1 Abteilung Virologie, Universitätsklinikum, Albert-Einstein-Allee 11, 89081 Ulm, Germany
2 Clinic for Infectious Diseases, Clinical Hospital Centre, Cambierieva 17, 51000 Rijeka, Croatia
3 Harvard Medical School, NRB 836, Department of Pathology, 77 Avenue Louis Pasteur, Boston, MA 02115, USA
Correspondence
Thomas Mertens
thomas.mertens{at}medizin.uni-ulm.de
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ABSTRACT |
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INTRODUCTION |
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To our knowledge, this is the first study of the HCMV UL78 gene. We identified the transcription start point and the start of the polyadenylated tail. We found differences in the regions carrying polyadenylation signals for UL78-like mRNAs in rodent and human CMVs. Specifically, we generated and characterized a UL78-deficient mutant and showed that the absence of the UL78 ORF hampered neither virus replication in fibroblasts or an organ-culture system, nor the expression of different viral proteins in fibroblasts, although the predicted pUL78 was quite conserved among clinical HCMV isolates.
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METHODS |
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Virus titration.
Quantification of viral titres was performed by standard plaque assays with end-point dilutions in triplicate. Virus yield assays (single-cycle replication) were performed on HFFs. Briefly, HFFs were grown in 48-well plates and infected 2 days later. Following adsorption, the monolayers were washed and fresh culture medium was added. At various days post-infection (p.i.), cell-culture supernatants were harvested and titrated in triplicate on HFFs. Cells were fixed and stained with a mAb against the viral pp65 (pUL83). Viral titres were determined by counting the number of antigen-positive cells.
Plasmid construction and bacterial artificial chromosome (BAC) mutagenesis.
Plasmid cloning was performed by using standard methods (Sambrook et al., 1989). Construction of the shuttle plasmid pSTdelUL78 was done by insertion of DNA fragments amplified with the Expand High Fidelity PCR system (Roche). For generation of the shuttle plasmid, two regions were cloned separately. Firstly, a fragment of 1·97 kbp containing 113 bp of the 5'-UL78-coding region was amplified with the primer pair Hind-78 (5'-CGGGTGCGCAAACGGaATtcGCGTCAG-3') and Xba78a (5'-TAGCCGATGtcTAGATCCGAGGCGG-3') [nucleotides mutated to create the restriction sites (underlined) are in lower case], cut with HindIII and XbaI and inserted into pUC19, resulting in plasmid pUCUL78-5'. Secondly, a 2·1 kbp fragment containing 159 bp of the 3'-UL78-coding region was amplified with primers Xba78b (5'-AACCACGTtctAGAAAGCCACGTTG-3') and Eco-78 (5'-GACGGCGGgaATTCCGCGGGAAGA-3'), cut with XbaI and EcoRI and subcloned into XbaI/EcoRI-digested pUCdel78-5'. From the second plasmid, the entire insert was excised by EcoRI/HindIII digestion and inserted into the shuttle plasmid pST76K (Pósfai et al., 1997
), resulting in pSTdelUL78.
Generation and reconstitution of the HCMV recombinant.
Mutagenesis of the HCMV BAC plasmid pHB5 with the shuttle plasmid pSTdelUL78 was performed in Escherichia coli strain CBTS as described previously (Borst et al., 1999; Wagner et al., 2000
). Selection and identification of BAC plasmids lacking most of the UL78-coding region were performed as described previously (Wagner et al., 2000
). Maxi-preparations of BAC plasmid DNA and reconstitution of infectious virus were performed as described elsewhere (Borst et al., 1999
; Casarosa et al., 2003
). As an important control, we used the virus RVHB5, obtained from transfection and reconstitution of BACmid pHB5. Cells were passaged 7 days after transfection and cultured until cytopathic effect became visible.
Viral nucleic acid isolation and analysis.
Fibroblasts were infected at an m.o.i. of 1 and harvested 3 days p.i. Extraction of total DNA from infected cells and Southern blot analysis were performed as described previously (Wagner et al., 2000; Casarosa et al., 2003
). The probe used represented nt 112520112921 of the HCMV genome, comprising the 3'-terminal region of the UL77 ORF and the 5' region of the UL78 ORF (AD169 strain, GenBank accession no. X17403).
