Gene mutational analysis of the binding and dispersion of ND10 by IE1
The ability to disperse ND10 is one of the properties of IE1 (Ahn & Hayward, 1997
; Ishov et al., 1997
; Kelly et al., 1995
; Korioth et al., 1996
; Tang & Maul, 2003
). To determine the IE1 motif required for this property, several IE1 deletion mutants were produced (, left; Supplementary Methods). Since the mAbs used to identify IE1 recognize an epitope between amino acids 430 and 530, some mutants with deletions in this area were detected using a GFP tag at the amino terminus of all constructs. A Western blot was performed to confirm the expression of mutant IE1 products after transfection into NIH/3T3 cells. After stripping, the same membrane was probed first with anti-IE1 antibody (top) and then with anti-GFP antibody (bottom). The products of the IE1 mutants were always seen as multiple bands, regardless of which antibody was used as a probe (, right). It is not clear whether these products arose from differential splicing or were post-translational effects.
Fig. 1. (Left) Schematic outline of the IE1 deletion mutants produced. (Middle) Different activities tested. Numbers in the ‘dispersion’ column indicate whether the respective mutants dispersed ND10 after transfection; numbers in the ‘binding’ (more ...)
We then analysed the ability of the mutants to bind to and disperse ND10 (, centre, which summarizes the data from 50 analysed cells). We transfected each mutant IE1 plasmid into NIH/3T3 cells overnight. After fixation and permeabilization, cells were stained for ND10 by using anti-PML antibody. shows that wild-type (wt) IE1 colocalizes with ND10 when less IE1 is produced (, rightmost cell; seen in 10/50 cells) and disperses ND10 (left cell) when IE1 is present at higher concentrations (seen in 40/50 cells). The fact that ND10 was dispersed in all cells that were transfected with wt IE1 for 72 h (not shown) confirmed our notion that the 20
% of cells where IE1 co-localized with ND10 had lower levels of IE1 expression. Deleting exon 2 (dl1–36, ) and nearly 200 aa at the carboxy terminus (dl400–595, ) had no effect on the normal co-localization and dispersion of ND10.
Fig. 2. Immunohistochemical analysis of ND10-dispersion capabilities of IE1 deletion mutants in transfected NIH/3T3 cells. Cells were stained for the GFP-tagged IE1 deletion mutants (green) and PML (red) as an indicator of ND10 integrity. (a) Cells transfected (more ...)
Any deletions made between aa 37–400, except the deletion of aa 135–141, resulted in the loss of both binding and dispersion capabilities (). Most deletions appeared to affect both binding and dispersion. Interestingly, the small 7 aa histone 2B homology-region deletion mutant (pgfpie1_dl135–141, ) retained ND10 binding ability [as shown in the nucleus on the right top of , the lower part of the nucleus is magnified in the boxes to show the co-localization of the IE1 dots and ND10], but did not, even when highly overexpressed, disperse this structure. These findings suggest that two domains, aa 37–99 and aa 350–400, determine the ND10 binding ability of IE1, and the domain affected by the 7 aa deletion (aa 135–141) determined the dispersion of ND10. Also, the binding of IE1 to PML (or intermediaries) seems necessary for the dispersion of ND10, but it is not sufficient for this dispersion.
Pinpointing the IE1 motif within the H2B homology region that is essential for ND10 dispersion
Previous studies of IE1 function by our and other groups have shown its activities of binding to non-specific DNA, in vitro
interaction with histones, interaction with ND10 proteins and dispersion of ND10, and transactivation of cellular genes (such as the thymidylate synthase and ribonucleotide reductase genes) involved in dNTP biosynthesis (Gribaudo et al., 2000
; Lembo et al., 2000
; Münch et al., 1988
; Tang & Maul, 2003
; Wilhelmi et al., 2008
). None of these functions have been mapped to the H2B homology region of IE1. Here we show the importance of the H2B homology region of IE1 for dispersing ND10 (). We further confirmed that IE1 with deletions in the H2B homology region also lost the ability to disperse other ND10 components, such as SP100 (), Daxx (—h), and ATRX (—l).
Fig. 3. Immunohistochemical analysis of ND10 component dispersion capabilities of IE1 with deletion of H2B homology domain (aa 135–141) in transfected NIH/3T3 cells. (a–d) Effect of IE1dl135–141 on SP100; (e–h) effect of IE1dl135–141 (more ...)
Since the H2B homology region contains 7 aa (NDIFERI), we used deletion mutations to further map out the minimal region required for dispersing ND10 (, and Supplementary Table S1, available in JGV Online). We made a series of deletion mutants by using overlapping PCR ( and Supplementary Table S1), and immunofluorescence assays (IFA) were performed to analyse the effects of these smaller deletion mutations on ND10 dispersal. As can be seen in , pgfpie1_dl137–139 lost the ability to disperse ND10, whereas the dual deletion mutations pgfpie1_dl137–138 and pgfpie1_dl138–139 did not. Therefore, the minimal region that is required for dispersing ND10 was mapped to aa 137–139 (IFE).
