The Human Cytomegalovirus IE2 and UL112-113 Proteins Accumulate in Viral DNA Replication Compartments That Initiate from the Periphery of Promyelocytic Leukemia Protein-Associated Nuclear Bodies (PODs or ND10)

Cell cultures and virus infection.

Permissive human diploid fibroblasts (HF cells) and Vero cells were grown in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum. HF cells at passages 7 to 13 were used for virus infection. Some samples as indicated were incubated in the presence of PAA at 200 μg/ml after infection.

The IE1-defective mutant HCMV(CR208) and its parent virus, HCMV(Towne), were provided by Edward Mocarski (Stanford University, Stanford, Calif.). The virus stocks used were prepared as described by LaFemina et al. (26). For experiments using indirect immunofluorescence assays (IFA), cells were seeded into four-well chamber slides (0.4 × 10⁵/well), and the subconfluent cells were infected with HCMV(Towne) or HCMV(CR208) at various multiplicities of infection (MOI; PFU per cell). In all cases, input supernatant virus was diluted with serum-free medium and was adsorbed for 1.5 h at 37°C, and then the inoculum was replaced with fresh warmed medium at time zero.

Expression plasmids and transient DNA transfection.

All replication machinery expression plasmids used in this work except pMP18 (IE2) and pJHA309(Flag/UL112-113) were described previously (47). For the six replication fork core machinery proteins, pRTS22 (UL44; processivity factor), pRTS6 (UL54; DNA polymerase), pRTS7 (UL57; SSB), pRTS9 (UL70; primase subunit), pRTS29 (UL102; primase-associated factor), and pRTS10 (UL105; DNA helicase subunit) were used. For the auxiliary components and stimulatory factor, p302 (UL36-38), pRTS18 (IRS1), pMP18 (IE2) (40), pRTS26 (UL112-113), pRTS5 (UL84), and pRTS28 (UL69) were used.

To generate pJHA309 expressing 5′ flag epitope (F)-tagged UL112-113 protein products, the 2.2-kb XbaI-BglII fragment was isolated from pRTS26, converted to a blunt-end form, and inserted into the BglII site of pJH272, an F-tag expression vector derived from pSG5 (16).

Plasmid pRL45, expressing both IE1 and IE2 under their natural transcriptional and splicing signals, plasmids pRL62, pMP88, pMP14, pMP83, and pRL94, expressing both wild-type IE1 and mutant IE2, and plasmids pRL72 and pRL84, expressing in-frame fusions between IE1 and IE2, were all described previously (26, 40).

All IE2 mutant expression plasmids containing deletions between codons 290 and 542 were generated in the pMP18 background for mammalian expression purposes. To do so, the XhoI site upstream of the major IE (MIE) enhancer/promoter region in pMP18 was first destroyed by Klenow fill-in (pJHA100), and the XhoI-StuI sites of IE2 exon 5 in pJHA100 were subsequently used to replace a 760-bp wild-type fragment (containing codons 290 to 542) with the equivalent XhoI-StuI fragments containing deletion mutations as described previously (2). This approach generated pCJC110(IE2Δ290-313), pCJC108(IE2Δ313-346), pCJC109(IE2Δ346-358), pJHA104(IE2Δ358-379), pJHA106(IE2Δ376-404), pJHA105(IE2Δ403-419), and pJHA107(IE2Δ487-518).

For DNA transfection experiments, Vero cells were seeded into two well-slide chambers (0.8 × 10⁵/well) and DNA (3 μg/well) was introduced for transient expression assays in subconfluent cells, using the N,N-bis-(2-hydroxyethyl)-2-aminoethanesulfonic acid-buffered saline version of the calcium phosphate procedure described previously (38).

Antibodies.

