Human herpesvirus 6 (HHV-6) alters E2F1/Rb pathwaysand utilizes the E2F1 transcription factor to expressviral genesEyal Sharon1, Ludmila Volchek1, and Niza Frenkel2

Department of Cell Research and Immunology and the S. Daniel Abraham Institute for Molecular Virology, Tel Aviv University, Tel Aviv 69978, Israel

Edited* by Bernard Roizman, The University of Chicago, Chicago, IL, and approved November 13, 2013 (received for review May 10, 2013)

E2F transcription factors play pivotal roles in controlling theexpression of genes involved in cell-cycle progression. Differentviruses affect E2F1/retinoblastoma (Rb) interactions by diversemechanisms releasing E2F1 from its suppressor Rb, enabling viralreplication. We show that in T cells infected with human herpes-virus 6A (HHV-6A), the E2F1 protein and its cofactor DP1 increased,whereas the Rb protein underwent massive degradation withouthyperphosphorylation at three sites known to control E2F/Rbassociation. Although E2F1 and DP1 increased without Rb sup-pression, the E2F1 target genes—including cyclin A, cyclin E, anddihydrofolate reductase—were not up-regulated. To test whetherthe E2F1/DP1 complexes were used for viral transcription, wescanned the viral genome for genes containing the E2F bindingsite in their promoters. In the present work, we concentrated onthe U27 and U79 genes known to act in viral DNA synthesis. Weconstructed amplicon-6 vectors containing a GFP reporter genedriven by WT viral promoter or by promoter mutated in the E2Fbinding site. We found that the expression of the fusion U27 pro-moter was dependent on the presence of the E2F binding site. Testof the WT U79 promoter yielded >10-fold higher expression of theGFP reporter gene than the mutant U79 promoter with abrogatedE2F binding site. Moreover, by using siRNA to E2F1, we found thatE2F1 was essential for the activity of the U79 promoter. Thesefindings revealed a unique pathway in HHV-6 replication: The viruscauses Rb degradation and uses the increased E2F1 and DP1 fac-tors to transcribe viral genes.

Human herpesvirus 6A (HHV-6A) and HHV-6B infect >90%of children by the age of 2 y (1). Recent studies have found

that ∼1% of children are born with chromosomally integratedHHV-6, suggesting that the virus is vertically transmitted ina Mendelian manner (2). HHV-6B is the causative agent of ro-seola infantum, a brief febrile infection with skin rash (3). Ina minority of patients, there are neurological complications upto lethal encephalitis (4). After productive infection, HHV-6Benters into latency from which it can be reactivated—e.g., fol-lowing bone marrow and hematopoietic stem cell trans-plantations. Viral reactivation can result in delayed transplantengraftment and severe complications up to lethal encephalitis(5). Furthermore, transplantation of solid organs—includingkidney, liver, lung, and heart—results in high rates of HHV-6reactivation, although only 1% of transplant recipients werefound to develop severe complications (6). There is no acutedisease known to be caused by HHV-6A, but recent studies havesuggested potential involvement in multiple sclerosis (MS) ag-gravation (7). HHV-6 was found more often in MS plaques thanin MS normal-appearing white matter or non-MS brains. HHV-6reactivation has been reported during MS clinical relapses (7).Recent evidence (8) has suggested the association of HHV-6A with Hashimoto thyroiditis, the most common of all thyroiddiseases.E2F1 acts as a transcription factor of genes involved in cell-

cycle progression, DNA replication, DNA repair and apoptosis(9, 10). The heterodimerization of E2F1 and its cofactor DP1 isessential for binding to promoters that carry the E2F binding site