Primer extension.
Primer extension was performed as described previously (Michel et al., 1993). Briefly, HFFs were infected with HCMV strain AD169 at an m.o.i. of 1. Cells were harvested at 24 h p.i. and total RNA was extracted by using a Midi RNA Extraction kit (Qiagen) as recommended by the manufacturer. Total RNA (20 µg) was annealed for 5 h to 100 ng of a UL78-specific oligonucleotide (5'-CCGACATAGAGTAGCCTTGC-3') spanning nt 113026113007, labelled with [32P]ATP by using T4 kinase. The nucleic acid was precipitated and resuspended in 300 µl 50 mM Tris/HCl (pH 8·3), 50 mM KCl, 6 mM MgCl2, 100 µM each dNTP, 5 mM dithiothreitol (DTT), 30 µg actinomycin ml1, 50 µg BSA ml1, 1 U RNasin µl1 and incubated for 30 min at 42 °C with 67 U avian myeloblastosis virus (AMV) reverse transcriptase. The extension products were resuspended in 10 µl double-distilled water and separated on a 6 % polyacrylamide gel.
Identification of the UL78 3' end.
For cDNA synthesis, total RNA from HCMV-infected fibroblasts was hybridized with the primer mix 5'-GTAAAACGACGGCCACTTTTTTTTTTTTTTTTTN-3', where N stands for A, G or C. The 5' region of this primer is complementary to the M13 universal primer. The reaction mix consisted of 3 µg RNA, 10 µM primer, 1/5 vol. first-strand buffer (Gibco-BRL), 200 µM each dNTP, 50 mM DTT, 100 U RNasin (Promega) and AMV reverse transcriptase. After incubation for 30 min at 37 °C, synthesis was stopped by heating to 94 °C. A 5 µl aliquot of the reaction mix was used for amplification with the M13 universal primer and the UL78-specific primer 1160 (5'-TCTGGTCGAGAGATGTCAGC-3') or 1360 (5'-TGTTGGTAACGACAACCACG-3'), hybridizing 355 and 159 bp, upstream of the translation stop codon, respectively. Amplicons were sequenced and the obtained sequences were aligned with the sequence of strain AD169.
Sequencing of UL78 regions from clinical isolates.
Total DNA was extracted from infected fibroblasts by proteinase K digestion as described previously (Michel et al., 2001). UL78 sequences were amplified by PCR. Primers were designed on the basis of data from strain AD169 (GenBank no. CAA353511). For amplification, the primers 5'-GGGTATATTCGTTCGGCGAG-3' and 5'-TATCTGCCACTTTTCTCCCCG-3' were used, both hybridizing outside the UL78-coding region. All amplifications were done in duplicate. PCR was performed as follows: 2 min at 94 °C, followed by 35 cycles of 15 s at 94 °C, 15 s at 58 °C and 120 s at 72 °C, using a GeneAmp 9700 PCR cycler (Perkin-Elmer) and the Expand High Fidelity PCR system (Roche). Amplification products were purified by using a GFX PCR DNA and Gel Band Purification kit (Amersham Biosciences). Direct sequencing was performed as described previously (Michel et al., 2001
). Briefly, for sequencing, an ABImed Cycle Sequencing kit (Applied Biosystems) was used according to the manufacturer's instructions (Roche Diagnostics) on the GeneAmp 9700 PCR cycler (Perkin-Elmer), performing the program as recommended by the manufacturer and using the sequencing primers UL78-675P (5'-AGACGCGCGAAAACGCCGG-3'), UL78-1195M (5'-GAATGCCAAGACGCATGGTG-3'), UL78-250M (5'-CCCTTGGACAACATGGTGG-3'), UL78-500 (5'-ATAGGGATCTGACGACAGCCTAGC-3'), UL78-outP (5'-GGGTATATTCGTTCGGCGAG-3'), UL78-outM (5'-TATCTGCCACTTTTCTCCCCG-3'), 1160 (5'-TCTGGTCGAGAGATGTCAGC-3'), 1360 (5'-TGTTGGTAACGACAACCACG-3') and UL78-970 (5'-CTACTACTGCTTCAGAGTCCT-3'). DNA sequences were separated in a 310 Genetic Analyser (Applied Biosystems).