Mouse monoclonal antibodies (MAbs) 6E1 and 12E2 against HCMV IE1 (UL123; exon 4) and IE2 (UL122; exon 5), respectively, were obtained from Vancouver Biotech (Vancouver, British Columbia, Canada). MAb CH810, which detects epitopes presented in both IE1 and IE2 (exons 2 and 3), was purchased from Chemicon (Temecula, Calif.). MAbs against ppUL44 (UL44) and BrdU were obtained from Advanced Biotech Inc. (Gaithersburg, Md.) and Becton Dickinson (San Jose, Calif.), respectively. A MAb against the F epitope was purchased from Eastman Kodak Company (New Haven, Conn.). The rabbit antipeptide polyclonal antibody (PAb) referred to as anti-PML(C), directed against amino acids at positions 484 to 498 of PML, was described previously (1, 3). Rabbit antipeptide PAb UL112-113(C), directed against the C terminus of HCMV UL112-113, and a rat antipeptide PAb against HCMV SSB (UL57) were prepared as described previously (39). The epitopes for the UL112-113(C) and SSB antibodies were NH₂-AGNGRRRGPRFLEDGL-COOH and NH₂-RTRLPVVPKQPKKEPS-COOH, respectively.

IFA.

For IFA, both virus-infected and DNA-transfected cells were fixed by either the methanol or paraformaldehyde procedure. For the methanol procedure, the cells were washed in Tris-buffered saline (TBS), then permeabilized with absolute methanol at 20°C for 10 min and rehydrated in ice-cold TBS for 5 min. For the paraformaldehyde procedure, the cells were washed in phosphate-buffered saline (PBS), fixed with 1% paraformaldehyde solution in PBS at 20°C for 5 min, and then permeabilized in ice-cold 0.2% Triton X-100 solution in PBS for 20 min before incubation with primary antibodies. Mouse MAbs 6E1 (for IE1), 12E2 (for IE2), CH810 (for both IE1 and IE2), and ABI44 (for ppUL44) were used at 1:200 dilutions. Mouse MAb for BrdU was used at a 1:100 dilution, and MAb for the F epitope was used at a 1:600 dilution. Rabbit PAb anti-PML(C) was used at a 1:1,000 dilution, and PAb anti-UL112-113(C) was used at a 1:800 dilution. Rat PAb against SSB was used at a 1:50 dilution. The antibody incubations were carried out in TBS at 30°C for 1 h, followed by incubation with fluorescein isothiocyanate (FITC)-labeled donkey anti-mouse immunoglobulin G (IgG) or rhodamine-coupled donkey anti-rabbit (or anti-rat) IgG antibody at a 1:100 dilution at 37°C for 45 min. For double labeling, mouse monoclonal and rat or rabbit polyclonal antibodies were incubated together. Slides were screened and photographed on a Leitz Dialux 20EB epifluorescence microscope with Image-Pro software (Media Cybernetics, Silver Spring, Md.). For confocal microscopy, a Noran OZ CLSM confocal microscope system with Intervision software (Noran Inc., Madison, Wis.) was used.

For BrdU labeling, the cells were pulse-labeled with 10 μM BrdU for 30 min at 37°C and were fixed and permeabilized as described above. The cells were then treated with 4 N HCl for 10 min at 20°C to expose the incorporated BrdU residues and washed three times for 5 min each with PBS to neutralize the acid before incubation with mouse anti-BrdU MAb.

Western blot analysis.

Cells were washed with PBS and lysed with 0.2 ml of ice-cold lysis buffer (50 mM Tris-HCl [pH 8.0], 150 mM NaCl, 1.0% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate [SDS]). Equal amounts of clarified cell extracts were separated by electrophoresis on SDS–8% polyacrylamide gels followed by electroblotting onto nitrocellulose. The blots were blocked by incubation for 1 h at 20°C in PBS plus 0.1% Tween 20 containing 5% nonfat dry milk. The blots were then washed twice with PBS-Tween 20 for 15 min and incubated with appropriate antibodies at a dilution of 1:3,000 for 1 h at room temperature. After three 10-min washes with PBS-Tween 20, the blots were incubated with horseradish peroxidase-conjugated goat anti-mouse IgG (Bio-Rad) for 1 h at 20°C. The blots were washed three times, and reacting protein bands were detected with an enhanced chemiluminescence system (Amersham ECL RP2106) using Kodak XAR film.

