Human Cytomegalovirus IE2 Research Articles

Human Cytomegalovirus IE1-72 Activates Ataxia Telangiectasia Mutated Kinase and a p53/p21-Mediated Growth Arrest Response

Human cytomegalovirus (HCMV) encodes several proteins that can modulate components of the cell cycle machinery. The UL123 gene product, IE1-72, binds the Rb-related, p107 protein and relieves its repression of E2F-responsive promoters; however, it is unable to induce quiescent cells to enter S phase in wild-type (p53(+/+)) cells. IE1-72 also induces p53 accumulation through an unknown mechanism. We present here evidence suggesting that IE1-72 may activate the p53 pathway by increasing the levels of p19(Arf) and by inducing the phosphorylation of p53 at Ser15. Phosphorylation of this residue by IE1-72 expression alone or HCMV infection is found to be dependent on the ataxia-telangiectasia mutated kinase. IE2-86 expression leads to p53 phosphorylation and may contribute to this phenotype in HCMV-infected cells. We also found that IE1-72 promotes p53 nuclear accumulation by abrogating p53 nuclear shuttling. These events result in the stimulation of p53 activity, leading to a p53- and p21-dependent inhibition of cell cycle progression from G(1) to S phase in cells transiently expressing IE1-72. Thus, like many of the small DNA tumor viruses, the first protein expressed upon HCMV infection activates a p53 response by the host cell.

Downregulation of GFAP, TSP‐1, and p53 in human glioblastoma cell line, U373MG, by IE1 protein from human cytomegalovirus

Human cytomegalovirus (HCMV) is a member of the beta-herpesvirus family, which has tropism for glial cells. It was recently reported that HCMV might play important roles in the pathogenesis of malignant glioma. In this study, we investigated the effects of the HCMV IE1 protein on the gene expression profile in the human glioblastoma cell line, U373MG by employing cDNA microarray technology. Using DNA chips containing approximately 1,000 human cDNAs, RNA samples from U373MG cells stably expressing IE1 were compared with those from the control cells lacking IE1 cDNA. Fluorescence intensities of 13 genes were significantly decreased in IE1-expressing cells, while one gene was found to be upregulated. Among these 14 genes, we chose to work further on glial fibrillary acidic protein (GFAP), thrombospondin-1 (TSP-1), and p53, because of their previously known involvement in tumorigenesis. The mRNA levels of all these genes were found to be decreased in IE1-expressing glioblastoma cells by real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) as well as Northern blot analysis. The decreased expression of these genes was also observed at protein levels as measured by immunocytochemistry or fluorescence-activated cell sorting (FACS) analysis. Our data strongly suggested that HCMV IE1 could modulate the expression of cellular genes that might play important roles in the pathogenesis of glial tumors.

Inhibition of Cell Division by the Human Cytomegalovirus IE86 Protein: Role of the p53 Pathway or Cyclin-Dependent Kinase 1/Cyclin B1

The human cytomegalovirus (HCMV) IE86 protein induces the human fibroblast cell cycle from G(0)/G(1) to G(1)/S, where cell cycle progression stops. Cells with a wild-type, mutated, or null p53 or cells with null p21 protein were transduced with replication-deficient adenoviruses expressing HCMV IE86 protein or cellular p53 or p21. Even though S-phase genes were activated in a p53 wild-type cell, IE86 protein also induced phospho-Ser(15) p53 and p21 independent of p14ARF but dependent on ATM kinase. These cells did not enter the S phase. In human p53 mutant, p53 null, or p21 null cells, IE86 protein did not up-regulate p21, cellular DNA synthesis was not inhibited, but cell division was inhibited. Cells accumulated in the G(2)/M phase, and there was increased cyclin-dependent kinase 1/cyclin B1 activity. Although the HCMV IE86 protein increases cellular E2F activity, it also blocks cell division in both p53(+/+) and p53(-/-) cells.

