747.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Molecular, Microbial and Structural Biology, The University of Connecticut Health Center, 263 Farmington Ave., Farmington, CT 06030. Phone: (860) 679-2310. Fax: (860) 679-1239. E-mail: weller{at}nso2.uchc.edu Back

{triangledown} Published ahead of print on 23 April 2008. Back


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Journal of Virology, July 2008, p. 6324-6336, Vol. 82, No. 13
0022-538X/08/$08.00+0     doi:10.1128/JVI.00455-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



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