Western blot analysis.
Western blot analysis was performed as described previously (Michel et al., 1996, 1998
) using mouse mAbs directed against the HCMV proteins IE1, pUL69, pp65 and pp28 (kindly provided by M. Mach, Universität Erlangen, Germany) and a polyclonal antiserum against pUL97 (Michel et al., 1996
). Briefly, fibroblasts were infected at an m.o.i. of 1 with the indicated virus strains and total cell lysates were extracted at different time points p.i. SDS-PAGE was performed according to standard protocols. Proteins were visualized with an enhanced chemiluminescence (ECL) reaction according to the manufacturer's recommendations (Amersham Biosciences).
Northern blot analysis.
HFFs were infected at an m.o.i. of 1 and harvested at the indicated time points. To arrest virus replication in the immediate-early or early stage, the metabolic inhibitors cycloheximide (CHX, 100 µg ml1) and phosphonoacetic acid (PAA, 250 µg ml1) were added to the infected fibroblasts as described previously (Michel et al., 1996). Total RNA was extracted by using a Midi RNA Extraction kit (Qiagen). Total RNA (10 µg) was separated as described by Sambrook et al. (1989)
. RNA was transferred on to a nylon membrane and immobilized by incubation for 2 h at 60 °C. For subsequent hybridization, DNA probes specific for UL78 and UL77 or for the cellular glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene were amplified by PCR. Membranes were stripped for 20 min with 0·1x SSC and 0·1 % SDS for rehybridization. Probes were labelled with [32P]dCTP by using a random priming kit (Pharmacia). Hybridization was carried out at 42 °C for 18 h in the presence of 50 % formamide, 2x Denhardt's solution, 5x SSPE, 0·05 % SDS and 200 µg salmon sperm DNA ml1. Filters were washed to a stringency of 0·2x SSC, 0·1 % SDS at 65 °C.
Renal artery organ-culture system.
Human renal arterial segments were obtained after informed consent of the patients. Segments were cultivated according to Voisard et al. (1999) and Reinhardt et al. (2003)
in a mixture of Ham's F-12/L-glutamine medium and Waymouth medium (1 : 2) (BioWhittaker) supplemented with 15 % FCS, 1 % penicillin/streptomycin at 37 °C and 5 % CO2. Segments were inoculated with 5x105 cell-free virus particles ml1 of either AD169 or the recombinant HCMV viruses, or were mock-infected. After 24 h, the arterial segments were washed three times with PBS and further cultivated as described above. Once a week up to day 56 p.i., aliquots of the culture supernatant were quantified for infectious virus. All of the tested supernatant aliquots from mock-infected segments were always negative for HCMV, irrespective of whether the donor was seropositive or seronegative (Reinhardt et al., 2003
).
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RESULTS |
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Northern blot analysis was performed to investigate the effect of the deletion on UL78/UL77 transcription. Total RNA was extracted at 36 h p.i. from cells infected with either AD169 or the deletion mutant. Membranes with the separated RNAs were hybridized sequentially with probes specific for the UL78 ORF, UL77 ORF and the cellular GAPDH gene. As shown in Fig. 4(c), different effects on UL78 transcription could be observed. As mentioned above, hybridization of mRNA from AD169-infected fibroblasts showed clearly that the 1·7 kb RNA contained the UL78-coding region, whereas the later-appearing longer RNA consisted of both UL77 and UL78. No signal was detected in RNA samples derived from cells infected with the HCMV UL78-deficient mutant after hybridization with a UL78-specific probe. In the RNA sample from cells infected with HCMV-delUL78, a weak signal at approximately 3 kb was detected after hybridization with the UL77-specific probe, suggesting that the UL77 gene might still be active to some extent. The UL77 mRNA level may have been affected by the absence of the UL78 region, which could result in increased instability of the mRNA molecules.