The Carboxyl-Terminal Region of Human Cytomegalovirus IE1491aaContains an Acidic Domain That Plays a Regulatory Role and a Chromatin-Tethering Domain That Is Dispensable during Viral Replication

The human cytomegalovirus major immediate-early (alpha) protein IE1(491aa) plays an important role in controlling viral gene expression at low multiplicities of infection. With a transient complementation assay, full-length IE1(491aa) enhanced the growth of ie1 mutant virus CR208 20-fold better than a deletion mutant lacking 71 carboxyl-terminal amino acids (IE1(1-420aa)). A 16-amino-acid domain between amino acids 476 and 491 was both necessary and sufficient for chromatin-tethering activity; however, this domain was completely dispensable for complementation of CR208 replication. The proximal 55-amino-acid acidic domain (amino acids 421 to 475) was found to be most important for function. A deletion mutant lacking only this domain retained chromatin-tethering activity but failed to complement mutant virus. Interestingly, serine phosphorylation (at amino acids 399, 402, 406, 423, 428, 431, 448, 451, and 455) was not required for complementation. These results show that IE1(491aa) is composed of at least two domains that support replication, a region located between amino acids 1 and 399 that complements ie1 mutant virus replication to low levels and an acidic domain between amino acids 421 and 479 that dramatically enhances complementation.

The putative zinc finger of the human cytomegalovirus IE2 86-kilodalton protein is dispensable for DNA binding and autorepression, thereby demarcating a concise core domain in the C terminus of the protein.

The IE2 86-kDa gene product is an essential regulatory protein of human cytomegalovirus (HCMV) with several functions, including transactivation, negative autoregulation, and cell cycle regulation. In order to understand the physiological significance of each of the IE2 functions, discriminating mutants of IE2 are required that can be tested in a viral background. However, no such mutants of IE2 are available, possibly reflecting structural peculiarities of the large and ill-defined C-terminal domain of IE2. Here, we revisited the C-terminal domain by analyzing IE2 mutants for transactivation, DNA binding, autoregulation, and cell cycle regulation in parallel. We found it to contain an unexpectedly concise core domain (amino acids 450 to 544) that is defined by its absolute sensitivity to any kind of mutation. In contrast, the region adjacent to the core (amino acids 290 to 449) generally tolerates mutations much better. Although it contributes more specific sequence information to distinct IE2 activities, none of the mutations analyzed abolished any particular function. The core is demarcated from the adjacent region by the putative zinc finger region (amino acids 428 to 452). Surprisingly, the deletion of the putative zinc finger region from IE2 revealed that this region is entirely dispensable for any of the IE2 functions tested here in transfection assays. Our work supports the view that the 100 amino acids of the core domain hold the key to most functions of IE2. A systematic, high-density mutational analysis of this region may identify informative mutants discriminating between various IE2 functions that can then be tested in a viral background.

Ability of the human cytomegalovirus IE1 protein to modulate sumoylation of PML correlates with its functional activities in transcriptional regulation and infectivity in cultured fibroblast cells.

In one of the earliest events in human cytomegalovirus (HCMV)-infected cells, the major immediate-early (IE) protein IE1 initially targets to and then disrupts the nuclear structures known as PML oncogenic domains (PODs) or nuclear domain 10. Recent studies have suggested that modification of PML by SUMO is essential to form PODs and that IE1 both binds to PML and may disrupt PODs by preventing or removing SUMO adducts on PML. In this study, we showed that in contrast to herpes simplex virus type 1 (HSV-1) IE110 (ICP0), the loss of sumoylated forms of PML by cotransfected IE1 was resistant to the proteasome inhibitor MG132 and that IE1 did not reduce the level of unmodified PML. Reduced sumoylation of PML was also observed in U373 cells after infection with wild-type HCMV and proved to require IE1 protein expression. Mutational analysis revealed that the central hydrophobic domain of IE1, including Leu174, is required for both PML binding and loss of PML sumoylation and confirmed that all IE1 mutants tested that were deficient in these functions also failed both to target to PODs and to disrupt PODs. These same mutants were also inactive in several reporter gene transactivation assays and in inhibition of PML-mediated repression. Importantly, a viral DNA genome containing an IE1 gene with a deletion [IE1(Delta290-320)] that was defective in these activities was not infectious when transfected into permissive fibroblast cells, but the mutant IE1(K450R), which is defective in IE1 sumoylation, remained infectious. Our mutational analysis strengthens the idea that interference by IE1 with both the sumoylation of PML and its repressor activity requires a physical interaction with PML that also leads to disruption of PODs. These activities of IE1 also correlate with several unusual transcriptional transactivation functions of IE1 and may be requirements for efficient initiation of the lytic cycle in vivo.

