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Our goal is to improve children's health by inspiring parents to become knowledgable partners who can work with their children's physicians in new and rich ways. The immediate-early 2 (IE2) protein of human herpesvirus 6 (HHV-6) is a potent transactivator of multiple cellular and viral promoters. Human herpesvirus 6 (HHV-6) is a betaherpesvirus initially isolated from human immunodeficiency virus (HIV)-infected individuals and from patients suffering from lymphoproliferative disorders (218).
It has been reported that the IE2 protein is a potent transcriptional activator of heterologous promoters (76, 96).
Transcriptional activators must possess at least two functional domains, a DNA binding domain that allows attachment of the transactivator to its target sequence within a gene promoter, and an activation domain that promotes the transcription of the target genes. Mapping studies have revealed that HCMV IE2 contains two distinct acidic activation domains, one at the N terminus and one at the C terminus (Figure 4.1B).
In the present work, we used deletion mutants to map functional transactivation domains of HHV-6A IE2 using complex and minimal promoter sequences. The HSB-2 and Molt-3 leukemia human T-cell lines were cultured in MegaCell RPMI-1640 medium supplemented with 10% foetal bovine serum (Sigma-Aldrich Canada, Oakville, ON) and M-plasmocin (InvivoGen, San Diego, CA) to prevent mycoplasma contamination. In order to map the transactivation domains of IE2, the following constructs were generated by single-base mutagenesis following the QuikChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA) instructions.
GA CAT TCG GAC GTA AAG-3a€™ and its complementary oligonucleotide (mutated base is italicized).
I digesting pcDNA4-IE2 into 3 fragments and ligating back together the two larger fragments (5854 and 2430 pb). Transfections of HEK293T were performed using the calcium phosphate precipitation procedures. Molt-3 cells were transfected by electroporation using a Gene Pulser apparatus and capacitance extender (Bio-Rad Laboratories, Hercules, CA).
For western blot analysis, cells were washed in PBS, lysed in an appropriate volume of Laemmli buffer and boiled. The main function currently known of HHV-6A IE2 is to promiscuously promote transcriptional activation (96). The various constructs encoding for IE2 deletion mutants were transfected into Molt-3 or HEK293T cells together with luciferase reporter plasmids. We first performed transfection of promoter constructs in Molt-3 T cells, a cell line efficiently infected by HHV-6.
On the other hand, deletion of the central R1 repeat region inhibited full transactivation of the reporters without completely abolishing the activity of IE2.
Interestingly, deletion of a short C-terminal stretch of 70 amino acids (1397-1466) drastically reduced IE2 activity on the whole array of reporter constructs, either minimal or complex.
The low transactivating activity of all IE2 deletion mutants could potentially be caused by poor expression or defective intracellular localization. Because IE2 needs to reach the nucleus in order to act as a transactivator, we had to confirm whether the inactive mutants were properly transported to the nucleus. We subsequently analyzed the intranuclear localization of the different IE2 mutants by immunofluorescence. Interestingly, the IE2 2-1396 C-terminal tail mutant has a visibly different punctate nuclear distribution. Since we have shown that IE2 is able to transactivate both its own promoter and the viral polymerase promoter, we sought to further characterize the consensus sequences required for efficient transactivation. The HHV-6A immediate-early locus A promoter has been tentatively identified as a sequence of circa 4 kbp between ORFs U90 and U94 (170).
The IE2 protein of HHV-6A is able to functionally transactivate diverse promoters (96, 170).
The significative transactivating activity of deletion mutants IE2 2-1131 and 2-1030 on the putative HHV-6A IE promoter was observed for this promoter only.
The sequence similarity between the C-terminal domains of HHV-6A IE2 and HCMV IE2 could entail similar functions for the two domains.
Interestingly, the 544-579 residues of HCMV IE2 have been defined as an acidic activator domain (204).
Another important functional difference between the IE2 proteins of HHV-6A and HCMV is here underscored. Our analysis of IE2 activation of multiple promoters failed to pinpoint specific transcription binding sequences unconditionally required for efficient transactivation.
Promoter transactivation by HHV-6A IE2 is possibly the result of several non-exclusive mechanisms of action.
Table 4.1 Summary of phenotypical and functional differences between HCMV, HHV-6A and HHV-6B.