Both IE2 and UL112-113 localize adjacent to PODs at very early times after infection.

When HF cells were infected with wild-type HCMV(Towne), the localization of IE2 adjacent to PODs was observed at 2 h after infection by confocal double-label IFA for IE2 and PML (Fig. 1a). This association is very transient and was detected in a only a few infected cells because IE1 normally rapidly displaces PML from the PODs (3). However, in HF cells infected with mutant HCMV(CR208), which lacks all of IE1 exon 4 but still has the ability to replicate efficiently at high MOI (15), the PODs remain intact because of the lack of functional IE1 protein (1). Therefore, HF cells were also infected with HCMV(CR208) at an MOI of 2.0 and observed by confocal double-label IFA for IE2 and PML. Under these conditions, most of IE2 was localized as an overlapping pattern in close proximity to but not identical to PODs in almost all infected cells even at 6 h after infection (Fig. 1b).

We also raised a rabbit anti-UL112-113 PAb that detects the 84-kDa form of the UL112-113 protein (see Fig. 8) and investigated the distribution pattern of UL112-113 in virus-infected HF cells. In both wild-type and ΔIE1 mutant virus-infected cells, UL112-113 gave a small nuclear punctate pattern at 6 h (Fig. 1c and d; see also Fig. 4) but was not detected at 2 h (data not shown). Because this punctate pattern of UL112-113 resembled PODs, confocal double-label IFA for both UL112-113 and PML was performed. The result showed that like IE2, at least the 84-kDa form of UL112-113 was also distributed adjacent to or touching PODs at 6 h in ΔIE1-infected cells (Fig. 1c). In contrast, when confocal double-label IFA was performed for both IE2 and UL112-113 in wild-type virus-infected cells at 6 h, the two proteins often colocalized and were much more closely associated with each other than either was with PML (Fig. 1d). These results clearly show that both IE2 and UL112-113 initially localize together and adjacent to or touching PODs at very early times after infection, although apparently IE2 targets to PODs at earlier times than UL112-113. They also demonstrate that these localization patterns for both IE2 and UL112-113 are not dependent on the expression of IE1 or on the loss of PML from the PODs.

Evaluation of the IE2 protein domains required for punctate POD association.

In DNA-transfected HF or Vero cells, the IE2 protein is distributed as a mixed nuclear pattern combining a background diffuse distribution with punctate bodies which exactly colocalize with PML in the PODs (ND/P pattern) (3). In contrast, a fully punctate distribution that only touches PODs is found at early times during HCMV infection of HF cells. To determine which regions of IE2 are required for the nuclear punctate distribution and for colocalization with PML in the absence of other viral proteins, we transfected Vero cells with a variety of IE2 deletion mutants followed by double-label IFA. The results are summarized in Fig. 2A and B. Two independent nuclear localization signals of IE2 have been mapped previously to regions between codons 145 and 151 and codons 321 and 328 (39), and all deletion mutants tested retained at least one of these motifs. When cells were transfected with plasmids expressing either IE2 alone (pMP18) or both IE1 and IE2 (pRL45), the typical wild-type mixed ND/P pattern for IE2 was detected as described previously (3). When cells were transfected with plasmids expressing IE2(Δ86-290) (pRL84) or IE2(Δ136-290) (pMP88), IE2 was distributed as a uniform nuclear diffuse form only, whereas cells transfected with pRL72, expressing IE2(Δ86-135) as a fusion protein (with an insertion of IE1 amino acids 87 to 131), showed a novel modified micropunctate pattern with numerous tiny punctate bodies (MP/P pattern). These IFA results demonstrate that several segments from the region between codons 87 and 290 are required for the normal punctate distribution of IE2. In addition, two mutant proteins, IE2(Δ542-579) lacking the C terminus in pMP14 and IE2(Δ290-492) lacking a large internal region in pMP83, both gave a totally diffuse nuclear distribution without showing any association with punctate bodies.

