1 MRC Virology Unit, Institute of Virology, Church Street, Glasgow G11 5JR, UK
2 Department of Virology, Royal Free and University College Medical School, Royal Free Campus, Rowland Hill Street, Hampstead, London NW3 2QG, UK
3 Section of Infection and Immunity, University of Wales College of Medicine, Tenovus Building, Heath Park, Cardiff CF14 4XX, UK
Correspondence
Andrew Davison
a.davison{at}vir.gla.ac.uk
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ABSTRACT |
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The GenBank accession numbers of the HCMV sequences reported in this paper are AY169795AY169800.
Published ahead of print on 20 February 2003 as DOI 10.1099/vir.0.18952-0
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MAIN TEXT |
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The upper part of Fig. 1(A) depicts the arrangement of ORFs UL131UL128 as predicted by Chee et al. (1990)
, and the lower parts show alternative predictions based on comparisons between the AD169 and CCMV sequences. UL130 is unaltered, while spliced genes replace UL131 upstream and UL129 plus UL128 downstream. One of these genes is named UL131A because it occupies the same region as UL131 but does not share any encoded amino acid sequence, since the first exon is in a different reading frame from UL131. The other spliced gene retains the designation UL128 because it shares amino acid sequence with the original UL128 but not with UL129. Fig. 1(B, C)
shows detailed alignments of the AD169 and CCMV sequences in these regions. Protein-coding regions were proposed from conservation of encoded amino acid sequences, and conceptually linked together via candidate splice donor and acceptor sites. This led to the hypothesis that UL131A and UL128 comprise two and three exons, respectively.
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A range of experiments was performed in order to investigate the expression patterns of UL131A and UL128, using various RNA preparations, primers and probes. A selection of results is shown in Fig. 2 and relevant primers and deduced transcriptional features are included in Fig. 1(B, C)
. RNA was prepared from human foetal fibroblasts mock-infected or infected with AD169 at an m.o.i. of 5. Infections were carried out under immediate early (1 h preinfection then 24 h in 200 µg cycloheximide ml-1), early (48 h in 300 µg phosphonoacetic acid ml-1) and late conditions (72 h with no inhibitor). RNA was extracted using TRIzol (Life Technologies), and the polyadenylated fraction was isolated using oligo(dT)cellulose and quantified by spectrophotometry. RNA integrity was assessed by Northern blotting using a cellular mRNA probe (not shown), and the absence of detectable viral DNA was confirmed by PCR. RT-PCR was carried out using a Titan kit (Boehringer Mannheim) and RACE using a SMART RACE kit (Clontech). 5'-RACE involved extension of an oligo(dT)-containing primer by a reverse transcriptase that adds a tract of C residues at the 3'-end of the cDNA, second strand cDNA synthesis primed by the SMART oligonucleotide which has a tract of G residues at the 3'-end, and PCR using SMART-specific and gene-specific primers. 3'-RACE involved reverse transcription using an oligo(dT) primer extended at its 5'-end by the SMART sequence, followed by PCR. This approach results in 5'-RACE and 3'-RACE products that are 30 and 55 bp longer, respectively, than the cognate transcribed sequences. All RT-PCR and RACE products relevant to locating mRNA ends and introns were cloned into pGEM-T (Promega), and several clones of each product sequenced. Northern blotting was performed using strand-specific RNA probes prepared using Lig'nScribe and MAXIscript kits (Ambion).
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The transcript mapping data support the expression pattern of UL131A and UL128 anticipated in Fig. 1(A), with both genes in the late kinetic class. Although UL131A consists of two exons and UL128 of three, unspliced and partially spliced RNAs were also detected. Our results are somewhat at variance with those of Chambers et al. (1999)
, who, using microarray technology, classified transcripts from UL131, UL130 and UL128 as defined by Chee et al. (1990)
in the late, early-late and early classes, respectively. However, kinetic class assigned from microarray data differs from that deduced from Northern blot data for a significant number of genes (Chambers et al., 1999
).