This work was supported by grant 14437 from the Canadian Institutes of Health Research (CIHR) to Louis Flamand. One feature they all share in common is their ability to cause dormant infections that stay in infected children for the rest of their lives. Deletion mutants of HHV-6 variant A IE2 allowed us to map functional transactivation domains acting on complex and minimal promoter sequences. Links between HHV-6 infection and other pathologies such as meningo-encephalitis (124), organ transplant rejection (32, 268) and AIDS (143, 215) have also been suggested.
Moreover, cotransfection experiments in T cells indicated that IE2 variant A can induce the transcription of a complex promoter such as the one present in the HIV LTR, as well as simpler promoters, whose expression is driven by a unique set of responsive elements (CRE, NF-AT, NF-I?B) (69, 96). For HHV-6A IE2, the activation and DNA binding domains have not been defined yet, but clues pertaining to the nature of its functional domains could perhaps be deduced from studies of human cytomegalovirus (HCMV) immediate-early protein IE2. Both domains are required for transactivation of most target reporter genes within the context of wild-type IE2 (204). This mapping allowed us to determine that both the N-terminal and C-terminal domains of IE2 are required for efficient transactivation, and that deletion of the C-terminal (1397-1466) tail of IE2 drastically reduces both transactivation and the nuclear patchy distribution of IE2. HEK293T human epithelial kidney cell line was cultured in MegaCell Dulbeccoa€™s modified Eaglea€™s medium (Sigma-Aldrich) supplemented with 3% foetal bovine serum and M-plasmocin, and passaged every two days. Construct pHHV-6 IE was generated by PCR amplification of the putative IE-A promoter sequence from pR56 (kindly provided by Dr. Samples were electrophoresed through a SDS-polyacrylamide gel, transferred to polyvinylidene fluoride membranes, and blotted for 1 h at room temperature. IE2 expression was detected with a mouse anti-His monoclonal antibody (Amersham Biosciences), followed by a rabbit anti-mouse antibody coupled to the Alexa 568 red dye (Molecular Probes, Eugene, OR). IE2 being a large protein, we generated various deletion mutants in order to determine which domains are essential for transactivation.
Because promoter consensus sequences could have an influence on which IE2 domains are required for efficient transactivation, complex (multiple transcription factor binding sequences) and minimal (single known binding sequence) viral and cellular promoter reporters were tested.
This region is not capable of transactivation by itself, as we have shown that the C-terminal (945-1466) domain of IE2 is transcriptionally inactive.
In order to determine whether the IE2 mutants are properly expressed upon transfection in the HEK293T cell line, we analyzed their relative expression by western blotting using anti-His and anti-IE2 antibodies.
A computer search of nuclear localization sequences using PSORT II (185) has revealed four recognizable putative NLS in HHV-6A IE2. Transfected HEK293T cells were prepared for microscopy and probed using an anti-His antibody.

Unlike wild-type IE2, IE2 2-1396 does not form large patches but numerous small dot-like structures. Left panels represent GFP distribution (GFP), while second column panels show the same fields colored with DAPI nuclear stain (DAPI). HEK293T cells were transfected with pcDNA4-IE2 or deletion mutant constructs and processed for immunofluorescence as described under materials and methods. To better characterize the IE2 transactivation domains, we generated deletion mutants of various regions of the protein and tested them for activation of both complex and minimal promoter reporter constructs.
An explanation for this residual activity may be linked to the numerous repeats (28 copies) of AP2, NF-I?B and PEA3 binding sequences present within this promoter.
The IE2 1397-1466 region aligns with residues 486-568 of HCMVa€™s 579 amino acid IE2 protein (Figure 4.1B).
Indeed, although HCMV and HHV-6 share high levels of genetic identity, both viruses have found different ways to exploit similar genetic elements.
IE2 is able to transactivate via the TATA box and the basal transcriptional machinery, but can also activate promoters that are TATA-less (such as the HHV-6A polymerase promoter) through yet unidentified transcription factor binding sites.
Availability of a specific promoter is determined by chromatin remodeling, and transcription factors have access to only a fraction of the cellular promoters at any moment.
He appears frequently in the media including such venues as the The New York Times, the TODAY Show, Good Morning America, & the Dr. This mapping showed that both the N-terminal and C-terminal domains of IE2 are required for efficient transactivation, and that deletion of the C-terminal (1397-1466) tail of IE2 drastically reduces both transactivation and the intranuclear distribution of IE2. Evidence for a co-factorial role of HHV-6A in AIDS come from several observations, including that co-infection of T cells with HIV and HHV-6 leads to the activation of HIV long terminal repeat (LTR)-directed viral gene expression and accelerates cytopathic effects (162).