(10). E2F1 activity is regulated by the tumor suppressor retino-blastoma (Rb) protein. The binding of hypophosphorylated Rbto E2F1 leads to inhibition of E2F1 transcription activity andcell-growth arrest. In the G1 phase, Rb protein is inactivatedfollowing its phosphorylation by cyclin D/CDK-4/6 and cyclinE/CDK-2 complexes, resulting in its dissociation from E2F1/DP1heterodimer and cellular entry into the S phase (11, 12). In mid-late S phase, the cyclin A/CDK-2 complex phosphorylates E2F1/DP1 complex, reducing their DNA binding capacity (10, 11). TheE2F1/Rb interactions were found to be targeted by differentviruses, including the adenovirus oncoprotein E1A, the papillo-mavirus E7 protein, and the SV40 large T antigen (13, 14). Theseviral proteins disassemble the E2F1/Rb complexes, resultingin the release of E2F1. A number of human herpesvirusesalso target the Rb protein (15). It has been shown that humancytomegalovirus (HCMV) tegument protein pp71 induces Rbdegradation (16) and that HCMV kinase protein UL97 phos-phorylates Rb protein (17). We have shown (18) that the in-fection of SupT1 T cells with HHV-6A was associated with cell-cycle arrest at the G2/M phase. Such arrest might be advanta-geous for viral replication and the production of the typicalHHV-6 cytopathic effect, consisting of multiple fused infectedcells. To test the mechanism(s) by which HHV-6A manipulates thecell cycle, we analyzed alterations of the E2F1/Rb pathway knownto be a checkpoint in the control of cell-cycle progression. Wedescribe a unique strategy used by HHV-6A inducing the degra-dation of Rb, so as to exploit the released E2F1 transcription

Significance

Human herpesvirus 6A (HHV-6A) and HHV-6B are prevalentviruses causing mild up to grave lethal diseases. Viruses mod-ulate cell-cycle progression so as to use cellular replicationmachinery. The retinoblastoma (Rb) tumor suppressor proteinrepresses the E2F1 transcription factor. This induces entryto the S phase, enabling efficient replication. Viruses employdifferent mechanisms modulating the Rb–E2F1 pathway.However, the mechanism(s) used by HHV-6 are unknown. Wehave found that HHV-6A infection induced Rb degradationwhile producing elevated levels of active E2F1 and DP1. Sur-prisingly, several cellular E2F target genes were not up-regu-lated, whereas active E2F1 was used to transcribe selected viralgenes containing the E2F binding site in their promoters. Theseviral genes play significant roles in viral DNA synthesis.

Author contributions: E.S., L.V., and N.F. designed research; E.S. and L.V. performed re-search; E.S., L.V., and N.F. analyzed data; and E.S., L.V., and N.F. wrote the paper.

The authors declare no conflict of interest.

*This Direct Submission article had a prearranged editor.

Freely available online through the PNAS open access option.1E.S. and L.V. contributed equally to this work.2To whom correspondence should be addressed. E-mail: nfrenkel@post.tau.ac.il.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1308854110/-/DCSupplemental.

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factor to transcribe selected cellular genes as well as viral genescontaining E2F binding site in their promoters.

ResultsHHV-6A Infection Leads to Rb Degradation. During lytic infection,herpesviruses synthesize viral DNA by using viral or cellularreagents required for nucleotide metabolism and DNA replica-tion. Cellular enzymes are encoded by E2F1-target genes. Thesegenes are negatively regulated by Rb protein, a major target forthe intervention of herpesviruses in the cell cycle (15). Disrup-tion of the E2F1/Rb complexes by phosphorylation and/or Rbdegradation can induce entry into the S phase, enhancing viralreplication. There are 13 Ser/Thr sites potentially phosphory-lated in Rb during G1/S transition. Accumulation of multiplephosphorylated Rb residues can result in the disruption of E2F1/Rb complexes (19). We tested Rb phosphorylation at residuesSer-780, Ser-807, and Thr-821 because they represent consensussites known to be phosphorylated during E2F1/Rb disassembly.SupT1 T cells were mock infected or infected with two tissueculture infectious dosage for infecting 50% of the cells (TCID50)per cell. Protein samples were prepared from the cytoplasmicand nuclear fractions at 24, 48, and 72 h postinfection (h.p.i.).Blots were reacted with antibodies to total Rb; phosphorylatedRb at amino acids S780, S807/811, T821; and the eIF2α, asloading control. The progression of infection was tested by ac-cumulation of the P41 protein reactive with 9A5 monoclonalantibody (20, 21).The results revealed (Fig. 1 A and B) that in the infected cells,