When mutant IE2 proteins containing smaller deletions within the internal region between codons 290 and 542 were investigated, four distinct deletions that removed codons 290 and 313, 313 and 346, 346 and 358, or 358 and 379 (pCJC110, pCJC108, pCJC109, and pJHA104) all still showed the typical wild-type punctate (ND/P) staining pattern for IE2 and retained the association with PML. However, three different internal deletions located at further C-terminal positions between codons 376 to 404 (pJHA106), 403 to 419 (pJHA105), and 487 to 518 (pJHA107) all lost the typical punctate pattern but remained positive by IFA. Interestingly, the PML distribution pattern in cells expressing the IE2(Δ376-404) and IE2(Δ403-419) mutants showed a large nuclear aggregated form of IE2 with either only a few remaining PODs or numerous micropunctate spots of PML, whereas the IE2(Δ487-518) mutant gave a nuclear and cytoplasmic diffuse pattern without affecting the punctate PML pattern. Representative patterns for the IE2(Δ136-290), IE2(Δ86-135), IE2(Δ290-313), IE2(Δ376-404), and IE2(Δ403-419) mutant proteins and the PML patterns in the same fields are shown in Fig. 2C.

Therefore, regions from both the nonconserved N terminus (codons 87 to 290) and the conserved DNA binding/dimerization domain in the C terminus (codons 379 to 579) of IE2 exon 5 contribute to both the punctate characteristics of IE2 and its association with PODs, although interestingly the central portion from codons 290 to 379 does not. This association is believed to be indirect because unlike IE1, IE2 does not interact with PML in yeast two-hybrid assays (1).

IE2 is incorporated into viral DNA replication compartments at late times after infection.

The IE2 protein is required for maximal efficiency of viral DNA replication in transient DNA cotransfection assays (35, 47). To further study the role of IE2 in viral DNA replication, the localization pattern of IE2 was also investigated at later times after infection with wild-type HCMV. Interestingly, the IE2 staining pattern at 96 h after infection of HF cells at high MOI showed large intranuclear structures that resembled viral DNA replication compartments (Fig. 3). Some of the infected cells showed two separate structures within the nucleus (arrow), but in most cells these had become enlarged and combined to form the fully developed irregular oval or kidney-shaped viral DNA replication compartments that nearly completely fill the nucleus (but sparing the nucleolus). These patterns observed by IE2 staining are very similar to those detected by staining for the SSB (UL57), UL44, and UL112-113 proteins at late times after infection (37) as well as for UL44 in functionally active HCMV replication compartments assembled in the cotransfection assay system (47).

To confirm the specificity of incorporation of IE2 into viral DNA replication compartments, we investigated the localization patterns of IE1, IE2, the polymerase processivity factor (UL44), UL112-113, and SSB (UL57), as well as newly incorporated pulse-labeled BrdU, at 6, 24, and 72 h in HF cells infected with HCMV(Towne) at a low MOI (<1.0). At 6 h, IE1 was distributed as a uniform nuclear diffuse pattern (Fig. 4a) and IE2 showed a nuclear punctate pattern (Fig. 4b) as previously described (3). Neither UL44 nor SSB was detected at 6 h (Fig. 4c and d), but when rabbit PAb UL112-113(C) directed against the C terminus of UL112-113 was used, a punctate staining pattern was obtained as described above (Fig. 4e). At 24 h after infection, IE1 remained as a uniform nuclear diffuse pattern, whereas IE2, UL112-113, and SSB all showed punctate structures larger than those at 6 h. UL44 was detected as both a punctate pattern in some cells and a nuclear diffuse pattern in most cells at 24 h, although it gave only a uniform nuclear diffuse form at 12 h after infection (not shown). At 72 h after infection, IE2 as well as UL44, UL112-113, and SSB all accumulated into large globular nuclear structures, including some that resembled complete viral DNA replication compartments, while IE1 still remained in a nuclear diffuse pattern. The cytoplasmic signals detected in virus-infected cells at late times (24 h and 72 h) by the rabbit PAb raised against UL112-113 were also detected with preimmune serum (not shown) and appear to result from the nonspecific binding of rabbit antibodies to virus-induced immunoglobulin Fc receptor-like proteins. This has also been seen for most other rabbit PAbs and has been described by other investigators (14). When double-label IFA for IE2 and UL44 was performed with rabbit antipeptide PAb against IE2 (P3) (38) and mouse MAb for UL44, the proteins were colocalized to the same structures (not shown).