HCMV IE2-mediated inhibition of HAT activity downregulates p53 function.

Targeting of cellular histone acetyltransferases (HATs) by viral proteins is important in the development of virus-associated diseases. The immediate-early 2 protein (IE2) of human cytomegalovirus (HCMV) binds to the tumor suppressor, p53, and inactivates its functions by unknown mechanisms. Here, we show that IE2 binds to the HAT domain of the p53 coactivators, p300 and CREB-binding protein (CBP), and blocks their acetyltransferase activity on both histones and p53. The minimal HAT inactivation region on IE2 involves the N-terminal 98 amino acids. The in vivo DNA binding of p53 and local histone acetylation on p53-dependent promoters are all reduced by IE2, but not by mutant IE2 proteins that lack the HAT inhibition region. Furthermore, the p53 acetylation site mutant, K320/373/382R, retains both DNA binding and promoter transactivation activity in vivo and these effects are repressed by IE2 as well. Together with the finding that only wild-type IE2 exerts an antiapoptotic effect, our results suggest that HCMV IE2 downregulates p53-dependent gene activation by inhibiting p300/CBP-mediated local histone acetylation and that IE2 may have oncogenic activity.

Human Cytomegalovirus-Induced Upregulation of Intercellular Cell Adhesion Molecule-1 on Villous Syncytiotrophoblasts

Human cytomegalovirus (HCMV) is secreted apically from villous trophoblasts, thus congenital infection is not likely to occur by basal release across the basement membrane. As an alternative route, we hypothesize that an HCMV-infected villous syncytiotrophoblast (ST) upregulates intercellular adhesion molecule (ICAM)-1, causing blood monocytes to bind to the ST and induce apoptosis. Purified (>99.99%) populations of human villous trophoblasts were differentiated into an ST-like culture, infected with HCMV strain AD169, and assessed for ICAM-1 expression by immunofluorescence. Infection strongly upregulated ICAM-1 24 h after challenge. ICAM-1 was also stimulated by transfection with viral genes IE2-55, IE1-72, and IE2-86, but not by UV-inactivated virus. Infection with a green fluorescent protein recombinant virus allowed infection and ICAM-1 expression to be topographically located. We found that ICAM-1 was expressed on both infected and noninfected cells. Furthermore, antibody to tumor necrosis factor (TNF)alpha and, to a lesser extent, interleukin (IL)1 beta inhibited ICAM-1 upregulation on noninfected cells but not on infected cells. We conclude that HCMV IE proteins stimulate ICAM-1 expression on villous trophoblasts by paracrine release of TNF alpha and IL1 beta, as well as by a direct effect on infected cells.

Small internal deletions in the human cytomegalovirus IE2 gene result in nonviable recombinant viruses with differential defects in viral gene expression.

The human cytomegalovirus (HCMV) IE2 86-kDa protein is a key viral transactivator and an important regulator of HCMV infections. We used the HCMV genome cloned as a bacterial artificial chromosome (BAC) to construct four HCMV mutants with disruptions in regions of IE2 86 that are predicted to be important for its transactivation and autoregulatory functions. Three of these mutants have mutations that remove amino acids 356 to 359, 427 to 435, and 505 to 511, which disrupts a region of IE2 86 implicated in the activation of HCMV early promoters, a predicted zinc finger domain, and a putative helix-loop-helix motif, respectively, while the fourth carries three arginine-to-alanine substitution mutations in the region of amino acids 356 to 359. The resulting recombinant viruses are not viable, and by using quantitative real-time reverse transcription-PCR and immunofluorescence we have determined the location of the block in their replicative cycles. The IE2 86 Delta 356-359 mutant is able to support early gene expression, as indicated by the presence of UL112-113 transcripts and UL112-113 and UL44 proteins in cells transfected with the mutant BAC. This mutant does not express late genes and behaves nearly indistinguishably from the IE2 86R356/7/9A substitution mutant. Both exhibit detectable upregulation of major immediate-early transcripts at early times. The IE2 86 Delta 427-435 and IE2 86 Delta 505-511 recombinant viruses do not activate the early genes examined and are defective in repression of the major immediate-early promoter. These two mutants also induce the expression of selected delayed early (UL89) and late genes at early times in the infection. We conclude that these three regions of IE2 86 are necessary for productive infections and for differential control of downstream viral gene expression.

