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Journal of Virology, August 1999, p. 6265-6270, Vol. 73, No. 8
Department of
Biology,1 Department of
Pediatrics,3 Center for AIDS
Research,4 and Center for Molecular
Genetics,5 University of California, San Diego,
La Jolla, California 92093-0116, and Axys Pharmaceuticals,
La Jolla, California 920372
Received 6 January 1999/Accepted 12 April 1999
We show the presence of numerous short tandem repeats in the human
cytomegalovirus (HCMV) genome and assess their usefulness as molecular
markers. The genome is shown to contain at least 24 microsatellite
regions that exhibit length polymorphisms. Insertion-deletion polymorphisms at these short tandem repeats are common (80% of repeats
examined are polymorphic among two laboratory strains and 10 clinical
isolates). This is the first report of widespread microsatellite length
polymorphism in a viral genome. Some regions are highly polymorphic:
one was revealed by DNA sequencing to contain length variants at five
closely linked sites, which combined resulted in 10 variants for this
region among the 12 strains and isolates examined. This study not
only provides a new molecular marker system for this virus but also
extends our understanding of microsatellite polymorphism in two
important ways. First, variable-length repeats in HCMV can be
considerably shorter than polymorphic repeats previously found in other
organisms. Second, highly variable microsatellite repeats are not
confined to prokaryotes and eukaryotes, as previously assumed. This
variation provides a useful marker system for distinguishing viral
isolates, and similar markers are also likely to be found in other
large-genome DNA viruses.
Short tandem repeats, or
microsatellites, defined as iterations of short motifs made up
of one to six bases, are known to be hot spots of length mutation in
higher (42) and lower (14) eukaryotes. Even
prokaryotic species have short iterations of short tandem repeats
(14, 15) that can vary in length (16, 33), as
well as longer hypermutable repeats that regulate virulence factors
(28). Insertions and deletions at microsatellite loci are
generally considered to be due to replication slippage errors (36,
42). Polymorphisms that result from these slippage errors are
valuable in pathogenic species because of their use as epidemiological markers (13, 26, 27).
Microsatellites are unusual for the following reasons. First, because
of replication slippage, they undergo a much higher rate of both
insertion and deletion mutations than nonrepetitive regions. Second,
unlike point mutations and insertions-deletions in nonrepetitive DNA,
microsatellite mutations are highly reversible, with back mutation
rates similar to forward mutation rates (8, 25, 46, 48). In
some cases, microsatellites can provide pathogenic microorganisms with
functional variation adaptive in rapidly changing environments
(28, 29).
It has been suspected that viral genomes may also be prone to the
accumulation of replication slippage errors (23). Variation has been found at the minisatellite level (defined as repeats of a
motif of 11 to 60 bp [30]) in herpes simplex virus,
although no clinical correlates of this variation have been discovered (44). Such large, minisatellite repeats have also been found within Epstein-Barr virus (4, 20, 21, 24, 46) and have proven useful in epidemiological studies of this virus (12, 37). These mutations affect very long repetitive
regions, but other mutations that affect repeats of
very short motifs have been found.
Several examples of functional microsatellite tracts have been
found among different classes of viruses. Functional
mononucleotide repeats have been found in mengovirus (10,
22), vesicular stomatitis virus (VSV) (2), hepatitis C
virus (50), and human respiratory syncytial virus
(18). Changes in the length of trinucleotide and
hexanucleotide repeats at the hemagglutinin cleavage site in avian
influenza virus have been associated with increased virulence (31,
49). While these microsatellite tracts function in different ways
within each virus, their presence in a variety of lengths in several
types of viral populations demonstrates that mutational processes
leading to expansion and contraction of nucleotide repeats do occur in viruses.
Although some effects of repetitive DNA sequences within viruses have
been demonstrated, the full extent of naturally occurring variation at
a variety of repeat loci, particularly at short repeats, has not been
investigated for any virus. Studies in humans have shown that repeats
less than five units long are typically monomorphic, and it has been
generally assumed that extremely short iterations do not undergo
replication slippage (47). Long repeats have been shown
experimentally to be more mutable than short ones in yeast
(43). Extremely short repeats, however, have not been well
investigated in any organism. In this study, we have investigated the
prevalence of length polymorphisms at short tandem repeats in human
cytomegalovirus (HCMV). HCMV is a large double-stranded DNA virus of
clinical importance. HCMV infection is considered endemic throughout
the world, with seroprevalence in adults ranging from 50 to 90%, and
infections are associated with a diverse range of clinical syndromes
(39). We have examined the potential of insertion-deletion
events at short tandem repeats to differentiate clinical viral
isolates. It might seem unlikely that the short repeats in this genome
would be sites of length polymorphisms. However, abundant length
polymorphisms were discovered at many of these repeats.