the Rb levels were significantly decreased (P ≤ 0.001). Rb deg-radation was found at 48 and 72 h.p.i., as seen in the cytoplasmicand nuclear fractions. Tests of Rb phosphorylation at three sitesrevealed no significant alterations in the phosphorylation level ofthese residues (Fig. 1 A and C). Thus, HHV-6A infection led todramatic Rb degradation without Rb phosphorylation.

The E2F1 and Its Cofactor DP1 Are Elevated During HHV-6A Infection.Heterodimers of E2F and DP family members are used in geneexpression of E2F target genes. This complex reduces the re-pression by the Rb protein family members (22). The release ofE2F1 induces its own expression as well as its cofactor DP1. Totest whether Rb degradation induced E2F1 and DP1 expression,nuclear protein lysates from mock-infected cells or infected cellswere prepared (Fig. 2) and were analyzed in Western blots, byusing the E2F1 and DP1 antibodies. The eIF2α antibody servedas loading control. Whereas in the mock infection the E2F1 levelwas low and steady, there was a significant increase in E2F1expression, starting from 48 until 96 h.p.i. A similar increase wasfound in the DP1 protein expression. The coordinated increasein E2F1 and DP1 proteins coincided with Rb degradation andE2F1 release.

In HHV-6A Infection, There Was No Induction of Expression of E2F1Target Genes.E2F1 in association with DP1 function as sequence-specific transcriptional activators of genes involved in cell pro-liferation (10). Because HHV-6A infection induced elevation offree E2F1, we tested the expression of the target genes cyclin A,cyclin E, and Dihydrofolate reductase (DHFR). Protein lysates ofmock-infected or HHV-6A–infected cells were prepared at 48and 96 h.p.i. Test with antibodies to E2F1, cyclin A, cyclin E,DHFR, and eIF2α (Fig. 3) revealed the following: (i) There wasincreased E2F1 protein, confirming the earlier results; (ii) therewas no up-regulation of cyclin A and DHFR; and (iii) cyclin Ewas found to be decreased at 96 h.p.i. Thus, during HHV-6Ainfection, there was no up-regulation of genes involved in S-phaseprogression, regardless of the induction of E2F1.

Viral Transcription Was Affected by the E2F Binding Site. Because theE2F1-responsive genes cyclin A, cyclin E, and DHFR were not

up-regulated, it was of interest to test whether the free E2F1 wasused to transcribe viral genes. We scanned the HHV-6A genomefor the consensus E2F binding site TTTSSCGC, where S is eithera G or a C. Several viral genes were found to have promoterswith E2F binding site, including the following: (i) U18, a tran-scription regulatory gene, part of the IE-B/E genes; (ii) U33,a viral tegument protein, found to be a critical mediator ofmetabolic stress; (iii) U52, which promotes the accumulation oflate transcripts, E gene; and (iv) U74, part of the helicase/pri-mase complex, E gene. In the present work, we have focused on

Fig. 1. Regulation of Rb and phosphorylated Rb during the HHV-6A in-fection. (A) Western blots of cytoplasmic (Left) and nuclear (Right) lysates ofmock-infected and HHV-6A–infected SupT1 T cells tested with antibodies toRb; phosphorylated Rb at amino acids S780, S807, and T821; HHV-6 P41; andeIF2α as a loading control. (B) Graphic representation of the Rb/eIF2a resultsrelative to the highest level of Rb/eIF2α in the cytoplasmic (Left) and nuclear(Right) fractions. Results are shown as mean ± SD, based on three in-dependent experiments for the cytoplasmic and nuclear fractions. The dif-ferences in Rb levels in the mock-infected vs. HHV-6–infected cells werestatistically significant as determined by t tests. ***P ≤ 0.001. (C) Graphicrepresentation of the phosphorylation levels of pRbS780/Rb, pRbS807/Rb,and pRbT821/Rb in the cytoplasmic (Left) and nuclear (Right) fractions. Ineach case, the results are calculated based on mean ± SD from three in-dependent experiments. The results are displayed relative to the highestlevel of phosphorylated Rb in the nuclear fraction. **P ≤ 0.01.