RNase P ribozyme as an antiviral agent against human cytomegalovirus.

Human cytomegalovirus (HCMV) represents one of the most medically important human viruses and causes a wide spectrum of human diseases, including birth defects and mental retardation in newborns, common opportunistic infections in acquired immunodeficiency syndrome (AIDS) patients (e.g., CMV-associated retinitis and pneumonia), and possibly cardiovascular diseases such as atherosclerosis. This chapter describes the utilization of RNase P ribozyme-specifically, M1GS ribozyme, as a gene-targeting agent for blocking HCMV gene expression and growth. The target for the RNase P ribozyme is the overlapping region of the mRNAs that code for HCMV major transcription factors IE1 and IE2, which are essential for viral gene expression and replication. The methods described in this chapter focus primarily on i) construction of the retroviral vector for expression of M1GS ribozymes in cultured cells, ii) generation of stable cell lines expressing ribozymes, iii) determination of the expression of M1GS RNAs in human cells, and iv) evaluation of the efficacy of ribozymes in inhibiting HCMV IE1/IE2 expression and viral growth. Using these methods, we successfully constructed M1GS RNAs against the IE1/IE2 mRNA sequence and recently showed that a reduction of up to 150- to 3000-fold in HCMV growth is found in cells that express the ribozymes.

RICK activates a NF-kappaB-dependent anti-human cytomegalovirus response.

The adapter kinase receptor interacting protein-like interacting caspase-like apoptosis regulatory protein kinase (RICK, also called RIP2 and CARDIAK) was found to be elevated at both the protein and RNA levels during human cytomegalovirus (HCMV) replication, suggesting either that the virus may require RICK for replication or that RICK is part of an unsuccessful host attempt to inhibit HCMV replication. It is demonstrated here that forced expression of RICK in either a kinase active or inactive form activates nuclear factor (NF)-kappaB by means of its intermediate domain and potently blocks HCMV replication in human fibroblasts. Importantly, NF-kappaB activation, which exerted a modestly positive effect on the early phase of infection, clearly had a strongly negative impact during later viral steps. A stable inhibitor of NF-kappaB (IkappaB) reverses the RICK inhibitory effect, and activation of NF-kappaB by IkappaB kinase beta expression is inhibitory to HCMV, demonstrating that NF-kappaB activation is part of a potent anti-HCMV response. Supernatant transfer experiments identified interferon-beta as a downstream component of the RICK inhibitory pathway. RICK expression was found to synergize with HCMV infection in the induction of interferon-beta expression. This study identifies an endogenous RICK-activated, NF-kappaB- and interferon-beta-dependent antiviral pathway that is either inhibited or faulty under normal HCMV replication conditions; efforts to bolster this pathway may lead to novel anti-viral approaches.

PIAS1 enhances SUMO-1 modification and the transactivation activity of the major immediate–early IE2 protein of human cytomegalovirus

The protein inhibitor of activated STAT1 (PIAS1), known to be a small ubiquitin-like modifier (SUMO) E3 ligase, was found to interact with the human cytomegalovirus IE2 protein. We found that the sumoylation of IE2 was markedly enhanced by wild-type PIAS1 but not by a mutant containing a Cys to Ser substitution at position 351 (C351S) within the RING finger-like domain. In target reporter gene assays, wild-type PIAS1, but not the C351S mutant, enhanced the IE2-mediated transactivations of viral polymerase promoter and cellular cyclin E promoter and this augmentation required the intact sumoylation sites of IE2. Our results suggest that PIAS1 acts as a SUMO E3 ligase toward IE2 and that it may regulate the transactivation function of IE2. To our knowledge, IE2 is the first viral target found to be regulated by a SUMO E3 ligase.