Computer survey of the genome.
A computer survey of the HCMV
AD169 genome (7) (GenBank entry HEHCMVCG) was carried out by
using a program written in True Basic for the Macintosh and available
from D.F. on request (12a). The program searches for all
possible mono- through hexanucleotide repeats and has been previously
described (14). Expected repeat distributions are derived
from the mean distribution of repeat sequences in randomly generated
genomes of identical length and base content (the HCMV genome is 57%
G+C). We targeted our attention on sites that had at least four
trinucleotide repeat units or five dinucleotide repeats or where the
mononucleotide tract was nine or more bases long. This resulted in 85 potentially polymorphic repeat sites.
Source of strains.
A total of 12 isolates were used to test
for polymorphisms at short repeats. HCMV laboratory strains AD169
(34) and Towne (32) were obtained from the
American Type Culture Collection (Rockville, Md.). Of the 10 clinical
isolates examined, 8 were cultured from five urine specimens and three
blood specimens of seven individuals diagnosed with AIDS-related CMV
retinitis. Two isolates were cultured from bronchial alveolar lavage of
bone marrow transplant recipients who were receiving treatment for CMV
pneumonitis. CMV cultures were expanded on human foreskin fibroblasts
maintained in Dulbecco's modified Eagle's medium supplemented with
10% fetal bovine serum, 2 mM L-glutamine, 100 U of
penicillin per ml, 0.01% streptomycin, and 20 µg of amphotericin B
per ml. DNA was purified from fully cytopathic CMV-infected human
foreskin fibroblasts by phenol extraction and ethanol precipitation as described previously (38).
PCR and analysis.
Primer pairs were designed to amplify 42 of the 85 longest mono-, di-, and trinucleotide repeats in the HCMV
genome. A total of 30 primer pairs were manually designed, each pair
amplifying a product containing from one to four target repeats (Table
1) and purchased from Research Genetics
(Huntsville, Ala.). PCR was performed as previously described
(13), with the addition of 0.1 µl of fluorescence-labeled
dCTP from Perkin-Elmer (Foster City, Calif.). A Perkin-Elmer 2400 thermocycler was used for the reactions, with a PCR program of 40 cycles of 1-min denaturation at 94°C, 1-min annealing at various
temperatures depending on primer (Table 1), and 1-min extension at
72°C. All products were analyzed on an AB1 373 automated DNA
sequencer (Applied Biosystems, Inc.) using labeled dCTP incorporation.
Genescan software was used to search for polymorphisms by length. All
polymorphisms were confirmed by analysis of independently amplified PCR
products. Sequencing to confirm length changes was carried out on an
ABI 373 sequencer, and all reported sequences were determined twice in
each direction. Before sequencing, all reaction products were cleaned
by using a QiaQuick PCR Clean-up kit from Qiagen (Santa Clarita,
Calif.).
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Numerous Length Polymorphisms at Short Tandem
Repeats in Human Cytomegalovirus
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
TABLE 1.
Primer pairs found to be polymorphic in this survey
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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The computer survey revealed that the HCMV genome contains
only repeats with very short iterations (two to five units), and there
is a slight excess of the longer mono-, di-, and trinucleotide repeats
compared to randomized genomes of equivalent nucleotide composition and
size (14). Observed and expected repeat distributions are
compared in Table 2. For all repeat
classes, longer repeats are more common than expected; however, in the
HCMV genome the maximum number of repeat units of a di- or
trinucleotide motif is only five, and the longest mononucleotide tract
is 13 bases.
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We surveyed 42 of the longest tandem repeats in 12 HCMV isolates, using 30 PCR primer pairs. Despite the short lengths of the repeats analyzed, 24 of 30 PCR primers (80%) amplified multiple length forms (Table 1), demonstrating repeat polymorphism, in this small group. All 12 isolates could be uniquely identified by using the 24 polymorphic regions. Figure 1 is a diagram of the HCMV AD169 genome showing positions of the polymorphic and monomorphic microsatellites that have been identified in this study.
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Variability in the number of repeat units at some of the targeted short tandem repeats was confirmed by sequencing, as shown in Fig. 2. In most cases, for the di- and trinucleotide repeats, more variants were revealed by sequencing than by size separation. For example, separation by length at the TRAN3 locus showed 4 length variants, while sequencing revealed a total of 10 different variants among the 12 strains. The microsatellites in UL69 and TRAN3 are both strikingly complex, with polymorphisms in more than one contiguous or neighboring repeat. Further, some of the polymorphic repeats in these clustered microsatellites (13) have as few as two repeat units, similar to the pattern seen in short clustered microsatellites in Candida albicans (27).