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the U27 and the U79 genes, both functioning in viral DNAsynthesis. The U27 gene encodes the P41 viral DNA polymeraseprocessivity factor (23). The U79–80 early gene encodes a familyof nuclear proteins possessing common amino acid termini and isgenerated by different alternative splicing. They were found tobe essential for viral DNA replication (24) and colocalize withcomponents of the viral DNA replication machinery.To test whether E2F1 is used in the expression of U27 and

U79 genes, we placed the promoters as well as site-specificmutations of the E2F binding site within the amplicon-6-GFPvector (pNF1194) containing the viral oriLyt, the pac-1–pac-2packaging signals, and the transgene GFP driven by the HCMVpromoter (25). The primer sequences are listed in Table S1. Wereplaced the HCMV promoter with the U27 or the U79 pro-moters containing the WT E2F binding site TTTGCCGC, as wellas a mutant sequence TGTGGATC. To monitor transfectionefficiency, RFP driven by the HCMV promoter was also placedin the vectors. The new plasmids (Fig. S1) were designated asamplicon-6-U27-promoter-GFP-RFP; amplicon-6-mutant-U27-pro-moter-GFP-RFP; amplicon-6-U79-promoter-GFP-RFP; and ampli-con-6-mutant-U79-promoter-GFP-RFP. Parallel 2 × 106 SupT1 T-cell

cultures were transfected with the amplicon plasmids. At 24 h aftertransfection, thecellsweremonitored forGFP and redfluorescentprotein (RFP) fluorescence (Fig. 4). Aliquots of the cultures wereused to prepare protein lysates, whereas additional aliquots weresuperinfected with 2 TCID50 per cell of HHV-6A. At 66 h post-superinfection, fluorescence images were obtained and proteinlysatesweretested inWesternblots,byusingantibodies toGFP,RFP,andeIF2αprotein.Theresultshaveshownthefollowing: (i)TheRFPexpression was detected in all cultures, demonstrating the successfultransfection (Fig. 4 A–D); (ii) GFP expression was not detected incultures that were solely transfected with the U27 WT or mutantpromoters (Fig. 4A); (iii) superinfection with HHV-6A led to GFPexpression in cultures that were transfected with the WT promoter,but not in cultures receiving the mutant U27 promoter (Fig. 4 A, B,and E); (iv) GFP fluorescence was observed in the cultureswhichwere transfectedwithU79WTormutantpromoters (Fig.4C);in cultures that received the WT U79 promoter, there was 10-foldhigherGFP expression than in the cultures that received themutantpromoter (Fig. 4F); and (v) superinfection with HHV-6A led toa stronger expression of GFP in cultures transfected with WT U79promoter (Fig. 4D,H, and I). Moreover, there was ∼17-fold higherGFP expression in the cultures that were transfected with theWTpromoter compared with cultures transfected with the mutantpromoter (Fig. 4I). All together, these results reveal a previouslyundescribed role of E2F binding site in the regulation of HHV-6gene expression.