Interactions between human cytomegalovirus IE1-72 and cellular p107: functional domains and mechanisms of up-regulation of cyclin E/cdk2 kinase activity.

Previous work has demonstrated that the human cytomegalovirus IE1-72 protein is able to bind to the N terminus of p107, and IE1-72 alone is sufficient for alleviation of p107-mediated cell growth suppression. However, the mechanism of this alleviation is unclear. Here, we show that IE1-72 can alleviate p107 inhibition of cyclin E/cdk2 kinase activity. We cotransfected various IE1-72 and p107 constructs into C33A cells and demonstrated that IE1-72 could activate the kinase activity of cyclin E/cdk2. Conversely, IE2-86 did not activate this activity, suggesting that the interaction between p107 and IE1-72 and the subsequent kinase activation are specific. By the use of a series of deletion and point mutants of IE1-72 and p107, we observed that a mutation of the loop region of helix-loop-helix-turn-helix in exon 3 of IE1-72 as well as a mutation of the leucine zipper-2 region in exon 4 of IE1-72 abolished binding to p107. In addition, these two IE1-72 mutants did not alleviate p107 inhibition of cyclin E/cdk2 kinase activity and also failed to alleviate p107 inhibition of the E2F-responsive promoter. Meanwhile, deletion of the N-terminal aa 1 to 175 of p107 abolished both p107 binding with IE1-72 and p107 inhibition of cyclin E/cdk2 kinase activity. This result confirms that the N-terminus aa 1 to 175 region of p107 is a common region where both IE1-72 protein and cyclin E/cdk2 bind. We propose a mechanism in which binding of IE1-72 to p107 displaces cyclin E/cdk2 from p107. Once released from p107, cyclin E/cdk2 is able to function as an active kinase.

A CTL epitope from human cytomegalovirus IE1 defined by combining prediction of HLA binding and proteasomal processing is the target of dominant immune responses in patients after allogeneic stem cell transplantation

Objectives and methods In an attempt to define HCMV IE1-derived, HLA-A ∗0201-restricted epitopes, an advanced computer-based epitope prediction combining HLA binding and proteasomal cleavages in silico was performed. Results This prediction algorithm clearly confirmed VLEETSVML to be the most likely CTL epitope. By tetramer staining, HCMV pp65 NLVPMVATV-specific CD8 + T cells were detectable in 18/24 HCMV seropositive HLA-A ∗0201-expressing individuals (median frequency 0.58%; range 0.1%–4.7%), and IE1 VLEETSVML-specific CD8 + T cells in 5/24 (median frequency 2.1%; range 0.1%–4.3%), respectively ( p<0.01). Also in recipients of an allogeneic SCT, VLEETSVML- and NLVPMVATV-specific CD8 + T cells were detectable in comparable frequencies, but again the number of patients with detectable pp65-specific CD8 + T cells was higher ( p = 0.014). In 4/15 individuals, all demonstrating IE1 VLEETSVML-specific CD8 + T cells prior to peptide stimulation, VLEETSVML-specific T cell lines (purity of 42.6%–98.6% of all CD3 +/CD8 + T cells) were successfully generated after 2–4 weeks of culture using the IFN-γ secretion assay. Conclusion In conclusion, this novel prediction strategy efficiently predicted an immunodominant viral T-cell epitope.