Finally, the C-terminal domain encompassing the final 436 residues of HHV-6A IE2 was shown to bind a DNA fragment containing the transcription initiation site, TATA box and upstream sequence of the putative IE-A promoter (199). HCMV like HHV-6 is a betaherpesvirus and shares limited amino acid sequence similarities, immunological cross-reactivity and overall gene organization with HHV-6 (157, 186, 266). The activator domains of HCMV IE2 do not seem to fall within the conserved region with HHV-6A IE2. Top drawing represents HHV-6A genome including left and right direct repeats (DRL and DRR) and immediate-early loci (IE-A and IE-B).
Cells were transfected with 1 I?g of reporter plasmid and up to 7 Aµg of expression vector per well, and brought to a total of 8 Aµg of DNA per well for each condition with the pcDNA4 control plasmid. After three 10-min washes with TBST, the blots were incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h. We arbitrarily divided IE2 into three major domains: the N-terminal region (amino acids 1-720), the R1 repeat region (amino acids 721-944), and the C-terminal region (amino acids 945-1466). Surprisingly, none of the IE2 mutant constructs had transactivating activity similar to wild-type IE2, suggesting that the full-length protein is required for efficient transactivation. In the light of these results, we further investigated the impact of this small deletion on IE2 activity. As shown in Figure 4.4, all of the mutants are detected just as strongly or better than wild-type IE2. Monopartite four-residue pattern sequences are present at 355-358, 1046-1051 and 1210-1215, including potential overlapping sequences.
Both GFP-IE2 NLS fusion proteins displayed intense and exclusively nuclear localization patterns, as opposed to the diffuse, cytoplasmic distribution of the control GFP protein (see supplementary material, Figure 4.5).
In HHV-6A infected cells, IE2 shows a punctate pattern that evolves into large nuclear patches at 24-48 hours post-infection (96).
Moreover, all of the mutants lacking the C-terminal (1397-1466) tail also lose the patchy distribution of the wild-type protein. Another important element of the promoter is the R3 region, composed of 25-30 copies of approximately 105 bp repeat units containing putative transcription factors AP2, NF-I?B and PEA3 binding sites. Left panels represent cells reacted with anti-His antibody (His-tagged IE2 in red) and second column panels show same fields colored with DAPI nuclear stain (DAPI in blue).
Both the N-terminal and the C-terminal domains of IE2 are required for efficient transactivation of the tested reporters. It is possible that these IE2 mutants exhibit basal transactivating potential on minimal promoters but when the number of regulatory elements is high, a cumulative effect can be detected.
The HHV-6A IE2 C-terminal tail includes a short motif that is highly conserved among all betaherpesviruses, namely residues 1397-1417 (486-506 for HCMV IE2) (45).
Mammalian one-hybrid tests with the C-terminal tail of HHV-6A IE2 did confirm that this protein fragment cannot act as a transcriptional activator (data not shown). Table 4.1 presents a summary of phenotypical and functional differences between HCMV and HHV-6 variants A and B.
Interestingly, we have demonstrated that upon HHV-6A infection, IE2 localizes in the vicinity of promyelocytic leukemia-associated nuclear bodies (PML-NB) (Tomoiu et al., unpublished results). We have previously characterized the IE1 variant B (95) and IE2 variant A (96) proteins translated from spliced transcripts of the IE-A locus. HCMV gene UL122, encoding for protein IE2, is a positional homologue of HHV-6 ORF U86 (190), and corresponds to the C-terminal portion of HHV-6 IE2.
However, minimal dimerization and DNA binding domains have been identified in the HCMV IE2 region having significant similarity with HHV-6 (45). This and future characterization of IE2 should provide a better understanding of this complex viral protein.
Lower drawing represents IE-A locus including repeat regions (R1-R3), orientation and approximate position of major ORFs (arrows) and coding exons of key transcripts (IE1, IE2 and U95). The blots were then washed with TBST and the proteins were visualized with enhanced chemiluminescence (Perkin-Elmer, Boston, MA) using a PhosphorImager system (Fuji Medical Systems, Stamford, CT).
