Department of Medical Microbiology1 and Section of Infection and Immunity2, University of Wales College of Medicine, Tenovus Building, Heath Park, Cardiff CF14 4XN, UK
Cardiff School of Biosciences, Cardiff University, Cardiff CF10 3US, UK3
Author for correspondence: Gavin Wilkinson. Fax +44 29 20745003. e-mail WilkinsonGW1{at}cf.ac.uk
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Abstract |
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Introduction |
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Concomitant HHV-6 and human cytomegalovirus (HCMV) infections occur frequently in immunosuppressed organ transplant patients (Irving et al., 1990 ; DesJardin et al., 1998
) and, given that they infect overlapping cell types (Kondo et al., 1991
; Taylor-Wiedeman et al., 1991
), the potential exists for the two viruses to interact directly in vivo. The HHV-6 genome (162170 kb) is composed of a unique long segment bracketed by direct repeats. The unique long genomic elements of HHV-6 and HCMV are essentially collinear (Neipel et al., 1992
), with 67% of HHV-6A (U1102) genes having counterparts in HCMV (Gompels et al., 1995
). In common with other herpesviruses, HHV-6 operates a cascade system of gene regulation conventionally divided into the immediate-early (IE), early and late phases. A positional homologue of the HCMV major IE gene (IE1) exists in HHV-6 (within the IE-A region). The HCMV major IE gene interacts with cellular factors (e.g. p107, E2F) and encodes a transcriptional trans-activator that acts synergistically with HCMV IE2 (Lukac et al., 1994
). HCMV IE1 is essential for efficient productive infection at low m.o.i. in vitro, but is dispensable at high-input m.o.i. (Mocarski et al., 1996
). HCMV IE1 induces the disruption of PML-bodies and causes both IE1 and PML to become associated with mitotic chromatin (Lafemina et al., 1989
; Wilkinson et al., 1998
). PML-bodies (also known as ND10 or PODs) are punctate structures associated with the nuclear matrix that contain, or transiently associate with, an assortment of cellular proteins including SUMO-1, Sp100, Sp140, CBP, BLM, Daxx, pRB and p53 (Zhong et al., 2000
). PML is the defining marker for PML-bodies and is required for the recruitment of other proteins to the domain (Ishov, 1999
). PML modification by covalent linkage to SUMO-1 (a small ubiquitin-like molecule) is required for its migration to PML-bodies (Muller et al., 1998
). Both HCMV IE1 and IE2 are SUMO-modified and, in the case of IE2, this modification is necessary for its transactivating functions (Muller & Dejean, 1999
; Hofmann et al., 2000
).
There is no obvious amino acid sequence homology between HCMV IE1 and its positional homologue in HHV-6. Four transcripts are produced from the HHV-6 IE-A region of which only one, designated HHV-6 IE1, is produced under IE conditions (Schiewe et al., 1994 ; Mirandola et al., 1998
). The HHV-6A IE1 transcript is produced from five exons; translation is initiated in exon 3 and encodes an open reading frame (ORF) (2826 bp) predicted to encode a product of 104 kDa (IE1941aa). The designated HHV-6B IE1 ORF is slightly longer (3237 bp) and is predicted to encode a 120 kDa protein (IE11078aa) (Dominguez et al., 1999
). Interestingly, the IE-A region is the most variable part of the HHV-6 genome, with ORFs in this region showing only 63% nucleotide identity between HHV-6A and HHV-6B variants as compared with an overall identity for all ORFs of 94% (Isegawa et al., 1999
).
The herpesvirus IE genes exhibit extreme sequence variation and have diverse biological properties yet clearly play a pivotal role in both initiating and establishing the lytic phase of the replication cycle. We were intrigued by the elevated level of sequence variation in the IE1 region between the HHV-6 variants and its absolute divergence from its counterpart in HCMV. It is possible that while the sequence of this genetic element may have diverged during the evolution of the betaherpesviruses, many of its functions have been conserved. In this study we therefore sought to analyse the products of the HHV-6A and HHV-6B IE1 genes and, more specifically, to explore their potential interactions with components of PML-bodies.