Interactions between Human Cytomegalovirus IE1-72 and Cellular p107: Functional Domains and Mechanisms of Up-Regulation of Cyclin E/cdk2 Kinase Activity

Interactions between Human Cytomegalovirus IE1-72 and Cellular p107: Functional Domains and Mechanisms of Up-Regulation of Cyclin E/cdk2 Kinase Activity

Human cytomegalovirus IE2 protein interacts with transcription activating factors

The human cytomegalovirus (HCMV) IE86 cDNA was cloned into pGEX-2T and fusion protein GST-IE86 was expressed inE. coli. SDS-PAGE and Western blot assay indicated that fusion protein GST-IE86 with molecular weight of 92 ku is soluble in the supernatant of cell lysate. Protein GST and fusion protein GST-IE86 were purified by affinity chromatography. The technology of co-separation and specific affinity chromatography was used to study the interactions of HCMV IE86 protein with some transcriptional regulatory proteins and transcriptional factors. The results indicated that IE86 interacts separately with transcriptional factor TFIIB and promoter DNA binding transcription trans-activating factors SP1, AP1 and AP2 to form a heterogenous protein complex. These transcriptional trans-activating factors, transcriptional factor and IE86 protein were adsorbed and retained in the affinity chromatography simultaneously. But IE86 protein could not interact with NF-kappaB, suggesting that the function of IE86 protein that can interact with transcriptional factor and transcriptional trans-activating factors has no relevance to protein glycosylation. IE86 protein probably has two domains responsible for binding transcriptional trans-activating regulatory proteins and transcriptional factors respectively, thus activating the transcription of many genes. The interactions accelerated the assembly of the transcriptional initiation complexes.

Cell cycle arrest by human cytomegalovirus 86-kDa IE2 protein resembles premature senescence.

Primary human embryo lung fibroblasts and adult diploid fibroblasts infected by the human cytomegalovirus (HCMV) display beta-galactosidase (beta-Gal) activity at neutral pH (senescence-associated beta-Gal [SA-beta-Gal] activity) and overexpression of the plasminogen activator inhibitor type 1 (PAI-1) gene, two widely recognized markers of the process designated premature cell senescence. This activity is higher when cells are serum starved for 48 h before infection, a process that speeds and facilitates HCMV infection but that is insufficient by itself to induce senescence. Fibroblasts infected by HCMV do not incorporate bromodeoxyuridine, a prerequisite for the formal definition of senescence. At the molecular level, cells infected by HCMV, beside the accumulation of large amounts of the cell cycle regulators p53 and pRb, the latter in its hyperphosphorylated form, display a strong induction of the cyclin-dependent kinase inhibitor (cdki) p16(INK4a), a direct effector of the senescence phenotype in fibroblasts, and a decrease of the cdki p21(CIP1/WAF). Finally, a replicative senescence state in the early phases of infection significantly increased the number of cells permissive to virus infection and enhanced HCMV replication. HCMV infection assays carried out in the presence of phosphonoformic acid, which inhibits the virus DNA polymerase and the expression of downstream genes, indicated that immediate-early and/or early (alpha) genes are sufficient for the induction of SA-beta-Gal activity. When baculovirus vectors expressing HCMV IE1-72 or IE2-86 proteins were inoculated into fibroblasts, the increase of p16(INK4a) (observed predominantly with IE2-86) was similar to that observed with the whole virus, as was the induction of SA-beta-Gal activity, suggesting that the viral IE2 gene leads infected cells into senescence. Altogether our results demonstrate for the first time that HCMV, after arresting the cell cycle and inhibiting apoptosis, triggers the cellular senescence program, probably through the p16(INK4a) and p53 pathways.

Human Cytomegalovirus Immediate-Early Protein IE2–86, but not IE1–72, Causes Graft Coronary Arteriopathy in the Transplanted Rat Heart

Graft coronary arteriopathy (GCA) after heart transplantation is a major factor limiting the long-term survival of the recipients. Human cytomegalovirus (HCMV) infection is a possible cause of this disease which is characterized by diffuse intimal thickening resulting from smooth muscle cell migration and proliferation. It has been reported that HCMV immediate-early (IE) proteins, IE1 and IE2, could play an important role in the development of this disease; however, the precise in vivo role of these proteins in causing GCA has not been clarified. Excised Lewis rat hearts were transfected with HCMV IE1-72, IE2-86 or control plasmid by intra-coronary infusion of Hemagglutinating Virus of Japan-liposome, and transplanted into syngeneic recipients' abdomens. All cardiac grafts continued to beat well throughout the incubation period in the absence of immunosuppression. Exclusive expression of IE1-72 or IE2-86 protein in coronary artery walls was demonstrated after IE1-72 or IE2-86 gene transfection, respectively. Luminal occlusion as a consequence of intimal thickening of graft coronary arteries developed in the IE2-86 transfected hearts at day 21 after transplantation (30.1+/-3.4% occlusion, P<0.0001), compared with the IE1-72 and control transfected ones (8.2+/-1.6 and 6.8+/-1.1%, respectively). In contrast, there was no significant difference in luminal occlusion between the IE1-72 and control transfected hearts. We have demonstrated that expression of IE2-86 alone, but not IE1-72, causes intimal hyperplasia after cardiac transplantation. IE2-86 protein may therefore prove to be a useful target in therapies aimed at preventing HCMV-related GCA and improving the long-term result of cardiac transplantation.