Absence of UL78 does not modify expression of viral proteins representing the different stages of virus replication
In order to answer the question of whether the absence of the UL78 gene influences viral protein expression at different stages of virus replication, immunoblotting experiments were performed. HFFs were infected with AD169 or HCMV-delUL78. At different time points p.i., cells were harvested. Crude protein extracts were separated by SDS-PAGE and incubated with mAbs directed against IE1, pUL69, pp65 or pp28, or with a polyclonal pUL97 antiserum. These proteins represent different stages of the virus replication cycle. However, as shown in Fig. 5, no significant differences in protein expression could be detected. In individual experiments, there were some differences in band intensities (e.g. pUL69 at 6 h or pp28 at 48 h p.i.), but these were not reproducible in repeated experiments.
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UL78 is conserved among clinical isolates
In order to elucidate whether sequence variations in UL78 existed, as is observed in the US28 protein, 11 low-passaged clinical isolates from nine patients were investigated. The isolates were derived from different specimens (urine, throat wash and leukocytes). As summarized in Fig. 7, multiple sequence alignments of the predicted amino acid sequences revealed that the UL78 protein was highly conserved among the clinical isolates. However, several variations could be found in the N-terminal region and at the very end of the C-terminal region. Overall, when compared with AD169, the identity was 98·599·7 % at the nucleotide level and 98·2100 % at the amino acid level. On the basis of the secondary structure as predicted by the SWISS-PROT program on the ExPASy Proteomics Server (last release 1999) (Bairoch & Boeckmann, 1992
), the first 41 aa of UL78 might constitute the first extracellular part of the protein (Fig. 7
). Isolates from the same patient, but isolated from different specimens, exhibited no sequence variations.
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DISCUSSION |
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Here, we showed that the UL78 mRNA has an early/late transcription pattern similar to those of a number of CMV GPCR homologues, including US28 (Bodaghi et al., 1998; Zipeto et al., 1999
), RCMV R78 (Beisser et al., 1999
) and MCMV M78 (Oliveira & Shenk, 2001
), and is not suppressed by PAA. All of these mRNAs are transcribed early in the replication cycle and the transcripts are suppressed by CHX, but not by PAA. These findings are in accordance with the recently reported results of Wang et al. (2004)
concerning the function of HCMV UL76. These authors also found that the UL78 transcript was not detected under immediate-early conditions and was not inhibited by PAA. In early/late and late stages, these ORFs were part of longer transcripts consisting of US27/US28, R77/R78 or M77/M78, respectively. Our Northern blot analyses showed clearly that the longer mRNA, which is expressed preferentially during the late stage of HCMV replication, included UL77/UL78-coding sequences.
Detection of the polyadenylated tail of the UL78 mRNA and sequence alignments revealed significant differences among the CMVs of different species. Although the functional relevance of the putative polyadenylation signals in the CMVs of humans, chimpanzee, rat and mouse is unknown, this observation might reflect evolution in different hosts. In the T78T79 region of the tupaia herpesvirus, for example, no polyadenylation signal could be found, even though T78 has 25 and 26 % similarity to ChCMV UL78 and HCMV UL78, respectively. It should be noted that the region between T78 and T79 encodes a putative protein with homology to MCMV M59. Such a configuration has not been observed in the other CMV genomes.