To localize the sites for viral DNA synthesis in infected cells, BrdU pulse-labeled DNA was stained with anti-BrdU MAb at the indicated time points and shown as double labeled for both UL112-113 and BrdU by IFA (Fig. 4e and f). Incorporation of BrdU into large nuclear structures that contain UL112-113 and also resemble those detected by both IE2, UL44, and SSB staining was observed only at and after 72 h, whereas BrdU was not incorporated significantly into virus-infected cells (judged by positive UL112-113 staining) at either 6 or 24 h. This result clearly demonstrates that the globular structures containing IE2, UL44, SSB, and UL112-113 detected at 72 h represent viral DNA replication compartments. Importantly, formation of both the globular and larger kidney-shaped structures containing IE2, UL44, SSB, UL112-113, and BrdU was not detected at 72 h after infection in the presence of PAA. Instead, IE2 and UL112-113 gave small punctate bodies similar to those seen at 6 h, and UL44 was stained as a nuclear diffuse pattern with only very tiny punctate bodies in the presence of PAA. No BrdU signals were detected in these residual UL112-113-positive prereplication foci in either virus-infected S-phase or non-S-phase cells at 72 h in the presence of PAA, although the S-phase cells still showed BrdU incorporation into cellular replisomes (data not shown). These results demonstrate that IE2, but not IE1, is incorporated efficiently into viral DNA replication compartments at late times after infection and that the larger punctate structures containing IE2, UL44, SSB, and UL112-113 detected at 24 h appear to represent the initial stages of formation of viral DNA replication compartments, although viral DNA synthesis does not occur yet at this time point.

Viral DNA replication compartments develop from the periphery of PODs.

Our observations that (i) both the IE2 and UL112-113 proteins localize adjacent to PODs in small punctate domains at very early times after infection, (ii) these punctate domains become larger at later time points, and (iii) both IE2 and UL112-113 are ultimately incorporated very efficiently into viral DNA replication compartments led us to ask whether the peripheries of PODs where both IE2 and UL112-113 initially accumulate are also sites for the initiation of viral DNA replication compartment formation. Therefore, the localization pattern of IE2 and its association with PODs were further investigated in HF cells infected with HCMV(CR208/ΔIE1) at an MOI of 5.0 (Fig. 5). When the cells were fixed at 48 h after infection, double-label IFA for IE2 and PML showed that IE2 accumulated adjacent to PODs in some infected cells (Fig. 5a) and that budding structures containing IE2 were present at the periphery of PODs in some other infected cells (Fig. 5b). Furthermore, in some cells, larger structures bounded or flanked by several PODs were detected at the same time points (Fig. 5c and d). Therefore, the IE2-containing budding structures that initiate from the periphery of PODs appear to coalesce to make larger structures that are themselves still flanked by PODs. It should be noted that budding structures were not initiated from all IE2 POD-associated domains, suggesting that localization of IE2 at the periphery of PODs is not itself sufficient for initiation of the budding structures. At 72 h after infection, the bulk of the IE2 protein in most cells had accumulated into usually just two separate nuclear globular structures (Fig. 5e) that were presumably in the process of enlarging and assembling into the fully developed kidney-shaped viral DNA replication compartments that nearly completely fill the nucleoplasm by 96 h (Fig. 3).

