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Antimicrobial Agents and Chemotherapy, June 1999, p. 1500-1502, Vol. 43, No. 6
VA Medical Center, Portland,
Oregon1; Rush Medical Center, Chicago,
Illinois2; University of Colorado Health
Sciences Center, Denver, Colorado3; and
Beth-Israel Deaconess Medical Center, Boston,
Massachusetts4
Received 28 October 1998/Returned for modification 20 January
1999/Accepted 30 March 1999
The polymerase (pol) coding sequence was determined for
40 independent clinical cytomegalovirus isolates sensitive to
ganciclovir and foscarnet. Sequence alignments showed >98%
interstrain homology and amino acid variation in only 4% of the 1,237 codons. Almost all variation occurred outside of conserved functional
domains where resistance mutations have been identified.
The technical difficulty and
time-consuming nature of cell culture-based susceptibility assays have
resulted in a need for rapid genotypic assays for cytomegalovirus (CMV)
drug resistance (9). Development of such assays will depend
on knowledge about the relative frequency of various resistance-related
mutations and the extent of interstrain variation that occurs among
drug-sensitive clinical isolates.
The CMV DNA polymerase (pol; UL54) (reviewed in reference
1) and the UL97 phosphotransferase are two viral
genes where certain mutations have been linked to drug resistance
(9). Ganciclovir resistance results from mutations in UL97
(4), pol (11), or both
(13). All known foscarnet and cidofovir resistance mutations
map to the pol gene, and cidofovir-resistant pol
mutants are generally cross-resistant to ganciclovir (7, 8).
It is important to distinguish resistance mutations from natural
interstrain sequence variation. Some of the observed amino acid changes
in the pol genes of resistant clinical isolates (5, 6,
11, 13) have subsequently been validated as resistance markers by
transfer of mutations to laboratory strains of CMV, but others have
not. We performed a multicenter sequencing study to assess the range of
interstrain variation in the pol gene of drug-sensitive
clinical isolates and to compare the loci of variation with resistance
mutations that have been identified to date.
Clinical CMV isolates for this study were collected at four
geographically separate institutions from 40 unrelated subjects who had
received organ transplants or had human immunodeficiency virus
infection. Some patients had received prior anti-CMV therapy. Susceptibility to ganciclovir and foscarnet was determined by plaque
reduction in fibroblast cell culture. The 50% inhibitory drug
concentration (IC50) was determined, and an isolate was
considered sensitive if the ganciclovir IC50 was 6 µM or
less and the foscarnet IC50 was 400 µM or less
(10). For the 40 isolates selected, the ganciclovir
IC50 ranged from 0.8 to 5.8 µM and the foscarnet IC50 ranged from 60 to 280 µM. All isolates were
therefore considered sensitive to both drugs.
The entire 3.7-kb CMV pol coding sequence of all isolates
was amplified by PCR from DNA extracts of infected cell cultures. A
full-length PCR product was obtainable by using the GeneAmp XL PCR kit
(PE Applied Biosystems, Branchburg, N.J.) and the following primers
which flank the pol coding region (forward,
5'-GTCAGCCTCTCACGGTCCGCTAT-3'; reverse,
5'-CTCAGTCTCAGCAGCATCATCAC-3'). The PCR products were sequenced without cloning, by using primers internal to the amplified segment (11) that produced overlapping coverage of the
pol coding sequence. Sequencing reactions were performed by
dideoxy chain termination chemistry, with an automated sequencer (ABI
373 or 377) and fluorescent dideoxy terminators (AmpliTaq FS cycle
sequencing kit; PE Biosystems, Foster City, Calif.), or by manual
methods with the Thermo Sequenase 33P terminator cycle
sequencing kit (Amersham, Arlington Heights, Ill.).
Derived sequences of each isolate were aligned with the strain AD169
reference sequence (EMBL accession no. X17403) by using a sequence text
editor. Codons which differed from strain AD169 were assessed as to
validity of the raw sequence data, and repeat sequencing reactions were
performed to recheck any ambiguity. Amino acid differences were
tabulated, graphed, and compared with previously published
pol resistance mutations. Sequence homology calculations at
the nucleotide and amino acid levels were also performed to compare
each isolate with strain AD169.