We generated constructs lacking either one or more of the large domains, or smaller portions of the protein for a more detailed mapping (Figure 4.2A). Transfection efficiency being relatively low in T-cell lines, it was difficult to obtain consistent results with many of the reporters tested. On the whole, the results suggest that the complete IE2 protein is required for successful transactivation of all tested promoters in HEK293T cells. We have therefore identified at least two sequences from HHV-6A IE2 that act as functional NLS, and are most likely required for the proper nuclear targeting of IE2.
In transfected HEK293T cells, wild-type IE2 also shows a patchy nuclear pattern (Figure 4.6).
In contrast, the IE2A 714-1466 construct (lacking the N-terminal region but not the C-terminal tail) does show the patchy distribution of wild-type IE2.
For HHV-6B, it has been shown that the R3 repeats strongly enhance transcription of gene U95, downstream of the IE promoter and on the opposite side of R3 from the IE-A locus (241). Total lysates were analyzed by SDS-polyacrylamide gel electrophoresis and probed using anti-His or anti-IE2 antibodies. Localization of the IE2 protein versus nucleus is underscored in the merge panels, at 400A— total magnification. This, however, does not explain why similar results are not observed with the IE2 2-1396 and IE2 2-1289 mutants. This motif forms part of the required transactivation, autoregulation, dimerization and DNA binding domains of HCMV IE2 (45, 204). This suggests that other viral proteins are likely to contribute to the CD4 and HHV-6A polymerase transcriptional activation.

PML-NB have a role in chromatin dynamics and are thought to participate in the repression of gene expression, as reviewed in (71). The ability of IE2 to transactivate multiple promoters having distinct transcription factor responsive elements could suggest that the HHV-6A IE2 protein does not bind a specific DNA consensus sequence directly, but rather interacts with regulatory proteins common to many transcriptional units. These results contrast sharply to that of human cytomegalovirus (HCMV) IE2, which down-regulates its promoter. Furthermore, this study underscores important functional differences between HCMV and HHV-6A IE2 proteins. Transactivation was evaluated using luciferase reporter constructs, coding for the luciferase gene driven by various promoters.
The enhanced expression of most mutants relative to wild-type IE2 can be explained by the shorter length of the encoded transcripts and proteins that leads to increased transcriptional and translational efficiencies. IE2 mutants 2-1030 and 2-1131, lacking the C-terminal putative NLS at residues 1207-1215, have a diffuse distribution that appears to be mostly cytoplasmic, probably due to impaired nuclear shuttling of the mutant proteins. Because IE2A 714-1466 has very little transactivating activity, it appears that transactivation and patchy nuclear distribution of IE2 are not correlated. However, the R3 region has no significant effect on transactivation by HHV-6A IE1, a IE-A protein encoded downstream of R3 on the reverse strand (170).
Transactivation of the pLTR-Luc reporter was quantified by measurement of luciferase activity, expressed in fold transactivation relative to control (pcDNA-transfected cells). Transactivation of the reporters was quantified by measurement of luciferase activity, expressed in fold transactivation relative to control (pcDNA-transfected cells). The exact role of R1 in the transactivation of promoters by IE2 is as yet unclear, but its strong basic charge and unstructured sequence suggest the ability to non-specifically bind DNA and thus participate in the attachment of IE2 to the promoter binding sites. Perhaps differences in protein folding of the various mutants are responsible for the obtained results.
The conservation of this element in HHV-6A may indicate an important role in the IE2 protein activity. Upon HHV-6 infection the nuclear PML-NB distribution is altered (95), and a consequence of this alteration may be greater chromatin availability to viral transactivators such as IE2.
However, the existing evidence that the C-terminal domain of HHV-6A IE2 is able to bind the putative IE-A promoter (199) and that its homologue HCMV IE2 also has a DNA binding domain (45) suggests that HHV-6A IE2 acts as a transcriptional enhancer through DNA binding followed by the activation or recruitment of transcriptional unit complexes.
Our characterization of HHV-6 IE2 transactivating activity provides a better understanding of the complex interactions of this protein with the viral and cellular transcription machinery and highlights significant differences with the IE2 protein of HCMV. HCMV IE2 is a 86-kDa protein whose biological functions are well defined and include transactivation of heterologous promoters (205), repression of its own promoter (108), association with the viral DNA replication compartment (7), blocking of cell cycle progression (257) and modulation of apoptosis (275).