Due to the fact that UL78-like genes are present in all known -herpesviruses, it has been speculated that these genes may play a crucial role in the pathogenesis of infection. Despite the relatively low level of sequence similarity to known chemokine receptors, HHV-6A U51 was reported to bind several chemokines, e.g. CCL2 and CCL5, as well as vMIP-II, a chemokine encoded by HHV-8 (Milne et al., 2000
). Concerning HHV-6 U12, it has been reported that RANTES/CCL5 elicits calcium release (Isegawa et al., 1998
). However, to date, no ligand binding or signalling has been shown for HCMV pUL78. Some interesting differences have been observed following infection with mutant viruses deficient for UL78-like genes of CMVs. It has been shown that RCMV pR78 and MCMV pM78 serve important functions during in vivo infection (Beisser et al., 1999
; Oliveira & Shenk, 2001
). R78-deficient mutants are associated with a lower mortality rate in immunocompromised rats than in animals infected with wild-type virus, although no significant differences in virus yield could be found between strains with deletions and wild-type virus in vivo (Beisser et al., 1999
). Furthermore, pR78 of RCMV seems to influence virus replication in fibroblasts and smooth muscle cells, where the R78-deficient mutants were found to replicate 10- to 100-fold less efficiently than wild-type RCMV. The M78-deficient mutant of MCMV only exhibited slightly reduced growth kinetics in vitro, but the authors suggested that pM78 facilitated accumulation of immediate-early viral mRNA (Oliveira & Shenk, 2001
). The deletion of R78 led to an attenuated, syncytium-inducing phenotype, whereas no phenotype has been described after infection with an M78-deficient mutant (Oliveira & Shenk, 2001
). As shown in the present study, the UL78-deficient HCMV mutant also exhibited no phenotype either in fibroblasts or in the in vitro renal-artery model system. Again, these results lead to the assumption of functional differences of the UL78-like proteins in different CMV species. Our results showed that the UL78 gene product is not as important for virus replication as pR78 for RCMV or pM78 for MCMV, at least in vitro. Recently, we showed that, although MCMV pM97 and HCMV pUL97 shared strong homologies in their amino acid sequences, they exhibited different functional properties (Wagner et al., 2000
). Differences between in vitro and in vivo results were also obtained with other genes. Viruses deficient for pR33 or pM33 were found to replicate in vitro with efficiencies similar to those of the corresponding wild-type viruses (Beisser et al., 1998
; Davis-Poynter et al., 1997
). However, the replication of both pR33 and pM33 deletion mutants was found to be impaired in the salivary glands of infected animals. Functional differences among the homologous proteins R33 and UL33 have also been found (Casarosa et al., 2003
). Although both receptors constitutively activate phospholipase C via Gq/11, and partially via Gi/o-mediated pathways, they exhibited profound differences in the modulation of CRE activation pR33 constitutively inhibited, whereas pUL33 constitutively enhanced CRE-mediated transcription.
The lack of an HCMV in vivo model still represents a great obstacle for investigating the influence of particular genes on HCMV pathogenesis. Thus, it is still questionable whether pUL78 can be considered as a potential target for future development of novel antiviral strategies as suggested (Beisser et al., 1999).
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Bairoch, A. & Boeckmann, B. (1992). The SWISS-PROT protein sequence data bank. Nucleic Acids Res 20, 20192022.[Medline]
Beisser, P. S., Vink, C., Van Dam, J. G., Grauls, G., Vanherle, S. J. V. & Bruggeman, C. A. (1998). The R33 G protein-coupled receptor gene of rat cytomegalovirus plays an essential role in the pathogenesis of viral infection. J Virol 72, 23522363.
Beisser, P. S., Grauls, G., Bruggeman, C. A. & Vink, C. (1999). Deletion of the R78 G protein-coupled receptor gene from rat cytomegalovirus results in an attenuated, syncytium-inducing mutant strain. J Virol 73, 72187230.
Bodaghi, B., Jones, T. R., Zipeto, D., Vita, C., Sun, L., Laurent, L., Arenzana-Seisdedos, F., Virelizier, J.-L. & Michelson, S. (1998). Chemokine sequestration by viral chemoreceptors as a novel viral escape strategy: withdrawal of chemokines from the environment of cytomegalovirus-infected cells. J Exp Med 188, 855866.
Borst, E.-M., Hahn, G., Koszinowski, U. H. & Messerle, M. (1999). Cloning of the human cytomegalovirus (HCMV) genome as an infectious bacterial artificial chromosome in Escherichia coli: a new approach for construction of HCMV mutants. J Virol 73, 83208329.
Casarosa, P., Bakker, R. A., Verzijl, D., Navis, M., Timmerman, H., Leurs, R. & Smit, M. J. (2001). Constitutive signaling of the human cytomegalovirus-encoded chemokine receptor US28. J Biol Chem 276, 11331137.