The E2F1 Transcription Factor Regulates Viral Transcription as Shownby siRNA and Overexpression Treatments. The involvement of theE2F1 transcription factor in viral transcription was tested byaltering the E2F1 levels in the cells using siRNA as well as E2F1overexpression. Parallel six-well cultures of 293T cultures weretransfected with the following: amplicon-6-U27-promoter-GFP-RFP (U27 WT), amplicon-6-mutant-U27-promoter-GFP-RFP(U27 mutant), amplicon-6-U79-promoter-GFP-RFP (U79 WT),and amplicon-6-U79 mutant-promoter-GFP-RFP (U79 mutant).Each culture was transfected with 2 μg of the promoter plasmidsolely or cotransfected with one of following: 10 nM negativecontrol siRNA, 10 nM siRNA to E2F1, or 2 μg of E2F1 over-expression plasmid. At 48 h.p.i., the cells were extracted, and theE2F1 and γ-tubulin were measured. The expression of GFP wasnormalized to RFP expression to correct for transfection effi-ciency, as well as to γ-tubulin protein to correct for gel proteinloading. The experiments were repeated three times. The resultsshowed the following: (i) siRNA against E2F1 led to >50% re-duction in the E2F1 (Fig. 5 A, lanes 2, 6, 10, and 14; and B–E)compared with the siRNA control; (ii) the decreased E2F1 causeda mild decrease in the activity of U27 WT promoter and sharpdecrease in the activity of U79WT promoter (Fig. 5 A, lanes 2 and10; B; andD); (iii) the promoters of U27 and U79 mutants showedno significant difference in their activity (Fig. 5 A, lanes 6 and 14;C; and E); (iv) moreover, the mutant promoters showed veryweak GFP expression in comparison with the WT promoters;

Fig. 2. E2F1 and DP1 expression during HHV-6A infection. (A) Western blotsof nuclear protein lysates reacted with antibodies to E2F1 and eIF2α. (B)Graphic representation of the results relative to the highest level of E2F1/eIF2α in the nuclear fraction. (C) Western blots of nuclear protein lysatesreacted with antibodies to DP1 and eIF2α. Two exposures of the blots (lowand high) are shown for the DP1 protein. (D) Graphic representation of theresults relative to the highest level of DP1/eIF2α in the nuclear fraction.Results are indicated as mean ± SD based on three independent experi-ments. *P ≤ 0.05; **P ≤ 0.01.

Fig. 3. Expression of E2F1 and E2F1 target genes. (A) Western blots of proteins of mock-infected and HHV-6A–infected cell lysates reacted with antibodies toE2F1 cyclin E, cyclin A, DHFR, and eIF2α. (B–E) Graphic representation of the results compared with the highest level of protein/eIF2α. The results are presented asmean ± SD based on four independent experiments for E2F1 and the E2F1 target genes. E2F1 increased significantly at 48 and 96 h.p.i. *P ≤ 0.05; **P ≤ 0.01.

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and (v) overexpression of E2F1 induced >10-fold GFP expression(P < 0.05) by using the WT promoters (Fig. 5 A, lanes 4 and 12; F;and H). However, this effect was barely visible, with only mildincrease, using the U27 mutant promoter (Fig. 5 A, lane 4; andG).The GFP expression of the U79 mutant promoter could not bedetected (Fig. 5 A, lane 16; and I).

DiscussionIn analyzing the ability of HHV-6A to modulate the E2F/Rbpathway, we found massive degradation of the Rb protein and nophosphorylation of residues Ser-780, Ser-807, or Thr-821. Inuninfected cells in late G1 phase the heterodimers of E2F1 andDP1 proteins were reported to be up-regulated, stimulating

Fig. 4. Viral promoters are regulated by the E2F binding site. (A–D) GFP and RFP fluorescence at 24 h after transfection (A and C) and 66 h after super-infection (B and D) with HHV-6A. Transfections were done with amplicon-6-U27-promoter-GFP-RFP (U27 promoter), amplicon-6-mutant-U27-promoter-GFP-RFP (mutant U27 promoter), amplicon-6-U79-promoter-GFP-RFP (U79 promoter), and amplicon-6-mutant-U79-promoter-GFP-RFP (mutant U79 promoter). At24 h after transfection and at 66 h after superinfection, RFP expression was viewed in the microscope to estimate efficiency of transfection. GFP expressionwas viewed in the microscope to estimate promoter strength. (E) Western blots of cultures that were transfected with U27 WT or mutant promoters werefollowed by 66 h after superinfection with HHV-6A. The blot was tested by using GFP, RPF, and eIF2a antibodies. GFP expression at 24 h after transfection withamplicon-6-U27 WT or mutant promoters was not detected. The results are based on three experiments. (F and G) Western blots of cultures that weretransfected with the WT U79 promoter or the mutant U79 promoter for 24 h (F) as well as these cultures following HHV-6A superinfected for 66 h (G). (H and I)Graphic representation of the GFP expression in cells transfected with the WT or mutant U79 promoters at 24 h after transfection (H) or 66 h after superinfection (I).GFP accumulation was divided by RFP accumulation to correct for transfection efficiency and by eIF2α as a loading control. Results are shown as mean ± SD based onthree experiments.