Fig. 4. Determination of the minimal motif in the H2B homology region required for IE1 to disperse ND10 in transfected NIH/3T3 cells. (a) The H2B homology domain is shown with nucleotide sequence numbers 135–141 (top); nucleotides and their encoded amino (more ...)
Mutagenesis within the H2B homology region of IE1
Next, we were curious as to whether a single amino acid deletion or a point mutation could cause IE1 to lose its ability to disperse ND10. We first utilized an overlapping PCR method by designing internal primers, in which the desired single deletion or point mutation was included, to construct different plasmids, as shown as in . After transfecting the plasmids into NIH/3T3 cells overnight, the cells were fixed and an IFA was performed. We found that all of the single amino acid mutants of IE1 could still disperse ND10 (results summarized on the right of ).
Interaction with PML, but not dispersion of ND10, is important for the activation of the MCMV MIEP by IE1
Previously, we found that IE1 can interact with PML and other ND10 components (Tang & Maul, 2003
). To determine the domains that are important for this interaction with PML, we transfected our mutated IE1-expressing plasmids (including wt, dl1–36, dl135–141, dl95–153, dl137–139 and dl310–424) into NIH/3T3 cells. Twenty-four hours post-transfection, nuclear extracts were made, and anti-PML and anti-GFP antibodies were employed for co-immunoprecipitation (co-IP) assays. The expression of IE1 (or its mutants) and PML was detected by Western blot, as shown in . As can be seen in , PML interacts with the mutants of dl1–36, dl135–141 and dl137–139, but not dl95–153 or dl310–424; PML is able to pull down wt IE1 and the three longer mutants (), and conversely, only the three longer mutants are able to pull down PML (). Antibodies against GFP and PML were made from mouse, so mouse IgG (mIgG) was used as control (), showing that neither PML nor IE1 was pulled down by mIgG beads. Therefore, elements in regions 195–153 and 310–424 are important for interacting with PML, either by direct binding or via some intermediary protein(s).
Fig. 5. (a–d) Interaction of PML with different IE1 deletion mutants. NIH/3T3 cells were transfected with different IE1 deletion mutant-expressing plasmids, and nuclear extracts were prepared. (a) Five per cent of the input cell extract probed with anti-GFP (more ...)
Next, we tested whether these mutants of IE1 retain their ability to activate MIEP. We co-transfected the NIH/3T3 cells with the firefly (Photinus)-luciferase reporter plasmid (in which the luciferase expression is directed by the MCMV MIEP and pgfpie1 (wt) or its mutants, including dl1–36, dl135–141, dl95–153, dl137–139 and dl310–424. pGFP was used as a control against possible GFP-induced activation (, lane 1). As shown in , wt IE1 and all other mutants that are able to interact with PML can activate MIEP. However, the IE1 mutants that lost the ability to interact with PML failed to activate the promoter. The results suggest that the interaction of IE1 with PML, but not the dispersion of ND10, is important for the activating function of IE1.
Generation of MCMV mutants with IFE-deleted IE1
To determine whether the disruption of ND10 by IE1 could be important for viral replication, we generated mutant MCMV [by using MCMV in which IE3 was tagged with GFP at the carboxy terminus (Martinez et al., 2010
)] with IFE-deleted IE1 and its revertant by using the galK
counter-selection BAC system (Martinez et al., 2010
). MCMVdlIFE, MCMVIFERQ, MCMVdlIE1 and MCMVIE1RQ were prepared as described in Methods.
MCMVdlIFE fails to disperse ND10, and IFE-deleted IE1 is attracted to IE3 domains
As can be seen in , IFE-deleted IE1 did not disperse the ND10 of NIH/3T3 cells. To test whether a whole virus with the same deletion would also be unable to disperse ND10, we infected NIH/3T3 with MCMVdlIFE at an m.o.i. of 0.5 for 24 h, and ND10s were visualized by IFA using anti-PML antibody. As shown in , ND10s were not dispersed, and PML tended to be attracted to IE3 domains (), which have previously been demonstrated to be pre-replication compartments (Martinez et al., 2010
). The revertant virus (MCMVIFERQ) was consistent with the wt MCMV infection in mouse cells (), as ND10s were dispersed (). Thus, we found that for MCMV to disperse ND10, the H2B homology region (minimized to IFE) of IE1 is necessary.
Fig. 6. IFA to determine effects of MCMVdlIFE on ND10 in NIH/3T3 cells, and the distribution of IE1 and IE3. NIH/3T3 cells seeded on coverslips were infected with MCMVdlIFE (a)–(d) or revertant MCMVdlIFERQ (i)–(l) and (m)–(p) for 24 h. (more ...)
