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Journal of Virology, November 1999, p. 9655-9658, Vol. 73, No. 11
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Preferential Associations of Alleles of Three
Distinct Genes Argue for the Existence of Two Prototype Variants of
Human Herpesvirus 7
Michael
Franti,
Jean-Thierry
Aubin,
Agnes
Gautheret-Dejean,
Isabelle
Malet,
Annie
Cahour,
Jean-Marie
Huraux, and
Henri
Agut*
Laboratoire de Virologie, C.E.R.V.I., UPRES
EA 2387, Hôpital Pitié-Salpétrière, 75651 Paris
Cedex 13, France
Received 2 June 1999/Accepted 9 August 1999
 |
ABSTRACT |
We had previously described six distinct alleles of the
glycoprotein B (gB) gene of human herpesvirus 7 (HHV-7). The genetic changes corresponding to these alleles did not affect gB gene transcription or translation in in vitro assays. The study of distinct
HHV-7-positive human samples showed preferential associations of some
gB alleles with some alleles of two other genes, distantly located on
the HHV-7 genome, coding for the phosphoprotein p100 (p100) and the
major capsid protein (MCP). Two allele combinations, corresponding to
44 and 31% of the samples studied, respectively, were interpreted as
the genetic signatures of two major prototype HHV-7 variants.
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TEXT |
Human herpesvirus 7 (HHV-7) was
originally isolated from the stimulated CD4+ T cells of a
healthy individual (9) and was subsequently characterized as
a ubiquitous virus, infecting most human beings (5, 10, 17, 25,
27). This virus was classified in the
Betaherpesvirinae subfamily on the basis of its genetic
organization (3, 6, 15, 16). Infected saliva is generally
considered to be the main source of human transmission (4,
26). Although many diseases have been hypothetically related to
HHV-7 infection (1-3, 7, 12, 13, 18, 19, 21, 23, 24),
convincing proof and precise knowledge of its pathogenicity are still missing.
The entire genome of HHV-7 has been recently sequenced for two
reference strains: JI (16) and RK (15).
Comparison of these two strains has shown the high degree of
conservation of the HHV-7 genome. However, a previous study had found a
restricted polymorphism of the glycoprotein B (gB) gene: five critical
positions were identified as the sites of point nucleotide
substitutions, and the stable combination of specific changes at these
positions allowed us to define six alleles of the gene (8).
The distribution of gB alleles varied according to the geographical
origin of the samples, suggesting the possibility of using these
alleles as indirect markers for the study of population genetics. The
reasons why the different gB alleles have emerged and have been
maintained in human populations were not clear. The protein gB plays an
important role in the early events of virus-cell interaction (11,
20), but the genetic differences between gB alleles were silent
at the protein level and did not favor the concept of selection
pressure based on distinct phenotypic properties. However, subtle
modifications of replication properties due to conformational
differences or preferential nucleotide usage could not be ruled out. An
alternative and more likely hypothesis was that gB alleles were tightly
associated with specific alleles of other genes, these preferential
associations being stably transmitted through human generations.
We then decided to explore these two possibilities through recombinant
gB expression assays and novel genetic analyses of different
HHV-7-positive human samples. The preliminary results shown here
confirmed the high conservation of the HHV-7 genome with a limited
apparent impact of allele-specific changes on phenotypes. However,
these data allowed us to move from the concept of gene alleles to that
of HHV-7 variants.
Transcription and translation efficiency of HHV-7 gB alleles.
