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 Institut für Medizinische Virologie und Epidemiologie der Viruskrankheiten, Universität Tübingen, 72076 Tübingen, Germany
4 Section of Infection and Immunity, University of Wales College of Medicine, Tenovus Building, Heath Park, Cardiff CF14 4XX, UK
Correspondence
Andrew J. Davison
a.davison{at}vir.gla.ac.uk
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ABSTRACT |
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The GenBank accession numbers of the HCMV DNA sequences reported in this paper are listed in Table 1.
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INTRODUCTION |
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The complete DNA sequence of AD169 has been determined (Chee et al., 1990), and comparisons with data for other strains have shown that it is a multiple mutant. It contains frameshift mutations in three genes (RL5A, RL13 and UL131A; Akter et al., 2003
; Davison et al., 2003a
, b
; Yu et al., 2002
), and 19 genes at the right end of UL (UL133UL150) have been replaced by an inverted duplication of a sequence from near the left genome end, leading to a substantial expansion of RL (Cha et al., 1996
; Prichard et al., 2001
). Some stocks contain a deletion that affects two genes (UL42 and UL43; Dargan et al., 1997
; Mocarski et al., 1997
) or a nucleotide substitution that inactivates one gene (UL36; Skaletskaya et al., 2001
). A low passage strain, Toledo, retains sequences at the right end of UL, but inversion of a substantial segment is evident, resulting in disruption of at least one gene (UL128; Cha et al., 1996
; Davison et al., 2003a
; Prichard et al., 2001
). In addition, several other low passage strains have lesions in one or more of a group of three adjacently located genes (UL128, UL130 and UL131A; Akter et al., 2003
). These findings indicate that no laboratory strain can be assumed to be genetically intact.
In this paper, we report the genome sequence of a minimally passaged HCMV strain, Merlin. Together with partial sequences from other strains, including some that have not been passaged in cell culture, we assess the gene content and variability of wild-type HCMV.
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METHODS |
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A random library of Merlin DNA fragments was prepared using a Novagen M13mp18 Perfectly Blunt Cloning kit, and sequenced using ABI PRISM 377 and Beckman CEQ 2000XL sequencing instruments. The sequence database was compiled from electropherograms using Pregap4 and Gap4 (Staden et al., 2000) and Phred (Ewing & Green, 1998
; Ewing et al., 1998
). Regions of ambiguity were resolved by sequencing of PCR products. The final, edited sequence was subjected to thorough manual checking against the electropherograms. It was determined to a mean redundancy of 8·44, and 96·2 % was obtained on both strands. Owing to the presence of inverted repeats in the genome, the database sequence contained incomplete copies of TRL and TRS at the ends. The genome sequence was reconstructed after locating the genome termini, which were predicted approximately from similarities to AD169, and mapped experimentally as described by Davison et al. (2003a)
. Merlin DNA was flush-ended and ligated to a partially double-stranded adaptor as supplied in the Clontech Marathon kit, and each terminus was identified by PCR using an adaptor-specific primer plus a Merlin-specific primer. Internal forms of the termini are present in the genome in the form of the junction between two a sequences, and were amplified using the two Merlin-specific primers. Products were ligated into M13mp18 or pGEM-T (Promega) and the inserts sequenced.
The GenBank accession number of the Merlin genome sequence is AY446894. The sequence was analysed using the GCG programs (Accelrys), the ClustalW sequence alignment program (Thompson et al., 1994) and the Ptrans sequence translation program (Taylor, 1986
). Comparisons were carried out with the updated AD169 genome sequence (Chee et al., 1990
; Davison et al., 2003a
; BK000394) and regions at the right ends of UL in Toledo and Towne (Cha et al., 1996
; U33331 and U33332).
Sequencing of regions in the genomes of additional strains.
Specific regions of additional strains were analysed by sequencing cloned PCR products. PCR primers were designed initially on the basis of conservation between the AD169, Merlin and, where available, Toledo sequences. In the initial round of sequencing, vector-specific primers were used to sequence into the ends of the inserts. In subsequent rounds, HCMV-specific primers were designed on the basis of conservation in the strains analysed, obtaining data for both strands. As data accumulated, this approach resulted in iterative redesign of PCR and sequencing primers, utilizing aligned sequences at each step in order to produce a set of primers that would work with all strains. The most recent set of PCR primers used is given in Table 2. Some of the sequences at the left end of UL were so variable that it was not possible to design universal sequencing primers, and genotype-specific primers were then used. For several more strains, PCR products containing UL146 were sequenced directly.