Martin) (170) using forward primer 5a€™-TTG ACT TAC CAG ACT GCA ACG-3a€™ and reverse primer 5a€™-TTC CTA CCC AAG CGG GTT AG-3a€™. Luciferase activity was measured on a MLX Microtiter plate luminometer (Dynex Technologies, Chantilly, VA).
Mutant IE2 2-1030 could not be detected by the P6H8 anti-IE2 mAb since this antibody binds to a region located between amino acids 1030-1131 (Tomoiu et al., unpublished results).
Unexpectedly, the IE2 I”R1 construct (lacking the R1 region) also shows a diffuse distribution even though it retains the C-terminal NLS.
IE2 itself was shown to weakly transactivate an IE-A promoter construct lacking the R3 repeats (96). Results are meansA±SD from 3 distinct experiments of duplicate transfections, and were normalized for protein content in each sample.
Results are means+SD from 3 distinct experiments of duplicate transfections, and were normalized for protein content in each sample. Alternatively, the R1 deletion may have an influence on global IE2 folding and mask NLS sequences, change its ability to bind promoter regions or recruit transcription factors.
It is of note that even if these mutants are less efficiently transported to the nucleus, as seen by immunofluorescence, because of a missing NLS, cell fractionation assays have shown that a significant fraction of these mutant proteins are still imported to the nucleus and should be available for transactivation (data not shown).
Accordingly, we hypothesize a role in dimerization and DNA binding for the C-terminal tail of HHV-6A IE2. More characterization of the functional domains of IE2 is required for a better understanding of the mechanisms underlying the complex relationship between the viral protein and its host transcriptional machinery. The values obtained are means of 3 distinct experiments performed in duplicate, and were normalized for protein concentration in each sample, as determined by a BCA colorimetric assay (Pierce, Rockford, IL).
The N-terminal (IE2 2-719) or C-terminal (IE2 945-1466) domains have no significant transactivating activity by themselves on any of the tested promoters. Therefore, expression levels cannot account for the lower transactivating activity of the IE2 mutants.
It would thus appear that both the C-terminal NLS and the R1 repeat domain are important for proper nuclear localization of IE2. Interestingly, since the Pol promoter is TATA-less (5), there must exist other unidentified binding elements that promote IE2 transactivation of the viral promoter.
We looked into the relevance of the R3 repeat region for IE2 transactivation by generating a pHHV-6 IE-Luc construct encoding for the full 4 kbp putative IE-A promoter upstream of the luciferase gene. IE2 expression and protein loading were assessed by western blotting using anti-His, anti-IE2 and anti-actin antibodies. The N-terminal NLS possibly plays a role in the import of mutants missing the C-terminal NLS. Indeed, it has been shown that the C-terminal domain of IE2 demonstrates some DNA binding activity (199).
Marc Pouliot for the use of his microscopy and imaging systems, Marie-Eve Janelle for cloning the GFP-IE2 423-440 NLS fusion construct, and Nathalie Cloutier for assisting in the cloning of the HHV-6A IE promoter reporter constructs. Likewise, mutants lacking either the N-terminal (IE2A 714-1466) or the C-terminal (IE2 2-1030) domains are unable to transactivate most reporter constructs.
However, the diminished transactivation activity of some mutant IE2 proteins such as IE2 2-1396 and IE2 2-1289 does not correlate with impaired nuclear import, as these retain the C-terminal NLS and their localization pattern is still nuclear.
Transactivation of the pHHV-6 Pol-Luc reporter was quantified by measurement of luciferase activity, expressed in fold transactivation relative to control (pcDNA-transfected cells). Cells were transfected and processed for luciferase activity measurement as described above. The nuclear distribution of wild-type IE2 suggests that there is some form of nuclear aggregation or polymerization of the protein that is not present with truncated IE2 2-1396.
The only exception to this is the HHV-6A IE promoter who is significantly transactivated by the IE2 2-1131 and 2-1030 mutants. Figure 4.7B shows that full length IE promoter is much more efficiently transactivated by IE2 than the promoter lacking the R3 region.
However, an unambiguous proof that the punctate nuclear pattern of IE2 is linked to its ability to bind DNA or to form protein dimers remains to be produced.
This suggests that the AP2, NF-I?B and PEA3 d PEA3 binding sites may be important for IE2 transactivation of what likely constitutes its own promoter.

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