Casarosa, P., Gruijthuijsen, Y. K., Michel, D. & 9 other authors (2003). Constitutive signaling of the human cytomegalovirus-encoded receptor UL33 differs from that of its rat cytomegalovirus homolog R33 by promiscuous activation of G proteins of the Gq, Gi, and Gs classes. J Biol Chem 278, 5001050023.
Chee, M. S., Satchwell, S. C., Preddie, E., Weston, K. M. & Barrell, B. G. (1990). Human cytomegalovirus encodes three G protein-coupled receptor homologues. Nature 344, 774777.[CrossRef][Medline]
Davison, A. J., Dolan, A., Akter, P., Addison, C., Dargan, D. J., Alcendor, D. J., McGeoch, D. J. & Hayward, G. S. (2003). The human cytomegalovirus genome revisited: comparison with the chimpanzee cytomegalovirus genome. J Gen Virol 84, 1728.
Davis-Poynter, N. J., Lynch, D. M., Vally, H., Shellam, G. R., Rawlinson, W. D., Barrell, B. G. & Farrell, H. E. (1997). Identification and characterization of a G protein-coupled receptor homolog encoded by murine cytomegalovirus. J Virol 71, 15211529.[Abstract]
Gao, J.-L., Kuhns, D. B., Tiffany, H. L., McDermott, D., Li, X., Franke, U. & Murphy, P. M. (1993). Structure and functional expression of the human macrophage inflammatory protein 1/RANTES receptor. J Exp Med 177, 14211427.
Gompels, U. A., Nicholas, J., Lawrence, G., Jones, M., Thomson, B. J., Martin, M. E., Efstathiou, S., Craxton, M. & Macaulay, H. A. (1995). The DNA sequence of human herpesvirus-6: structure, coding content, and genome evolution. Virology 209, 2951.[CrossRef][Medline]
Isegawa, Y., Ping, Z., Nakano, K., Sugimoto, N. & Yamanishi, K. (1998). Human herpesvirus 6 open reading frame U12 encodes a functional -chemokine receptor. J Virol 72, 61046112.
Margulies, B. J., Browne, H. & Gibson, W. (1996). Identification of the human cytomegalovirus G protein-coupled receptor homologue encoded by UL33 in infected cells and enveloped virus particles. Virology 225, 111125.[CrossRef][Medline]
Megaw, A. G., Rapaport, D., Avidor, B., Frenkel, N. & Davison, A. J. (1998). The DNA sequence of the RK strain of human herpesvirus 7. Virology 244, 119132.[CrossRef][Medline]
Menotti, L., Mirandola, P., Locati, M. & Campadelli-Fiume, G. (1999). Trafficking to the plasma membrane of the seven-transmembrane protein encoded by human herpesvirus 6 U51 gene involves a cell-specific function present in T lymphocytes. J Virol 73, 325333.
Michel, D., Salamini, F., Bartels, D., Dale, P., Baga, M. & Szalay, A. (1993). Analysis of a desiccation and ABA-responsive promoter isolated from the resurrection plant Craterostigma plantagineum. Plant J 4, 2940.[CrossRef][Medline]
Michel, D., Pavi, I., Zimmermann, A., Haupt, E., Wunderlich, K., Heuschmid, M. & Mertens, T. (1996). The UL97 gene product of human cytomegalovirus is an early-late protein with a nuclear localization but is not a nucleoside kinase. J Virol 70, 63406346.[Abstract]
Michel, D., Schaarschmidt, P., Wunderlich, K., Heuschmid, M., Simoncini, L., Mühlberger, D., Zimmermann, A., Pavi, I. & Mertens, T. (1998). Functional regions of the human cytomegalovirus protein pUL97 involved in nuclear localization and phosphorylation of ganciclovir and pUL97 itself. J Gen Virol 79, 21052112.[Abstract]
Michel, D., Höhn, S., Haller, T., Jun, D. & Mertens, T. (2001). Aciclovir selects for ganciclovir-cross-resistance of human cytomegalovirus in vitro that is only in part explained by known mutations in the UL97 protein. J Med Virol 65, 7076.[Medline]
Milne, R. S. B., Mattick, C., Nicholson, L., Devaraj, P., Alcami, A. & Gompels, U. A. (2000). RANTES binding and down-regulation by a novel human herpesvirus-6 chemokine receptor. J Immunol 164, 23962404.