To investigate whether these intermediate-stage nuclear budding structures containing IE2 also contain other viral replication or accessory proteins, double-label IFA for UL112-113 and IE2, for UL112-113 and UL44, or for UL112-113 and PML was performed on ΔIE1-infected HF cells fixed at 48 h (Fig. 6). Again both IE2 and UL112-113 proved to be colocalized either in small punctate bodies or within the larger granular structures (Fig. 6a). UL44 was distributed as a mixture of nuclear diffuse and punctate forms, and the punctate forms were colocalized with UL112-113 (Fig. 6b). Importantly, both the UL112-113 nuclear structures budding from the periphery of PODs and POD-associated small punctate bodies that were detected by IE2 staining (Fig. 5) were also detected by double-label staining for UL112-113 and PML (Fig. 6c). These results demonstrate that the nuclear budding structures containing IE2 that initiate from the periphery of PODs also contain viral DNA replication proteins, including UL44 and UL112-113, and represent initial stages in assembly of viral DNA replication compartments.

To confirm that the large globular structures containing viral proteins that were detected at 48 h in HCMV(CR208/ΔIE1)-infected cells (Fig. 5 and 6) are also observed in wild-type virus-infected cells, and to show that viral DNA synthesis occurs in these structures, HCMV(Towne)-infected cells were pulse-labeled with BrdU and double-label IFA for both UL112-113 and BrdU was performed at 48 h. In most cells showing two or three large globular structures containing UL112-113, the newly incorporated BrdU signals were also colocalized in the same structures (Fig. 7c to f), although BrdU was not incorporated into cells with several smaller punctate UL112-113 domains, which probably represent earlier stages of infection (Fig. 7a and b). However, interestingly, in some cells showing two or three large globular structures containing UL112-113, BrdU incorporation was not detected in the same structures (Fig. 7g and h). These latter cells may reflect an intermediate stage in which all of the viral replication machinery proteins are not yet assembled sufficiently to allow viral DNA synthesis, or possibly they contain complete assembled pre-replication compartments that lack viral DNA templates.

The antipeptide PAb UL112-113(C) detects the intact 84-kDa form of the UL112-113 protein.

The UL112-113 locus expresses four differently spliced polypeptides of 34, 43, 50, and 84 kDa, as shown by immunoblot assays of extracts prepared from HCMV-infected HF cells (53). However, because the antipeptide UL112-113(C) PAb used in our study was raised against the C-terminal 16 amino acids of the UL112-113 transcription unit, it was expected to be specific for only the intact 84-kDa form of UL112-113. To confirm the specificity of the UL112-113(C) PAb, extracts of HF cells infected with HCMV(Towne) were prepared at 6, 24, 48, and 72 h after infection and the proteins were fractionated by SDS-polyacrylamide gel electrophoresis (PAGE) for immunoblot assay. As controls, extracts of Vero cells transfected with either a plasmid expressing 5′ F-tagged UL112-113 or the empty vector alone (pSG5) were also used for immunoblot assay with mouse MAb against the F epitope. The 5′ F tag in the UL112-113 expression vector is expected to be present in all four UL112-113-derived proteins. The Western blot results showed that only the 84-kDa band was specifically detectable with the UL112-113(C) antibody in virus-infected HF cells, whereas all four (34-, 43-, 50-, and 84-kDa) forms of UL112-113 were detected with anti-F antibody in DNA-transfected Vero cells (Fig. 8A). Consistent with previous observations (53), the 84-kDa UL112-113 protein was synthesized within 6 h and increased from early to late times during infection, and the 43-kDa UL112-113 protein was the most abundant among the four related protein products even in DNA-transfected cells.

To compare the distribution pattern of the 84-kDa version of UL112-113 to those of the smaller proteins encoded by the UL112-113 locus by IFA, Vero cells were transfected with plasmid DNA expressing the 5′ F-tagged version of UL112-113. In this case, the UL112-113 products detected by anti-F antibody were distributed as a mixture of both nuclear diffuse and typical small punctate patterns (Fig. 8B) as well as some punctate structures that were larger than those detected with the anti-UL112-113(C) PAb in infected cells. However, double-label IFA revealed that the staining pattern of the 84-kDa form of UL112-113 detected here with UL112-113(C) PAb was exactly the same as that detected by the MAb for the F epitope (Fig. 8C). Therefore, although the pattern in overexpressed transient assays contrasts somewhat with the small punctate pattern only as observed for the 84-kDa form in HF cells infected at a low MOI (Fig. 1c and d; Fig. 4d), the results suggest that all four forms may have the property of targeting to the PODs.