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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The 12 HCMV isolates examined were distinguished from one another by variability at 24 repeat regions. Therefore, this study provides a substantial panel of molecular markers with which to investigate evolutionary and epidemiological patterns in this important pathogen. Because the genome of HCMV is smaller than those of the eukaryotes and prokaryotes in which microsatellite variation has been previously utilized, it will be possible to survey virtually all microsatellite variation in HCMV populations. Our results suggest that this will also be a useful approach to the development of molecular markers in similar viruses.
The microsatellites screened here do not yet constitute an exhaustive survey of the HCMV genome; we surveyed only 49% of the total repeats longer than the chosen cutoff (tracts of four trinucleotide, five dinucleotide, or nine mononucleotide repeats), and these excluded the regions of the Toledo genome that are not found in Towne or AD169 (6). Therefore, a variety of additional length polymorphisms may be present throughout the rest of the genome. Examination of these additional repetitive regions will increase the likelihood of finding specific polymorphic markers that are linked to biological or pathological phenotypes.
A single set of primers often flanks multiple short polymorphic iteractions, and sequencing reveals that length variants from different isolates with the same overall length are often made up of more than one sequence (Fig. 2). This phenomenon, in which apparently equivalent length variants can be shown to have different mutational histories, is an example of homoplasy (40), and many cases of homoplasy in microsatellite length variants can be resolved by sequencing (11, 19, 27). Thus, variation in length offers only a first order of discrimination. Sequencing of alleles or viral length variants adds a second level and is necessary to obtain maximum resolution between viral isolates.
Sequencing to confirm length variation at tandem repeats also revealed a variety of point mutations. It has been shown that there are more point mutations in regions around microsatellites than in nonrepetitive regions (9). Targeting of microsatellites therefore permits the detection of more point mutations than would be found if random segments of the genome of the same length were sequenced.
Many of the HCMV repeats are found in noncoding regions and are associated with a high local level of base pair polymorphism. This suggests that these polymorphisms are evolutionarily neutral or nearly so. They are therefore likely to provide appropriate markers for epidemiology and strain identification. Other repeats are found in known or putative coding regions, promoters, operators, and other functional DNA. These may not be neutral and may therefore be limited in utility as markers. These microsatellites, however, may provide adaptive variation important to viral evolution and genetic variability, perhaps similar to the functionally important mononucleotide runs found in VSV (2) and respiratory syncytial virus (18). If changes in these microsatellites are found to directly influence the expression of a gene or function of a gene product, studies of these polymorphisms may provide novel insight into gene regulation in the virus.
While microsatellites are frequently used as markers in mapping, gene function, and population studies of higher eukaryotes (1, 5, 17), the full extent of such repeats has not been determined in any virus. Other classes of repeats have been examined. Many studies have used polymorphic restriction enzyme sites as epidemiological markers. Herpesvirus restriction enzyme polymorphisms have been examined in several studies, some of which have found relatively little variation (3, 45). Some restriction enzyme variants have been of use in distinguishing among different strains of VZV (41) and even in detecting different patterns of herpesvirus evolution in different parts of the world (35). Microsatellites are likely to evolve more quickly than restriction sites. This will make them useful for following changes in pathogen populations over short time scales.
None of the repeats examined in this study are long enough to have been included in a similar survey of a eukaryotic genome for microsatellite length variants (48). Despite previous assumptions that such polymorphisms should be extremely rare, this study demonstrates the potential of very short repeats to provide polymorphic molecular markers. It also reveals an important role for insertion-deletion mutations as a mechanism for producing genetic variation in viral genomes, as previously demonstrated for eukaryotic and prokaryotic genomes. This study provides an important new type of molecular marker with which to investigate questions of epidemiology and evolution within this virus. While these short tandem repeats are shorter than those frequently examined for studies of microsatellite variation, the insertion-deletion events at these repeats clearly classifies them as microsatellites.
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ACKNOWLEDGMENTS |
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This work was supported by grant 1-RO3 AI41370 from the National Institute of Allergy and Infectious Diseases (to C.W.). D.M. is an NSCORT graduate fellow, and D.F. is a Markey graduate fellow.
We are most grateful to Chris Lambros for making this study possible. We thank Frank Huang and Tamarah Westmoreland for assistance.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Biology 0116, University of California, San Diego, La Jolla, CA 92093. Phone: (619) 534-4113. Fax: (619) 534-7108. E-mail: cldavis{at}ucsd.edu and cwills{at}ucsd.edu.
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Journal of Virology, August 1999, p. 6265-6270, Vol. 73, No. 8
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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