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E2F1-dependent transcription and entry into the S phase (26).Both HHV-6A and -6B infections were reported to induce cell-cycle arrest at the G2/M phase during the first 48 h.p.i. (18, 27).As shown here in HHV-6A infection of SupT1 T cells, there waspronounced up-regulation of E2F1 and DP1 proteins by 48 h.p.i.Increased E2F1 expression was also described by using a micro-array assay of HHV-6B–infected adult T-cell leukemia cell line(28). Although E2F1 and DP1 levels were elevated, we foundthat cyclin A and DHFR were not up-regulated, and cyclin Edecreased at 96 h.p.i. Our results differ from the results of DeBolle et al., who found that late post–HHV-6A infection ofhuman cord blood mononuclear cells there was increased accu-mulation of cyclin A without up-regulation of cyclin E (27).Hence, the expression of E2F1 target genes during HHV-6Ainfection might vary depending on the cells or tissues used.To test the potential utilization of E2F1 in the transcription of

viral genes, we scanned the viral genome for E2F binding sites.We chose the U27 early (β) gene encoding the p41 DNA poly-merase processivity factor and the U79 immediate early (α) genealso active in viral DNA synthesis. DNA polymerase processivityfactors were found to be essential for the replication of severalherpesviruses. The HSV-1 mutant of UL42 DNA polymeraseprocessivity factor was found to be defective in viral replication(29, 30). The HCMV UL44 gene encoding the ICP36 proc-essivity factor is homologous to the HSV-1 UL42 gene and to theHHV-6 U27. By its association with viral DNA polymerase, itstimulates viral replication (31). We now show that a GFP re-porter gene is expressed if it is linked to the WT U27 promoterwith an intact E2F binding site. A three-nucleotide mutation in

the E2F1 binding site abolished the activation of the promoter.However, no expression of GFP was noted in cells transfectedwith WT U27 promoter in absence of superinfecting virus, in-dicating potential participation of additional viral protein(s) inmediating activation of the U27 promoter. Moreover, whereasthe treatment of the cells with siRNA to E2F1 resulted in milddecrease in the promoter activity, the overexpression of E2F1led to a substantial increase of the promoter activity. This resultindicates that other E2F factors may play a role in the inductionof U27 promoter.The mRNA transcripts of the U79 gene were found to encode

immediate early nuclear proteins playing a role in viral DNAsynthesis (24). We have found a strong activity of the U79 pro-moter even in the absence of additional viral factors. This activitydepended on the intactness of the E2F binding site becausemutation of the site led to 90% reduced activity. The involvementof E2F1 in U79 promoter activity was found by using siRNA andoverexpression of E2F1. This finding indicates that E2F1 is amajor factor in U79 expression.The involvement of E2F1 in transcription of the U27 and the

U79, β, and α genes, respectively, is a previously undescribedoutlook of the regulation of HHV-6 gene expression. It is ofinterest for several reasons: First, ∼30% of cellular genes containpromoters with E2F1 binding site. Analyses of ∼24,000 pro-moters revealed that >20% of the promoters were bound byE2F1. These results place the E2F1 as a factor that contributesto the regulation of a large fraction of human genes (32). Sec-ond, a functional distinction between the various E2F1 bindingsites could be caused by their association with different protein