IFE-deleted IE1 produced from MCMVdlIFE-infected cells was detected by anti-IE1 antibody (). Interestingly, IE1 with deleted IFE colocalized with IE3 in MCMVdlIFE-infected cells, as shown in . This is in stark contrast to wt MCMV infection in NIH/3T3 cells or the revertant (). Whether the ability to disperse ND10 is related to the colocalization of IE3 with IE1 needs to be explored further.
To determine the physical relationship of IE3 with IE1dlIFE and PML over the course of infection, we performed IFA to show the distributions of IE3, IE1dlIFE, and PML throughout the time course of infection. NIH/3T3 cells were infected with MCMVdlIFE at an m.o.i. of 0.5 and fixed at the indicated times post-infection (p.i.) (Supplementary Figures S1 and S2, available in JGV Online). As can be seen, IE3 diffused in the nucleus at an early time of infection [Supplementary Figs S1(A2) and S2(A2)], while IE1dlIFE also diffused in the nucleus [Supplementary Fig. S1(A1)]. At the early time of infection [4 h p.i.; Supplementary Fig. S1(A1–A4)], E1dlIFE diffused in all the nuclei of the IE1dlIFE positively expressed cells. No punctate dots were seen, which is different from what was seen in pIEdlIFE-transfected cells. Later, at 8 h p.i., IE3 formed small dots in the nucleus [bottom of Supplementary Fig. S1(B2), and top of Supplementary Fig. S2(B2)]. IE3 formed domains in the nucleus when the infection time was extended, as shown in Supplementary Fig. S1 C1–C4 (16 h p.i.) and D1–D4 (24 h p.i.). IE1 distributed as both diffusion and dots that co-localize with IE3 during the entire time of infection (Supplementary Fig. S1, B1–B4, C1–C4, and D1–D4). When IE3 formed small dots at 4 h p.i., it distributed side by side with ND10 (Supplementary Fig. S2(B1–B4)]. At a later time of infection, when IE3 formed domains in the nucleus, ND10 appeared to be partially attracted to IE3 domains. However, ND10 remained intact throughout the time course of infection.
Dispersing ND10 by IE1 is not important for MCMV gene expression or viral replication
The biological function of ND10 in relation to viral replication has been controversial. The observations that major components such as PML, Daxx and Sp100 have inhibitory effects on viral gene expression and replication support the notion that ND10s are defensive foci (Everett & Chelbi-Alix, 2007
; Everett et al., 2006
; Ling et al., 2005
; Tavalai et al., 2008
), but the fact that DNA viruses replicate DNA and transcribe important viral RNA at ND10 argues that ND10 might favour viral replication (Everett et al., 2004
; Tang et al., 2000
). If the dispersion of ND10 has positive effects on viral gene expression and viral replication, then MCMVdlIFE infection in NIH/3T3 cells should have significant defects in both viral gene expression and viral replication. We performed Western blot and p.f.u. assays to test these assumptions.
First, we infected MCMVdlIFE and its revertant (MCMVdlIFERQ) into NIH/3T3 cells at an m.o.i. of 0.5; the total-cell-lysate samples were collected at different time points p.i. (mock infected, 6, 12, 24, 48 and 72 h p.i., as shown in ), and then different viral products were detected by Western blot. The following proteins were detected: viral IE proteins, including IE1 and IE3; early proteins, including E1 (also called m112/113) and M44; and M25, containing three products (one is an early protein and the other two are late proteins). Tubulin was used as a sample loading control. Thus, no significant defect in viral gene production was observed between the infections of MCMVdlIFE and its revertant.
Fig. 7. Viral gene expression and replication. (a) NIH/3T3 cells were mock infected (lane 1 in each column) or infected with MCMVdlIFE or rescued revertant MCMdlIFERQ for 6 h (lane 2), 12 h (lane 3), 24 h (lane 4), 48 h (lane 5) and 72 h (lane 6) at an m.o.i. (more ...)
We then performed p.f.u. assays to detect viral replication. NIH/3T3 cells seeded in 24-well plates were infected with either MCMVdlIFE or wt MCMV at an m.o.i. of either 0.01 () or 0.1 (). Revertants were also infected as controls. The infected cells were collected (together with medium) at the number of days p.i. indicated in (x-axis). No significant differences were observed between MCMVdlIFE and MCMVdlIFERQ or wt MCMV regarding viral growth in cell culture. Therefore, we conclude that the dispersion of ND10 might not be important for the replication of MCMV in mouse cells. However, it is not clear whether the growth defect resulting from the failure of ND10 dispersal in MCMVdlIFE infection might be compensated for by that of the IE1dlIFE co-localized with IE3. In addition, it still remains to be determined whether the dispersion of ND10 might affect viral replication in vivo.