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Methods |
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DNA transfections were carried out using Polyfect (Qiagen) according to the manufacturers standard protocol. U373 cells expressing 6xHis-tagged SUMO-1 (U373-SUMO) were generated by puromycin (Invitrogen) selection following transfection with pIRES-HisSUMO, kindly provided by D. Bailey (Marie Curie Research Institute, Oxted, UK). pZ29-GFP and pU1102-GFP (see below) were transfected into U373 cells and cell lines generated following G418 sulphate selection (2mg/ml; Sigma).
Plasmid construction.
The HHV-6 IE1 ORFs from strains U1102 (Gompels et al., 1995 ) and Z29 (Dominguez et al., 1999
) were amplified by PCR from genomic DNA and cloned directly as C-terminal GFP fusion constructs in the transient mammalian expression vector pCT-GFP-TOPO (Invitrogen) to generate the plasmids pU1102-GFP and pZ29-GFP. HHV-6 template DNA was extracted from tissue culture preparations of the appropriate virus using a silica slurry method previously described (Boom et al., 1990
). For Z29 IE1 the 5' primer was Z29IE-CL (GATAAATTTGAGCATTTTCTTCG) and the 3' primer was A-IECL2 (GCGGTGTCTCAATTTGCATC). For U1102, the 5' primer A-IECL1 (GATAGATTTGAGCATTTTCTACG) and the 3' primer A-IECL2 were utilized. Primers were obtained from Oswel DNA services and HPLC-purified. The PCR conditions involved denaturation at 94 °C for 2 min; 9 cycles of denaturation (94 °C for 30 s), annealing (50 °C for 30 s) and incubation (68 °C for 7 min); a further 19 cycles in which the incubation period increased by 20 s per cycle; and finally incubation at 68 °C for 14 min. 100 pmol of each primer, 200 µM dNTPs (Amersham Biosciences), manufacturers buffer containing 1·5 mM MgCl2 and 1 unit Pwo polymerase (Hybaid) were used in a final reaction volume of 50µl. Thermocycling was undertaken in a PTC-100 thermocycler (GRI Ltd). All PCR-generated fragments for cloning were sequenced and constructs that matched the published sequence used for each variant.
A plasmid expressing a nuclear form of GFP was made by amplifying exons 2 and 3 of HCMV IE1 by PCR and inserting the fragment into the EGFP-N1 mammalian expression vector (Clontech) as an N-terminal GFP fusion.
Construction of a recombinant adenovirus.
The complete HHV-6 IE1 ORF from strain U1102 was amplified using primers RS1 (GGCCGTCGACGCGGTGTCTCAATTTGCATC) and RS2 (GGCCACTAGTACATCTAGGTTTCATCTAGCTA) with the same cycling conditions as for the generation of pU1102-GFP and pZ29-GFP. The DNA sequence of the clone used in expression studies matched the published sequence (Gompels et al., 1995 ). A replication-deficient adenovirus recombinant (RAdHHV-6) encoding HHV-6-IE1941aa under the control of the HCMV major IE promoter was generated as described previously (Wilkinson et al., 1992
).
Immunohistochemistry.
An anti-HHV-6 IE1-specific polyclonal antibody was generated by intraperitoneal murine immunization with the RAdHHV-6 recombinant as described previously (Tomasec et al., 2000 ) and diluted 1 in 100 for use in immunofluorescence and Western blots. Monoclonal antibodies to SUMO-1 (Zymed) and PML (Santa Cruz) and rabbit polyclonal antisera specific for PML (MBL) were diluted 1 in 100 for use in immunofluorescence. FITC- and Texas red-conjugated secondary antibodies were obtained from Sigma. Immunofluorescence and Western blotting conditions were performed as described previously (Sambrook & Russell, 2001b
). For co-immunofluorescence primary antibodies were mixed and applied together, as were secondary antibodies.
Immunoprecipitation.
Extracts were made from U373 cell lines by lysing centrifuged intact cells in 500 µl RIPA buffer (0·5%, v/v, NP-40, 0·5%, w/v, sodium deoxycholate, 0·5%, w/v, SDS, 150 mM NaCl, 10 mM Tris pH 7·4, 5 mM EDTA, 2 mM PMSF, 4 mM N-ethylmaleimide). Lysates were cleared by centrifugation at 10000 g (10 min). Rabbit polyclonal anti-GFP (2 µl; Abcam, UK) was added to 500 µl lysate and incubated with mixing overnight at 4 °C. Protein Aagarose (20 µl; Amersham Biosciences) was added for a further 3 h at 4 °C. Precipitated material was recovered by centrifugation (10000 g for 10 min) and washed four times in RIPA buffer. The sample was boiled for 5 min in 1x SDSPAGE running buffer containing 100 mM DTT and analysed in Western blot experiments (as above) following electrophoresis on a 6% polyacrylamide gel (Sambrook & Russell, 2001a ).