The sequences of UL131A and UL128 in AD169, which has been passaged many times in human fibroblast cell lines, were compared with those in six other HCMV strains. Four had been grown in human fibroblast cell lines: three of these (Merlin, 3157 and 6397) derived in Cardiff by three passages from urine samples from congenitally infected infants and one the widely used low passage Toledo strain (Quinnan et al., 1984). DNA from the Cardiff strains was obtained from purified virions and from Toledo as infected-cell DNA. DNA was also prepared directly from clinical material for two strains, one (W) from the lung of an HCMV-infected AIDS patient and one (3301) from the urine of a congenitally infected child. Two overlapping fragments of about 4 kbp were PCR-amplified from five of the DNA samples and cloned into pGEM-T. The primers used were 5'-TGCTTAAGCCAATCGCAGCG-3' (in UL147) and 5'-ATCCCGCGAATCTCAGCCGT-3' (UL128 exon 2), and 5'-AATGTTGCGAATTCATAAACGTCA-3' (UL128 exon 1) and 5'-ACTGGTCAGCCTTGCTTCTAGTCA-3' (UL123). For each strain, the inserts in four plasmids were sequenced on both strands and a consensus established to exclude PCR artefacts. Corresponding data for the sixth strain (Merlin) were obtained as part of shotgun cloning the entire genome in M13. Sequences were compiled using PREGAP4 and GAP4 (Staden et al., 2000
) and PHRED (Ewing & Green, 1998
; Ewing et al., 1998
). The region containing the genes of interest was analysed using the GCG suite (Accelrys), and the corresponding AD169 sequence was included.
The predicted arrangement of UL131A, UL130 and UL128 is shown for each strain in the upper part of Fig. 3(A). These genes were intact in two non-passaged strains (3301 and W), but disruptions were apparent in the five passaged strains. As explained above, AD169 has a frameshift mutation in UL131A exon 1 that would cause fusion of the N terminus of the UL131A protein to sequences encoded in another reading frame. Merlin has a C to T transition in UL128 exon 3 that introduces a stop codon and would cause premature translational termination. 3157 had a G to C transversion in the GT dinucleotide of the splice donor site at the end of UL128 exon 1, which would result in lack of splicing. 6397 has a 1 kbp deletion that would abolish expression of UL131A and UL130. In Toledo, inversion of a substantial region results in disruption of UL128 by introducing UL148A in place of UL128 exon 3. Fig. 3(A)
also recapitulates observations made by Davison et al. (2003a)
on the equivalent region in the genomes of two other primate cytomegaloviruses, CCMV and the Colburn strain of simian cytomegalovirus (SCMV), both of which have been passaged in human fibroblasts. CCMV UL128 exon 1 and SCMV UL128 exon 2 appear to be frameshifted, the former by the gain of a single nucleotide, as explained above, and the latter by a single nucleotide deletion. We conclude that passage of HCMV isolates in cell culture is associated with the loss of function of UL128 or UL131A or, perhaps, UL130. Indeed, the mutated UL128 in Merlin at passage 3 was also the only form detected at the end of passage 1, during which the virus underwent many rounds of replication, but was not detected in the urine sample from which the strain was isolated. Similarly, the deletion in 6397 was not detected in unpassaged material.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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![]() ![]() ![]() ![]() |
---|
Chambers, J., Angulo, A., Amaratunga, D. & 9 other authors (1999). DNA microarrays of the complex human cytomegalovirus genome: profiling kinetic class with drug sensitivity of viral gene expression. J Virol 73, 57575766.
Chang, Y., Jeang, K. T., Lietman, T. & Hayward, G. S. (1995). Structural organization of the spliced immediate-early gene complex that encodes the major acidic nuclear (IE1) and transactivator (IE2) proteins of African green monkey cytomegalovirus. J Biomed Sci 2, 105130.[Medline]
Chee, M. S., Bankier, A. T., Beck, S. & 12 other authors (1990). Analysis of the protein coding content of the sequence of human cytomegalovirus strain AD169. Curr Top Microbiol Immunol 154, 125169.[Medline]
Davison, A. J., Dolan, A., Akter, P., Addison, C., Dargan, D. J., Alcendor, D. J., McGeoch, D. J. & Hayward, G. S. (2003a). The human cytomegalovirus genome revisited: comparison with the chimpanzee cytomegalovirus genome. J Gen Virol 84, 1728.