Fig. 5. E2F1 is involved in viral transcription. The 293T cells were transfected with viral promoters including U27 WT, U27 mutant, U79 WT, and the U79mutant. The cells were treated several folds: siRNA control, siRNA to E2F1, and E2F1 overexpression vector. The cellular proteins were prepared from thecultures at 48 h.p.i. (A) Western blots of cultures that were reacted with antibodies to GFP, RFP, E2F1, and γ-tubulin. (B–E) Graphic representation of the GFPexpression in cells transfected with the viral promoters U27 WT (B), U27 mutant (C), U79 WT (D), and the U79 mutant (E) and cotransfected with siRNA controlor siRNA to E2F1. GFP expression (promoter activity) was divided by RFP expression to correct for the efficiency of transfection and by γ-tubulin as a loadingcontrol. (F–I) Graphic representation of the E2F1 expression in cells transfected with viral promoters U27 WT (F), U27 mutant (G), U79 WT (H), and the U79mutant (I) or cotransfected with E2F1 expression vector (O.E). E2F1 expression was divided by γ-tubulin as a loading control. Results are shown as mean ± SDbased on three experiments. *P ≤ 0.05; **P ≤ 0.01.

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complexes (33). The cellular E2F1 target genes were found to bemanipulated during the infection: Whereas E2F1 and DP1 werespecifically induced, there was no up-regulation of cyclin A,cyclin E, and DHFR expression. Hence, a possible mechanismfor the utilization of E2F1 transcription factor in viral replicationmight involve viral protein(s) recruiting the E2F1 and directing it toviral promoter(s) or selected cellular promoters. Examples for suchprocesses include the E2 adenovirus promoter that is activated by anE2F1/E4 complex (34). In addition, the bovine herpes virus 1 in-fection leads to increased E2F1 protein levels, and the activity of thebovine ICP0 early promoter is increased dramatically by E2F1 (35).

Materials and MethodsCells and Viruses. SupT1 CD4+ human T cells were obtained from the NationalInstitutes of Health AIDS Research Repository. The SupT1 Cells were cultured inRPMI 1640, 10% (vol/vol) FCS, and gentamycin (25 μg/mL). The 293T cells werepropagated in DMEM, 10% (vol/vol) FCS, and gentamycin (25 μg/mL). The HHV-6A (U1102) was obtained from Robert Honess (National Institute for Medical Re-search, London). Virus stocks were prepared from virus secreted into the mediumand concentrated by centrifugation at 65,000 × g for 2.15 h (4 °C). Titers weredetermined by using the endpoint assay, estimating the TCID50 per milliliter.

Plasmids. The details on plasmids are provided in SI Materials and Methods.

Transfection. A total of 2 × 106 cells were transfected with 13 μg of the testplasmid, by using microporator MP1000 (Bio Digital). The efficiency of trans-fection was determined by RFP expression. The 293T cells were transfected bycalcium phosphate.

siRNA Transfection. The siRNA duplexes were inserted by the calcium phos-phate method. The negative control duplex corresponds to a scrambledsequence that is absent in the human genome. The duplexes were purchasedfrom IDT (TriFECTa Dicer-Substrate RNAi kit).

Fluorescence and Light Microscopy. Fluorescence microscopy was performedby using the Nikon Eclipse TE200-S microscope with ACT-1 software.

Western Blotting. Protein extracts were prepared as described (18). The anti-bodies used in Western blotting are described in SI Materials and Methods.

ACKNOWLEDGMENTS. We thank Dr. Bala Chandran (Rosalind FranklinUniversity) for the gift of the 9A5 antibody recognizing the HHV-6 P41protein. The E2E1 expression plasmid was kindly gifted by Dr. Yoel Kloog(Tel Aviv University). We thank the National Institutes of Health AIDSResearch and Reference Reagent Program (Division of AIDS) for the SupT1Tcells from James Howxie. This work was supported by the Israel ScienceFoundation; the S. Daniel Abraham Institute for Molecular Virology; andthe S. Daniel Abraham Chair for Molecular Virology and Gene Therapy, TelAviv University.

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