Minisini, R., Tulone, C., Lüske, A., Michel, D., Mertens, T., Gierschik, P. & Moepps, B. (2003). Constitutive inositol phosphate formation in cytomegalovirus-infected human fibroblasts is due to expression of the chemokine receptor homologue pUS28. J Virol 77, 44894501.
Nicholas, J. (1996). Determination and analysis of the complete nucleotide sequence of human herpesvirus 7. J Virol 70, 59755989.[Abstract]
Oliveira, S. A. & Shenk, T. E. (2001). Murine cytomegalovirus M78 protein, a G protein-coupled receptor homologue, is a constituent of the virion and facilitates accumulation of immediate-early viral mRNA. Proc Natl Acad Sci U S A 98, 32373242.
Pósfai, G., Koob, M. D., Kirkpatrick, H. A. & Blattner, F. R. (1997). Versatile insertion plasmids for targeted genome manipulations in bacteria: isolation, deletion, and rescue of the pathogenicity island LEE of the Escherichia coli O157 : H7 genome. J Bacteriol 179, 44264428.
Rawlinson, W. D., Farrell, H. E. & Barrell, B. G. (1996). Analysis of the complete DNA sequence of murine cytomegalovirus. J Virol 70, 88338849.[Abstract]
Reinhardt, B., Vaida, B., Voisard, R. & 7 other authors (2003). Human cytomegalovirus infection in human renal arteries in vitro. J Virol Methods 109, 19.[CrossRef][Medline]
Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Streblow, D. N., Soderberg-Naucler, C., Vieira, P., Smith, J., Wakabayashi, E., Ruchti, F., Mattison, K., Altschuler, Y. & Nelson, J. A. (1999). The human cytomegalovirus chemokine receptor US28 mediates vascular smooth muscle cell migration. Cell 99, 511520.[Medline]
Vink, C., Beuken, E. & Bruggeman, C. A. (2000). Complete DNA sequence of the rat cytomegalovirus genome. J Virol 74, 76567665.
Vink, C., Smit, M. J., Leurs, R. & Bruggeman, C. A. (2001). The role of cytomegalovirus-encoded homologs of G protein-coupled receptors and chemokines in manipulation of and evasion from the immune system. J Clin Virol 23, 4355.[CrossRef][Medline]
Voisard, R., von Eicken, J., Baur, R., Gschwend, J. E., Wenderoth, U., Kleinschmidt, K., Hombach, V. & Höher, M. (1999). A human arterial organ culture model of postangioplasty restenosis: results up to 56 days after ballooning. Atherosclerosis 144, 123134.[CrossRef][Medline]
Wagner, M., Michel, D., Schaarschmidt, P., Vaida, B., Jonjic, S., Messerle, M., Mertens, T. & Koszinowski, U. (2000). Comparison between human cytomegalovirus pUL97 and murine cytomegalovirus (MCMV) pM97 expressed by MCMV and vaccinia virus: pM97 does not confer ganciclovir sensitivity. J Virol 74, 1072910736.
Waldhoer, M., Kledal, T. N., Farrell, H. & Schwartz, T. W. (2002). Murine cytomegalovirus (CMV) M33 and human CMV US28 receptors exhibit similar constitutive signaling activities. J Virol 76, 81618168.
Wang, S.-K., Duh, C.-Y. & Wu, C.-W. (2004). Human cytomegalovirus UL76 encodes a novel virion-associated protein that is able to inhibit viral replication. J Virol 78, 97509762.
Zipeto, D., Bodaghi, B., Laurent, L., Virelizier, J.-L. & Michelson, S. (1999). Kinetics of transcription of human cytomegalovirus chemokine receptor US28 in different cell types. J Gen Virol 80, 543547.[Abstract]
Received 9 July 2004;
accepted 19 October 2004.