Davison, A. J., Akter, P., Cunningham, C. & 7 other authors (2003b). Homology between the human cytomegalovirus RL11 gene family and human adenovirus E3 genes. J Gen Virol 84, 657663.
Ewing, B. & Green, P. (1998). Base-calling of automated sequencer traces using PHRED. II. Error probabilities. Genome Res 8, 186194.
Ewing, B., Hillier, L., Wendl, M. C. & Green, P. (1998). Base-calling of automated sequencer traces using PHRED. I. Accuracy assessment. Genome Res 8, 175185.
Fleming, P., Davis-Poynter, N., Degli-Esposti, M., Densley, E., Papadimitriou, J., Shellam, G. & Farrell, H. (1999). The murine cytomegalovirus chemokine homolog, m131/129, is a determinant of viral pathogenicity. J Virol 73, 68006809.
Lagenaur, L. A., Manning, W. C., Vieira, J., Martens, C. L. & Mocarski, E. S. (1994). Structure and function of the murine cytomegalovirus sgg1 gene: a determinant of viral growth in salivary gland acinar cells. J Virol 68, 77177727.[Abstract]
MacDonald, M. R., Li, X.-Y. & Virgin, H. W., IV (1997). Late expression of a chemokine homolog by murine cytomegalovirus. J Virol 71, 16711678.[Abstract]
MacDonald, M. R., Burney, M. W., Resnick, S. B. & Virgin, H. W., IV (1999). Spliced mRNA encoding the murine cytomegalovirus chemokine homolog predicts a chemokine of novel structure. J Virol 73, 36823691.
Nielsen, H., Engelbrecht, J., Brunak, S. & von Heijne, G. (1997). Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Eng 10, 16.[Abstract]
Penfold, M. E., Dairaghi, D. J., Duke, G. M., Saederup, N., Mocarski, E. S., Kemble, G. W. & Schall, T. J. (1999). Cytomegalovirus encodes a potent chemokine. Proc Natl Acad Sci U S A 96, 98399844.
Quinnan, G. V., Jr, Delery, M., Rook, A. H. & other authors (1984). Comparative virulence and immunogenicity of the Towne strain and a nonattenuated strain of cytomegalovirus. Ann Intern Med 101, 478483.[Medline]
Rawlinson, W. D., Farrell, H. E. & Barrell, B. G. (1996). Analysis of the complete DNA sequence of murine cytomegalovirus. J Virol 70, 88338849.[Abstract]
Revello, M. G., Baldanti, F., Percivalle, E., Sarasini, A., De-Giuli, L., Genini, E., Lilleri, D., Labò, N. & Gerna, G. (2001). In vitro selection of human cytomegalovirus variants unable to transfer virus and virus products from infected cells to polymorphonuclear leukocytes and to grow in endothelial cells. J Gen Virol 82, 14291438.
Saederup, N., Lin, Y.-C., Dairaghi, D. J., Schall, T. J. & Mocarski, E. S. (1999). Cytomegalovirus-encoded chemokine promotes monocyte-associated viremia in the host. Proc Natl Acad Sci U S A 96, 1088110886.
Saederup, N., Aguirre, S. A., Sparer, T. E., Bouley, D. M. & Mocarski, E. S. (2001). Murine cytomegalovirus CC chemokine homolog MCK-2 (m131129) is a determinant of dissemination that increases inflammation at initial sites of infection. J Virol 75, 99669976.
Sinzger, C., Schmidt, K., Knapp, J., Kahl, M., Beck, R., Waldman, J., Hebart, H., Einsele, H. & Jahn, G. (1999). Modification of human cytomegalovirus tropism through propagation in vitro is associated with changes in the viral genome. J Gen Virol 80, 28672877.
Staden, R., Beal, K. F. & Bonfield, J. K. (2000). The Staden package, 1998. Methods Mol Biol 132, 115130.[Medline]
Vink, C., Beuken, E. & Bruggeman, C. A. (2000). Complete DNA sequence of the rat cytomegalovirus genome. J Virol 74, 76567665.
Received 5 November 2002;
accepted 12 February 2003.