UL112-113 recruits the UL44 polymerase processivity factor into POD-associated sites in DNA transfection assays in the presence of core replication complex components.

The data presented above demonstrated that at least the 84-kDa form of UL112-113 associates with PODs at 6 h in virus-infected cells and later accumulates at the periphery of PODs together with IE2. To further investigate the association of the UL112-113 protein with PODs, Vero cells were transiently transfected with a plasmid expressing the 5′ F-tagged version of UL112-113 and double labeled for the F epitope and endogenous PML. The results confirmed that both the small punctate and larger globular patterns of UL112-113 were associated with PODs (Fig. 9a) and also demonstrated that even overexpressed UL112-113 product associated with PODs in the absence of other viral proteins. Because IE2 also colocalizes with PODs in transient DNA transfection assays (3), and both IE2 and UL112-113 are colocalized adjacent to PODs at 6 h as well as incorporated into viral DNA replication compartments at 72 h in virus-infected cells as described above, we also examined the distribution of the two proteins when transfected together in Vero cells. As expected, both proteins proved to colocalize within the same punctate and globular structures that presumably correspond to PODs (Fig. 9b).

Both IE2 and UL112-113 are expressed at very early times in virus-infected cells and are targeted to POD or to POD-associated domains before the replication fork proteins are synthesized. Therefore, we also examined whether expression of either IE2 or UL112-113 might affect the localization patterns of core replication machinery proteins in transient cotransfection assays. A mouse MAb against the HCMV polymerase processivity factor UL44 was used to track the localization and assembly of the replication proteins. As expected, UL44 was distributed as a uniform nuclear diffuse pattern in Vero cells when transfected alone, and this diffuse pattern was not affected in cells cotransfected with a plasmid expressing IE2 alone (not shown). Similarly, when all six viral core replication machinery proteins (polymerase, UL44, SSB, and the triplex helicase-primase complex) were coexpressed in Vero cells in the absence of the oriLyt plasmid or any accessory proteins, UL44 was still distributed as a typical nuclear diffuse form in all positive cells (Fig. 9c). However, when the same six viral core replication machinery proteins were expressed together with UL112-113, the distribution of UL44 was changed to include typical punctate patterns in a majority of the cells, and double labeling revealed that the reorganized punctate forms of UL44 were colocalized with endogenous PML in the PODs (Fig. 9d). Importantly, this alteration in the distribution of the replication proteins did not occur when they were all cotransfected together with IE2 (not shown). Similarly, even when all six core replication machinery proteins plus UL112-113 and all of the other auxiliary components including UL36-38, IRS1, IE2, UL84 and UL69 (but omitting IE1) were cotransfected together, UL44 was still very efficiently accumulated into POD-associated sites (Fig. 9e). Obviously, not all of these proteins are expected to be expressed in all cotransfected cells, but the results demonstrated that in the context of at least some of the other core replication machinery proteins, UL44 (and presumably the other replication fork complex proteins also) are recruited efficiently into POD-associated sites in the presence of UL112-113.

To evaluate whether all or only some of the core replication complex proteins are required for this altered localization pattern, UL44 and UL112-113 were cotransfected together with various subsets of the six core genes (not shown). The results revealed that although UL112-113 had a small effect on UL44 alone (12% of the doubly expressing cells showing punctate POD-associated UL44 patterns), the effect was greatly enhanced in the presence of either SSB (UL57) alone, polymerase (UL54) alone, or the three helicase-primase complex proteins together, with in each case more than 80% of the UL112-113- and UL44-coexpressing cells showing colocalization in the PODs. Although appropriate antibodies are not available for determination of whether only some or all of the other core replication proteins are similarly recruited to the PODs in infected or cotransfected cells, we conclude that UL112-113 plays an important role in viral DNA replication compartment formation at early stages during virus infection by at least recruiting the UL44 component of the viral DNA replication fork complex into the IE2-containing domains at the periphery of PODs.