1 Section of Infection and Immunity, University of Wales College of Medicine, Heath Park, Cardiff CF14 4XY, UK
2 Department of Medical Microbiology, University of Wales College of Medicine, Heath Park, Cardiff CF14 4XY, UK
Correspondence
Gavin Wilkinson
WilkinsonGW1{at}cardiff.ac.uk
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ABSTRACT |
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The GenBank accession numbers of the HCMV sequences reported in this paper are AY325309AY325312.
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MAIN TEXT |
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During productive HCMV infection 2.7 RNA can be detected in polysome preparations (Wathen & Stinski, 1982
; Wu et al., 1992
). Forced expression of an RL4 ORF epitope-tagged fusion protein derived from either strain Towne or AD169 generated a 24 kDa product that trafficked to the nucleoli (Bergamini et al., 1998
). However, despite its abundance, no
2.7-associated translation product has yet been detected during HCMV infection (Greenaway & Wilkinson, 1987
; Lord et al., 1989
).
Concern exists regarding the genomic integrity of the commonly used laboratory strains AD169 and Towne (Akter et al., 2003; Dargan et al., 1997
; Davison et al., 2003
; Goldmacher et al., 1999
; Mocarski et al., 1997
), in particular at the ends of the UL/b sequence boundary where the
2.7 gene resides (Cha et al., 1996
). We were particularly concerned that a number of genes within these regions exhibit a high degree of sequence variation between clinical isolates (Lurain et al., 1999
; A. Davison, personal communication). We wished to evaluate the integrity of the
2.7 gene within the laboratory strains and, more generally, sequence variation in the
2.7 gene. To this end, the complete gene from the Toledo strain and three HCMV clinical isolates was sequenced. Furthermore, in order to investigate the function of the
2.7 gene, an HCMV deletion mutant was generated in the Toledo strain.
The complete 2.7 gene was amplified by PCR from genomic DNA of the Toledo strain and three recent HCMV clinical isolates, 3157, 6397 and Merlin. Clinical isolates were cultured from urine samples of new-born infants and passaged on human foetal foreskin fibroblasts (HFFFs) fewer than three times prior to analysis. The gene was amplified as two separate overlapping fragments of 1215 and 1697 bp (covering 2421 nucleotides; 45752574 of the strain AD169 sequence, GenBank accession no. X17403) using an enzyme with proof reading activity (Dynazyme EXT; Finnzymes). PCR-generated products were cloned into pCR2.1-TOPO (Invitrogen) and sequenced using the BigDye Terminator Cycle Sequencing Kit (PE Applied Biosystems). Overall, the gene exhibited greater than 99 % conservation throughout its entire length compared to the strain AD169 sequence. However, the Toledo strain and each of the three clinical isolates exhibited variations in the sequence corresponding to the RL4 ORF leading to disruption and truncation of the predicted protein sequence (Fig. 1
A). Each of the sequence variations in RL4 was confirmed by sequencing directly three independently amplified PCR products from the same DNA samples.
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The putative protein products of the ORFs with the same start codon as RL4 in strain AD169 are aligned in Fig. 1(B). The protein products of the Toledo strain, 6397 and Merlin have homology with the amino terminus of the strain AD169 putative protein product; however, each of these viruses exhibits premature truncation of the postulated protein. The RL4 sequence encoded by isolate 3157 diverges from strain AD169 after only nine amino acids resulting in a major disruption and truncation of the postulated open reading frame. No major conserved ORFs were generated by changes detected in the gene sequence. The sequences corresponding to the two short upstream ORFs found in strain AD169 were conserved in strain Toledo and the clinical isolates (not shown).
To determine the copy number of the 2.7 gene in the Toledo strain and three clinical isolates, DNA was prepared from purified virus stocks. Virus harvested in supernatants from infected-cell monolayers was centrifuged at 12 000 g, resuspended in TE buffer (10 mM Tris, 1 mM EDTA) pH 8·0, 1 % NP40 and digested overnight at 37 °C with proteinase K (200 µg ml-1). DNA was then extracted gently with phenol/chloroform/isoamyl alcohol (25 : 24 : 1) before being precipitated and resuspended in TE buffer. Viral DNA was digested with HindIII and KpnI to identify the copy number of the gene. Digested DNA was run on 0·50·8 % agarose/1x TBE (Tris/borate/EDTA) gels. The DNA was then transferred to a nitrocellulose membrane by capillary action in 20x SSC overnight and fixed by baking at 80 °C for 2 h. The membrane was then hybridized with a DNA probe labelled with [
-32P]CTP using HighPrime (Roche) and washed as described previously (Phillips et al., 1998
) before autoradiography.