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Results |
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To examine the intracellular distribution of the HHV-6 IE1 gene product in eukaryotic cells, U373 cells were transfected with plasmids expressing GFP-tagged IE1 proteins from both A and B type HHV-6 variants. Proteins from both variant A (IE1941aa-GFP) and B (IE11078aa-GFP) were demonstrated during real-time imaging to traffic to and remain associated with punctate nuclear domains (data not shown). This observation was followed up with immunofluorescence experiments in which the endogenous fluorescence associated with expressed IE1941aa-GFP and IE11078aa-GFP was shown to co-localize with PML (Fig. 1). When expressed in isolation, both IE1941aa-GFP and IE11078aa-GFP clearly traffic to and form a stable interaction with the PML-associated nuclear domain. Unlike its positional analogue in HCMV, the HHV-6 IE1 gene product did not obviously perturb the intracellular distribution of PML nor associate with chromatin in cells undergoing mitosis.
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HHV-6 IE1 is SUMO-1-modified
To detect expression of the HHV-6 IE1 gene product, an antibody specific for GFP was used in Western blot experiments. Two discrete protein species with estimated molecular masses of approximately 235 and 260 kDa were detected in cells transfected with pZ29-GFP (Fig. 3.). These values are significantly larger than the 147·5 kDa predicted size of the fusion protein (IE11078aa is estimated at 121·5 kDa and GFP at 27 kDa). Interestingly, the HCMV major IE protein also migrates anomalously on SDSPAGE suggesting a significantly higher apparent molecular mass than that predicted from its sequence. The presence of a second higher molecular mass species detected in Western blot experiments would be consistent with HHV-6 IE1 being covalently conjugated to SUMO-1.
To further investigate expression of HHV-6 IE1, the gene derived from strain U1102 was inserted into a replication-deficient adenovirus vector. Mice were immunized with the adenovirus recombinant (RAdHHV-6) to generate limited amounts of HHV-6 IE1-specific polyclonal antibodies. Co-immunofluorescence using the murine antibody demonstrated that the native form of the HHV-6 IE941aa co-localized with PML-bodies when expressed following infection with RAdHHV-6IE1 (Fig. 4a). No cross-reactivity of either secondary antibody with the incorrect primary antibody was seen, nor was any staining observed on the green channel when the same immunofluorescence was carried out on cells infected with empty adenovirus vector (data not shown). Cell extracts prepared from U373 cells infected with RAdHHV-6IE1 were analysed by Western blot using the HHV-6 IE1-specific polyclonal antibody. A major protein species with an estimated molecular mass of around 160 kDa was identified (by SDSPAGE) (Fig. 4b
); thus the native protein also migrates with an anomalous mobility. Although only a single predominant species was clearly identifiable for the native form of the protein, this does not exclude SUMO-modification since the gross overexpression of the protein from the adenovirus recombinant has the potential to overwhelm sumoylation pathways and mask detection of low abundance SUMO-modified species on Western blots.
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Discussion |
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PML-bodies have been implicated in a wide range of cellular processes including transcriptional control, chromatin remodelling, apoptosis, cellular transformation and immune regulation. PML expression is stimulated by treatment with interferon and its enhanced association with its nuclear domain may act as a barrier to virus infection (Bonilla et al., 2002 ). Following infection, the HCMV genome traffics to PML-bodies and this is the site at which virus transcription and DNA replication are established (Ishov & Maul, 1996
; Maul et al., 1996
; Ahn et al., 1999
). Expression of the HCMV IE1 gene product promotes the disruption of PML-bodies, causing the PML protein to be dispersed throughout the nucleus and to associate with mitotic chromatin. It is not clear why DNA virus genomes are deposited at PML-bodies nor why viruses encode genes that compromise their integrity. Herpes simplex virus 1 ICP0 promotes both the dissolution of PML-bodies and the degradation of PML, EpsteinBarr virus BZLF1 promotes the nuclear dispersal of PML without its degradation and the adenovirus E4 ORF3 induces the formation of thread-like PML-structures (Adamson & Kenney, 2001
; Everett & Maul, 1994
; Carvalho et al., 1995
). HHV-6 IE1 is clearly functionally distinct from its positional analogue in HCMV in leaving PML-bodies superficially intact. Additionally, PML-bodies remain superficially intact in JJhan or Molt 3 lymphocytic cell lines and in PBMC cultures infected with either the HHV-6A or HHV-6B variant. HHV-6 infection is remarkable for not promoting the disruption or destruction of these nuclear domains.