We found >98% interstrain homology at both the nucleotide and the
amino acid level. In comparison to the AD169 sequence, the number of
nucleotide changes within the 3,729-bp pol coding sequence for each strain ranges from 25 to 63. The average number of nucleotide changes per strain is 46. There are a total of 282 variant nucleotides, of which >80% produce silent mutations. Among the 1,237 codons in the
pol coding sequence, each isolate has nine or fewer amino acid changes compared to the AD169 sequence. A change from the AD169
sequence was observed at 47 codons (Table
1). This represents 4% of the total
codons in pol. There are only four codons (655, 685, 897, and 1122) where 10 or more isolates have an amino acid configuration
different from the most common one. In most of the rest of the codons
that show variation, only one or two isolates show any change. Changes
that are not amino acid substitutions include a codon insertion at 885 and codon deletions at 1151 and 1156 (Table 1).
0066-4804/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Interstrain Variation in the Human Cytomegalovirus
DNA Polymerase Sequence and Its Effect on Genotypic Diagnosis of
Antiviral Drug Resistance
ABSTRACT
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TABLE 1.
Pol amino acid differences of clinical isolates from
strain AD169
Codons with amino acid changes are strongly clustered outside the previously defined conserved catalytic sequences of pol (12) (Fig. 1). The greatest concentration of variant codons is at 626 to 697, 868 to 899, and 1116 to 1162. Within the conserved regions I to VII and A (Fig. 1), variation occurs only at codon 697 (on the fringe of domain II). In the area between codon 492 and region A (also known as delta-region C) (7, 13), which includes some known resistance mutations (Fig. 1), one isolate has an amino acid change at codon 515. There is no overlap between the amino acid variation observed in these drug-sensitive isolates and the published pol drug resistance mutations that have been proven by marker transfer experiments. The list of codons where a single mutation confers a resistance phenotype upon transfer to strain AD169 or Towne includes 408, 412, 501, 513, 522, 545, 700, 715, 781, 802, 809, and 987 (2, 5-7, 12, 14).
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This study of drug-sensitive CMV clinical isolates demonstrates the very high level of sequence conservation of the DNA polymerase coding region, the strong clustering of the amino acid changes outside the functional conserved domains, and the absence of overlap between baseline sequence variation and the mutations that have been shown to be related to drug resistance. The absence of significant sequence divergence in pol is in contrast to the envelope glycoprotein gB gene which is directly adjacent to pol. In that gene, grouped variation results in sequence divergence of up to 12% among isolates, about 10-fold greater than that seen here in pol (3), and the variation affects functional domains and epitopes.
Increasing information is available concerning the pol mutations in clinical isolates that are markers of drug resistance (5-7) (Fig. 1). Dual resistance to ganciclovir and cidofovir has been mapped to pol region IV (codons 379 to 421), codon 987 in region V, and a region between codons 501 and 545 (5, 7, 8, 12, 13). The only baseline variation observed in these codon ranges is in the latter area (at codon 515). Foscarnet resistance has been mapped to codons 700, 715, 756, 781, 802, 809, and 821, involving regions II, III, and VI (codon range 696 to 845). Mutations in this region sometimes confer low-grade cross-resistance to ganciclovir and other drugs (5-8). No baseline amino acid variation was observed in this region except at codon 697. An amino acid change at codon 676 was previously reported in association with a ganciclovir-cidofovir-resistant isolate (13). The same change was found in two sensitive isolates in this study and has been found by marker transfer to be unrelated to resistance to ganciclovir and cidofovir (7).
Based on current knowledge, genotypic assays for resistance should focus on the pol codon ranges 379 to 421, 492 to 545, 696 to 845, and 978 to 988. The sequence database obtained from this study should be useful for optimization of these genotypic assays.
Nucleotide sequence accession numbers. Sequences determined in this study have been deposited in GenBank under accession no. AF133589 through AF133628.
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ACKNOWLEDGMENTS |
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We thank Matthew Shiveley, Ernest Winkfield, Julia Clark, and Joe Zhou for excellent technical assistance and W. Lawrence Drew and Richard C. Miner for providing nine CMV isolates and their sensitivity data.
This work was supported by subcontracts to the participating laboratories from Social and Scientific Systems based on an NIH/NIAID Cooperative Agreement (AI38858). Sequences obtained at the University of Colorado Health Sciences Center were provided by the University of Colorado Cancer Center DNA Sequencing and Analysis Core Facility, which is supported by the NIH/NCI Cancer Core Support Grant (CA46934). Identification of pol resistance mutations was supported by VA research funds and NIH/NIAID grant AI39938 to S.C. C.S.C. was supported by NIH grants AI41690 and U01 AI27659.
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FOOTNOTES |
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* Corresponding author. Mailing address: Infectious Disease Section P3ID, VA Medical Center, 3710 S.W. U.S. Veterans Hospital Rd., Portland, OR 97201. Phone: (503) 273-5185. Fax: (503) 273-5348. E-mail: chous{at}ohsu.edu.
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REFERENCES |
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