KpnI cuts 584 bp downstream of the transcriptional start site of the 2.7 gene and in the UL segment of the genome in strain AD169 (Chee et al., 1990
). When KpnI-digested strain AD169 DNA is hybridized against a PCR-generated probe specific for the first 527 bp of the gene, two DNA fragments are recognized owing to the presence of the gene in both copies of the repeats flanking UL (Fig. 2A
). However, when KpnI-digested DNA of each of the other isolates is similarly probed only a single band is recognized, indicating that the
2.7 gene is only single copy in each of these viruses. This was confirmed by digesting with HindIII and probing with a
2.7-specific sequence (Fig. 2B
). Digested DNA of strain AD169 produces six bands because HindIII cuts outside of the repeats and the genome inverts to yield four individual isomers (Fig. 2C
). When DNA from the other viruses is similarly hybridized the
2.7-specific probe anneals to only three fragments; two of these fragments are present in 0·25 M internal repeats and the other fragment detected denotes a 0·5 M terminal fragment. This indicates that the
2.7 gene of HCMV clinical isolates lies in the UL segment of the genome close to flanking repeats.
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A virus mutant containing the predicted recombination was confirmed by Southern analysis (Fig. 3). When EcoRV-digested DNA of Toledo and TOL
2.7 was probed with a
2.7 promoter fragment (nucleotides 54554576) a size difference of
1700 bp is detected, which is consistent with targeted replacement of 2421 bp of the
2.7 gene with the 720 bp of EGFP (Fig. 3A
). When similarly digested DNA of both viruses is probed with a 2466 bp PCR-generated fragment covering the complete
2.7 gene a band is only detected from Toledo DNA, confirming the complete deletion of
2.7 from the mutant virus (Fig. 3B
). Additional digests confirmed the predicted genomic structure of TOL
2.7 (not shown).
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The complete 2.7 gene sequence is clearly highly conserved not only in HCMV clinical isolates but also in the common laboratory strains, thus demonstrating that the gene sequence was not substantially modified during prolonged in vitro passage. In contrast, the major predicted ORF TRL4/IRL4 is not conserved, which would be consistent with the
2.7 gene not encoding a functional translation product. A number of viruses encode RNAs that are apparently not translated but function at the transcript level including the human herpesvirus-8 abundant 1·1 kb nuclear untranslated RNA (nut-1 RNA) (Zhong & Ganem, 1997
), the EBERs of EpsteinBarr virus, the LAT transcript of herpes simplex virus-1 and VA1 and VAII RNAs of adenovirus (Sharp et al., 1993
; Perng et al., 2000
). An interaction between the
2.7 transcript and ribonucleoproteins La and Ro has been reported (Lord et al., 1989
). However no major alteration could be detected in the subcellular localization or relative abundance of these proteins following infection with Toledo or TOL
2.7 (not shown).
The 2.7 kb transcript is found predominantly in the cytoplasm, does associate with polysomes and reporter genes cloned within the gene are expressed at an extremely high level (Gawn & Greaves, 2002; Geballe et al., 1986
; Spaete & Mocarski, 1985
) (this study, not shown). While it seems probable that the
2.7 transcript does not encode a functional translation product, one cannot yet preclude a potential role for translation products of the short conserved upstream 24 and 108 bp ORFs. However, a comparative analysis of Toledo- or TOL
2.7-infected cell extracts by MALDITOF mass spectroscopy was unable to detect peptides corresponding to either of the short predicted translation products (data not shown).
Each of the clinical isolates analysed contains only a single copy of the 2.7 gene with the size of the b sequence much reduced compared to the more commonly used laboratory strains and similar to those previously noted in strains Davis (DeMarchi, 1984
) and Toledo (Prichard et al., 2001
). Our study is consistent with the contention that
2.7 is a haploid gene that has become duplicated due to expansion of the b sequence in the heavily passaged strains AD169 and Towne. Although the
2.7 gene is clearly dispensable for efficient HCMV replication in fibroblasts in vitro, the observation that a duplication of this abundantly transcribed gene appears to have occurred independently in two heavily passaged strains is suggestive that the gene's function may not be exclusively restricted to a role in vivo.
While there is no obvious 2.7 gene homologue in any other characterized
-herpesviruses, it is interesting to note that the equivalent region of chimpanzee CMV (GenBank accession no. AF480884) does contain extended regions with significant sequence homology. However, the homology does not extend into the
2.7 promoter element and it has yet to be established if these sequences are transcribed in the chimpanzee virus. Finally, the high level of sequence conservation of the
2.7 gene combined with its abundant transcription makes it an extremely attractive target for nucleic acid-based detection methods of HCMV in clinical samples (Aono et al., 1998
).
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ACKNOWLEDGEMENTS |
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Received 16 April 2003;
accepted 26 June 2003.