In addition to PML, a large number of cellular proteins have been reported to either transiently or stably associate with PML-bodies, including p53, Rb, CBP, Daxx, Bax and SP100 (Zhong et al., 2000 ). Furthermore, a significant number of viral proteins traffic to PML-bodies without perturbing their integrity, e.g. HCMV IE2, EBV EBNA-5, HHV-8 K8, simian virus 40 T Ag, bovine papillomavirus E2, L1 and L2 (Ahn & Hayward, 1997
; Katano et al., 2001
; Murges et al., 2001
; Szekely et al., 1996
; Day et al., 1998
; Jiang et al., 1996
; Bonilla et al., 2002
). Currently, the HHV-6 IE gene product has not been assigned any specific function. However, the strong association revealed by immunohistochemistry and real-time imaging suggests that HHV-6 IE1 may act by affecting the function of PML-bodies. HHV-6 IE1 clearly has the potential to modify components, regulate the composition, or influence trafficking to and from PML-bodies without inducing a morphological change.
SUMO-1 is conjugated to proteins by a thioester bond formed between the C terminus of SUMO-1 and a lysine residue in the targeted protein. Unlike ubiqitination, which targets proteins for degradation, modification by SUMO-1 can have a variety of different effects. Most significantly, SUMO-1 modification of the PML protein is necessary for its localization to PML-bodies and the trans-activating function of HCMV IE2 (Hofmann et al., 2000 ; Rodriguez et al., 1999
). We observed that expression of the HHV-6 IE1-GFP fusion protein, but not GFP alone, was substantially enhanced in a U373-SUMO-1 cell line. It is therefore likely that sumoylation is required to stabilize the expressed HHV-6 IE1 gene product. This situation would not be unique; SUMO-1-modified I
B
and Mdm2 are resistant to ubiquitin-mediated degradation (Desterro et al., 1998
). Herpesvirus IE proteins are associated with establishment of a cellular environment compatible with the lytic phase of infection. SUMO-1 modification can be induced in response to stress. The heat-shock transcription factors 1 and 2 (HSF1 and HSF2) are both converted to their DNA-binding form by SUMO-1 modification in response to stress (Hong et al., 2001
), while DNA damage induces SUMO-modification of topoisomerase I and II (Goodson et al., 2001
; Mao et al., 2000
). It is possible that in both HCMV and HHV-6 infections the transition from latent to the lytic replication cycle may be promoted by the SUMO-1 modification of IE gene products. Sumoylation pathways may play an important role in determining the fate of betaherpesvirus infections.
On analysing the level of sumoylation of the HHV-6 IE protein three slower-migrating species were identified that would be consistent with the protein containing three sumoylation sites. This degree of sumoylation would be unusual, although PML has been demonstrated to have three SUMO-modification sites. However, analysis of the IE1 gene sequence revealed only a single consensus sequence for SUMO-1 modification (I/LKXE) in both U1102 and Z29 IE1 sequences at amino acids 665 and 802 respectively. Further cryptic sites may yet be present. Alternatively, conjugation with a single SUMO-1 molecule could potentially facilitate a second SUMO-1-independent modification resulting in reduced mobility, e.g. ubiquitination. Further analysis of the protein is required to determine the number of SUMO-1-modification sites it contains.
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Acknowledgments |
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The authors thank both Carole Rickards and Lynne Neale for their assistance throughout this work and Daniel Bailey for generously providing the plasmid pIRES-HisSUMO and for helpful discussions. G.W. was supported by funding from the Wellcome Trust.
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Received 17 April 2002;
accepted 17 July 2002.