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Data were compiled as for Merlin, omitting PCR primer sequences. The consensus for each sequence was established on simple majority of aligned readings, and nucleotides differing from the consensus were examined individually. PCR-induced errors were apparent as nucleotides in single PCR clones that did not match those in the other three. In a few instances of ambiguity, where two clones differed from the other two, either an independent PCR product was sequenced or the consensus was adjusted to agree with other strains at that position. The absence of mixtures of strains in the same sample was monitored by checking that regions of overlap between PCR products (67430 bp; Table 2) were identical and that no clones differed substantially in sequence from others for that fragment. Two samples in which the latter criterion was not met were analysed further as described.
GenBank accession numbers for the sequences determined are given in Table 1. The data were analysed as for Merlin. Phylogenetic analysis used the PHYLIP package (Felsenstein, 1989
).
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RESULTS AND DISCUSSION |
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Previously, we reinterpreted the gene layout in the genome of AD169 and at the right end of UL in Toledo by comparisons with the closest relative of HCMV, chimpanzee cytomegalovirus (CCMV), allowing, where appropriate, for the presence of genes unique to either genome (Akter et al., 2003; Davison et al., 2003a
, b
). The conclusions were extended to yield the predicted genetic content of Merlin illustrated in Fig. 1
. Our interpretations of the genetic content of AD169 and Toledo were essentially confirmed by Murphy et al. (2003)
. These workers also predicted 12 novel protein-coding open reading frames (ORFs) in AD169 using a bioinformatics tool (the Bio-Dictionary-based gene finder, BDGF), most of which are small or extensively overlap recognized genes. Given the modest level of discrimination achieved with this algorithm even for recognized genes, and the absence of additional data, we consider it premature to include these ORFs in the gene layout.
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In order to compare Merlin with other strains in regions where AD169 or Toledo is mutated, substantial sections at the left (18·4 kbp for five strains, containing RL1UL11) and right ends of UL (25·6 kbp for eight strains, containing UL120UL150) were sequenced as clones derived from overlapping PCR products. A high level of accuracy was evident in that the sequence at the right end of UL for Merlin determined by PCR was identical to that obtained by M13 sequencing of the genome, except for one homopolymeric tract, which contained nine T residues in the former analysis and ten in the latter. Evolutionary selection against homopolymeric tracts in protein-coding regions is apparent in the Merlin genome, as only one of the 47 tracts of nine or more residues is located in a coding region (UL88). This is presumably because slippage during viral DNA replication promotes a degree of length instability. Within the regions in Merlin for which sequence data were derived for other strains, there are seven such tracts at the left end of UL and two at the right end. All exhibited intra-strain length variation in cloned PCR products but not in cognate M13 clones obtained for sequencing the Merlin genome. This indicates that the degree of length instability occurring during PCR is much greater than any occurring during viral DNA replication, and that the lengths of tracts of nine or more residues derived by PCR must be viewed as approximate. This accords with Shinde et al. (2003), who described slippage during PCR of templates containing a tract of nine or more A residues.
The PCR-derived sequences were compared with data published previously for two of the strains analysed, and also facilitated a reassessment of gene content in the regions concerned. An 18 kbp segment of the sequence of the right end of UL in Toledo determined by Cha et al. (1996) differs from our version by five nucleotide differences and six single nucleotide insertions or deletions. Four of the latter cause frameshifting, in UL140, UL141, UL145 and UL150. A 2·2 kbp segment of the sequence published by Cha et al. (1996)
for Towne (short variant; see the footnote to Table 1
) differs at the 5' end of UL148 by three deletions of 13 bp and two substitutions. In our previous assessment of HCMV gene layout (Davison et al., 2003a
), UL3 was the most marginal gene listed by Chee et al. (1990)
to be retained. It lacks a counterpart in CCMV and partially overlaps UL2, which has a counterpart. Unusually, the termination codon for UL3 is variable in location between strains, and the UL3 reading frame contains an internal stop codon in one unpassaged strain (3301). Therefore, we have downgraded UL3 to an ORF unlikely to encode protein, and have omitted it from Fig. 1
. In updating the gene layout, we also noted that UL120 and UL121 are distantly related to each other, and therefore form a new gene family.
CCMV has three genes at the right extremity of UL that lack counterparts in Merlin (Davison et al., 2003a). In order to determine whether Merlin has suffered a deletion in this region, we attempted to identify the right end of UL in the sequences derived for other strains by PCR. This was approached by anchoring the sequences at both ends of UL within TRL or IRL using a PCR primer located completely within RL, and was successful for three strains. The results confirmed that Merlin has the same gene content at the ends of UL as two other low passage strains (3157 and 6397) and one unpassaged strain (W), with the junctions between UL and TRL or IRL located identically in each strain. Although the right end fragment of UL was amplified in two other strains (Toledo and 3301) using the primer within RL, the left end of UL was not, and it was necessary to use a primer marginally inside UL to amplify this region. It is not known whether this failure was technical or whether the ends of UL are located differently in these two strains. Nonetheless, we concluded that Merlin has not lost sequences at the ends of UL during passage and found no evidence for HCMV counterparts to the additional CCMV genes.