Identification and characterization of the gene products of open reading frame U86/87 of human herpesvirus 6

The human herpesvirus 6 (HHV-6) immediate early-A locus (IE-A) locates in the position analogous to the human cytomegalovirus (HCMV) major IE (MIE) locus that is well-known to play critical roles in viral infection. Similarly to HCMV MIE, HHV-6 IE-A consists of two genetic units, IE1 and IE2, corresponding to open reading frames U90-U89 and U90-U86/87, respectively. However, the HHV-6 IE-A locus exhibits limited sequence homology with the HCMV MIE locus. In this study, to characterize HHV-6 IE2 gene products, polyclonal antibodies against four domains of the U86/87 open reading frame were generated by immunization of rabbits with bacterially-expressed proteins. Three polypeptides derived from the U86/87 region with apparent molecular masses of 100, 85 and 55 kD were detected in HHV-6-infected cells 3 days after infection, while IE1 polypeptides with apparent molecular mass greater than 170 kD were detectable as early as 8 h. Mapping of the IE2 gene products with the antibodies suggests differential splicing and alternative translation initiation in the IE2 genetic unit. The IE2 products show a mixed cytoplasmic and nuclear localization pattern. In addition, the 437 amino acid carboxyl-terminus domain bound to a DNA fragment containing the putative IE-A promoter. These results suggest that HHV-6 IE2 plays a critical role in transcriptional regulation and viral growth as does HCMV IE2, although it is likely that HHV-6 IE2 has expression kinetics different from HCMV IE2.

Open reading frame UL26 of human cytomegalovirus encodes a novel tegument protein that contains a strong transcriptional activation domain.

A selection strategy, the activator trap, was used in order to identify genes of human cytomegalovirus (HCMV) that encode strong transcriptional activation domains in mammalian cells. This approach is based on the isolation of activation domains from a GAL4 fusion library by means of selective plasmid replication, which is mediated in transfected cells by a GAL4-inducible T antigen gene. With this screening strategy, we were able to isolate two types of plasmids encoding transactivating fusion proteins from a library of random HCMV DNA inserts. One plasmid contained the exon 3 of the HCMV IE-1/2 gene region, which has previously been identified as a strong transcriptional activation domain. In the second type of plasmid, the open reading frame (ORF) UL26 of HCMV was fused to the GAL4 DNA-binding domain. By quantitative RNA mapping using S1 nuclease analysis, we were able to classify UL26 as a strong enhancer-type activation domain with no apparent homology to characterized transcriptional activators. Western blot analysis with a specific polyclonal antibody raised against a prokaryotic UL26 fusion protein revealed that two protein isoforms of 21 and 27 kDa are derived from the UL26 ORF in both infected and transfected cells. Both protein isoforms, which arise via alternative usage of two in-frame translational start codons, showed a nuclear localization and could be detected as early as 6 h after infection of primary human fibroblasts. By performing Western blot analysis with purified virions combined with fractionation experiments, we provide evidence that pUL26 is a novel tegument protein of HCMV that is imported during viral infection. Furthermore, we observed transactivation of the HCMV major immediate-early enhancer-promoter by pUL26, whereas several early and late promoters were not affected. Our data suggest that pUL26 is a novel tegument protein of HCMV with a strong transcriptional activation domain that could play an important role during initiation of the viral replicative cycle.