Two samples (W and TB40/E) appeared to comprise mixtures of two strains, and additional clones were sequenced for the variable UL146 gene in order to estimate their proportions. In each case, one strain proved much more abundant than the other, allowing the sequence of the major strain to be assembled with confidence. The presence of a second strain in W was apparent from a minority of clones from several PCR products that differed significantly from the majority. In an analysis of 28 plasmids containing the UL146 gene, only one originated from the minor strain. Additional experiments using different PCR primers were carried out in order to confirm the fragment linkages. TB40/E was revealed as containing a second strain by the detection of a single diverged clone for one PCR product. The level of contamination was low, since all the UL146 sequences from 16 plasmids originated from the major strain.
The presence of multiple strains in W is characteristic of unpassaged samples from AIDS patients. For example, ML also yielded two UL146 sequences (Table 1). TB40/E has an extensive passage history (Sinzger et al., 1999
, 2000
), which involved culture of the original specimen (TB40) for five passages in HFFs, followed by transfer in parallel for 35 passages in HFF or human umbilical vein endothelial cell (HUVEC) cultures. By passage 20, the virus grown in HFFs had lost the ability to grow in HUVECs, whereas the virus grown in HUVECs retained the ability to grow in HFFs. At passage 35, each virus was plaque purified, yielding TB40/F (grown in HFFs) and TB40/E (grown in HUVECs). TB40/E subsequently retained the ability to grow in HUVECs even after 40 passages in HFFs. This is in contrast to most HCMV strains, which lose their ability to grow in HUVECs after adaptation to HFFs. The stock of TB40/E analysed in the present study had undergone a further 510 passages in HFFs after plaque purification in HUVECs. Comparative sequence data were also obtained for earlier passages, specifically the TB40-derived viruses from passage 22 during the 35 passages on HFFs and HUVECs. Unexpectedly, the only virus detected in these samples was the minor component of TB40/E, although it cannot be ruled out that the major component was also present at a low level. The involvement of more than one virus in the derivation of TB40/E cautions against its use as a general laboratory strain, and complicates any correlations between genotype and phenotype that may be made using it.
Sequence variation among HCMV strains has been documented previously, largely as the result of studies on single genes. The availability of the Merlin and AD169 sequences, plus the region in Toledo that is absent from AD169, afforded the opportunity to analyse variation across the complete gene set. Fig. 2 shows Ka values (a measure of nonsynonymous nucleotide substitution, which underlies amino acid sequence divergence) computed for pairwise alignments of coding sequences. A sizeable set of genes displays unusually high variation, in some cases to a very impressive degree; for example, the RL12 proteins are only 49 % identical in amino acid sequence. This set comprises many, but not all, genes that encode proteins known or predicted to be secreted or membrane-associated; all 25 genes with Ka values of 0·03 or more are in this category. This correlation suggests that high rates of divergence have been driven by exposure to components of the immune system. Certain regions considered not to encode protein also exhibit high sequence divergence, including RL and the region between US34A and TRS1.
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In comparison with the two unpassaged strains, all of the passaged strains analysed have visibly disabling mutations in UL128, UL130 or UL131A, and most have mutations in at least one member of the RL11 gene family. Strains that have been passaged in HFFs have been noted previously to contain mutations in one or more of UL128, UL130 and UL131A, of which the first and last are spliced (Akter et al., 2003; G. Hahn, M. Revello, M. Patrone, E. Percivalle, G. Campanini, A. Sarasini, M. Wagner, A. Gallina, G. Milanesi, U. Koszinowski, F. Baldanti & G. Gerna, unpublished results). Thus, Merlin, 3157 and Toledo are mutated in UL128, 6397 lacks UL130 and UL131A, and AD169 has a lesion in UL131A. UL128 is also mutated in CCMV and the Colburn strain of simian cytomegalovirus (Davison et al., 2003a
). The list of HCMV strains mutated in this region can now be extended to include Davis, which has a frameshift in the 5' region of UL130, and Towne, which has a frameshift near the 3' end of this gene (also shown by G. Hahn and others, unpublished results). Unusually, the coding regions for UL128, UL130 and UL131A are intact in TB40/E, but a cysteine codon, which is conserved between HCMV and CCMV in the region near the 3' end of UL130 that is compromised in Towne, is substituted by a serine codon. It is possible that this difference modifies the function of UL130 in a way that retains the ability of the virus to grow in HUVEC and HFF cells. In addition, TB40/E is frameshifted in UL141, although the presumed parent with this gene intact was also detected at low levels. With regard to the RL11 gene family, 3157 is frameshifted in RL13, 6397 is frameshifted in RL13 and has an in-frame termination codon in UL9 as a result of a nucleotide substitution, Toledo has an in-frame deletion in RL13 (Yu et al., 2002
) and is frameshifted in UL9, and AD169 is frameshifted in RL5A and RL13 (Davison et al., 2003a
, b
; Yu et al., 2002
). These lesions were confirmed by additional PCR experiments. It is also evident from the data of Cha et al. (1996)
that RL13 is frameshifted in Towne. Of the passaged strains examined, only Merlin appears to be wild-type in the RL11 gene family. In summary, the genotypes of strains for which data are available for both the RL11 gene family and the region containing UL128, UL130 and UL131A are: Merlin, UL128; 3157, RL13, UL128; 6397, RL13, UL9, UL128; Toledo, RL13 (although protein function might be maintained since the deletion is in-frame), UL9, UL128; and AD169, RL5A, RL13, UL131A (plus other mutations identified outside these regions).
It seems likely that the genes susceptible to ready mutation upon passage in cell culture have roles in tropism or pathogenicity. UL128, UL130 and UL131A encode predicted secreted proteins, and the UL128 protein has the sequence characteristics of a CC-chemokine (Akter et al., 2003; G. Hahn and others, unpublished results). Adaptation of HCMV to HFFs is associated with disruption of any one of these genes, and G. Hahn and others (unpublished results) have demonstrated that each gene is required for growth in HUVECs. The proteins encoded by the RL11 gene family are loosely related to each other and to proteins encoded by a gene family in the E3 region of human adenoviruses (Davison et al., 2003b
). Their functions are unknown, although the RL11 protein has been shown to bind the Fc domain of immunoglobulin G (Atalay et al., 2002
; Lilley et al., 2001
). Potential roles for members of the RL11 gene family in tropism have been indicated by Dunn et al. (2003)
from global mutational analysis of a bacterial artificial chromosome (BAC) of Towne. These workers reported that, in comparison with unmutated virus, a UL9 mutant grew better in HFFs and a UL10 mutant grew much better in an epithelial cell line. However, in a similar study done with a BAC of AD169, Yu et al. (2003)
did not note enhanced growth of UL9 mutants in HFFs. It is possible that this difference was due to the use of different parental viruses (both of which were multiple mutants at the outset). Also, the propensity for HCMV to mutate during passage prompts caution regarding the genotypes of engineered mutants, particularly in studies of tropism. The potential roles of genes in the RL11 family in growth and tropism thus remain to be defined.
The HCMV genome contains several extensive regions that apparently do not encode proteins (Fig. 1). Exploiting earlier studies in one of these regions (DeMarchi, 1983
; Jahn et al., 1984
; Nelson et al., 1984
; Wathen & Stinski, 1982
), Plachter et al. (1988)
mapped a leftward oriented 5 kb RNA that encompasses nearly all of the region between UL105 and UL111A. This transcript is unusual in that only a minority of the population is polyadenylated [as measured by the ability to bind oligo(dT)], and it is one of several present in virions (Bresnahan & Shenk, 2000
). Plachter et al. (1988)
concluded from sequence analysis that the 5 kb RNA is unlikely to encode a protein. In a later analysis summarized in Fig. 5
(C), Chee et al. (1990)
assigned six small ORFs to this region, though three (UL107, UL108 and UL110) would be even smaller if they were to commence at the first ATG codon, and one (UL111) entirely lacks ATG codons.
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General collinearity between HCMV and CCMV in the UL105UL112 region is apparent in Fig. 5(A), except for a discontinuity caused by the absence of UL111A from CCMV. However, in contrast to recognized protein-coding regions, collinearity in this region is characterized by many local insertions and deletions. Moreover, the sequence has unusual compositional features, consisting of short G+C-rich regions interspersed by longer A+T-rich regions (Fig. 5B
). As expected from their composition, the A+T-rich regions are abundant in termination codons (Fig. 5D
), and thus contain shorter ORFs than the G+C-rich regions where ORFs were defined by Chee et al. (1990)
(Fig. 5C
). A region of variable size (3·311·5 kbp) with similar compositional features is present in all cytomegaloviruses, and an equivalent non-coding region of 2·6 kbp is also present in human herpesviruses 6 and 7, which belong to a distinct genus of the Betaherpesvirinae (Roseolovirus). Conservation of this putative non-protein-coding region over substantial periods of evolution strongly implies an important role. At present, this role is unknown, though it may relate to a function of the 5 kb RNA or, perhaps less likely, to the presence of cis-acting elements.
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ACKNOWLEDGEMENTS |
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Received 9 December 2003;
accepted 26 January 2004.