In order to investigate whether the six gB alleles exhibited a
different capacity to be transcribed, in vitro transcription was
studied after each allele had been cloned in the plasmid pcDNA3.1 under
the control of the T7 promoter. The RNA transcripts, synthesized by
means of T7 RNA polymerase with the RiboMAX in vitro transcription kit
(Promega, Madison, Wis.) as reported previously (14), had an
apparent molecular length of 2,400 bp, as expected (20). Their concentration was estimated by spectrophotometry, and comparison of these concentrations showed no differences between the six alleles
(data not shown). Another question was the possibility of differences
in the translation of transcripts. To explore that point, a 3.8-kbp
chimeric gene consisting of a 2.4-kbp gB gene fused with a 1.4-kbp
luciferase gene (luc) (22), was constructed for
each of the six alleles and subcloned into the mammalian expression vector pcDNA. Optimal conditions for expression had been previously established by introducing the six-histidine-containing sequence ATGCCGCGGGGTTCT(CAT)6GGTATGGCTAGC
upstream of the luciferase gene and studying the expression of
(HIS)6-luciferase in plasmid-transfected CHO cells by means
of (HIS)6-sequence-specific immunofluorescence assay. CHO
cells were transfected with the SUPERFECT transfection reagent (Qiagen,
San Diego, Calif.) with plasmids containing the chimeric
gB-luc genes, and luciferase activity was determined by
using the luciferase assay reagent (Promega). Luciferase activity was
similar for the six different chimeric gB-luc genes,
suggesting that no difference in translation efficiency was detectable
between the six gB gene alleles (data not shown). These results showed that the allele-specific alterations of the gB gene which did not
induce any change in the predicted amino acid sequence had no apparent
effect on either the transcription or translation of this gene. This
result reinforced the general conclusion that the gB gene of HHV-7 was
highly conserved and led us to hypothesize that stable gB alleles were
related to a more general polymorphism of the HHV-7 genome rather than
to specific properties of gB.
Polymorphism of the p100, MCP, and gL genes.
Our study was
subsequently focused on three other genes located on distant parts of
the HHV-7 genome: the gene coding for the structural phosphoprotein
p100, known as open reading frame (ORF) U11 and located at position
15982 to 18249; the gene coding for the major capsid protein (MCP),
known as ORF U57 and located at position 83514 to 87551; and the gene
coding for glycoprotein L (gL), known as ORF U82 and located at
position 116038 to 116778 (Fig. 1).
Comparison of the JI (16) and RK (15) sequences revealed relevant differences between these three genes. In the p100
gene, an AAT-to-GAT substitution induced the presence of an
MboII restriction site in RK, this site being absent in the case of JI, and the predicted change of an asparagine residue (for JI)
into an aspartic acid residue (for RK). In the MCP gene, the codons ATC
and ACA (JI) at positions 763 and 774 were changed into ATA and ACG,
respectively (RK). Consequently, the corresponding MboII and
MunI restriction sites present in JI were absent and had
been replaced by an SspI site in RK. In the gL gene, a
TTA-to-TAA substitution at codon position 59 resulted in the appearance
of an additional ApoI cleavage site in RK. This substitution
would change a leucine residue (JI) into a stop codon (RK). We made the
hypothesis that the genetic changes at these critical positions supported the definition of alleles for the corresponding genes in the
same sense as we had interpreted gB gene polymorphism previously.
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FIG. 1.
Location and polymorphism sites of the p100, gB, MCP,
and gL genes of the HHV-7 genome. The HHV-7 genome (144,861 bp long) is
schematized at bottom, with the left and right terminal repeat
sequences (TRL and TRR, respectively) and the internal repeat sequences
(R1 and R2) noted. The different genetic sequences are presented at
each codon mentioned for the four genes. The codon number of each gene
corresponds to the published JI sequence (16). The five
codon sequences of gB support the definition of six gB alleles as
reported elsewhere (8).
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Fifty HHV-7-positive samples from unrelated individuals previously
analyzed with regard to gB alleles (
8) were then studied
at
the critical positions of the p100, MCP, and gL genes by PCR
and
further genetic analysis of amplified products. Briefly, samples
corresponding to 1 µg of DNA extracted from peripheral blood
mononuclear
cells were subjected to nested PCR as described previously
(
8)
by using the primers indicated in Table
1. Amplified products
were subjected to
both nucleoside sequence determination and restriction
endonuclease
digestion as described previously (
8). The digestion
with
the restriction endonucleases
MboII,
SspI,
MunI, and
ApoI
was done in accordance with the
manufacturers' instructions (Boehringer,
Mannheim, Germany; Ozyme,
Beverly, Mass.); positive digestion
controls were added in each
experiment when the loss of a unique
restriction site was suspected. In
the case of the p100 gene,
the two putative alleles corresponding to
the presence of AAT
and GAT at codon 721 were found in 21 (42%) and 29 (58%) of the
50 samples studied, respectively (Table
2). These two alleles
were arbitrarily
designated p100-A and p100-B. In the case of
the MCP gene, four
potential alleles, designated MCP-A, MCP-B,
MCP-C, and MCP-D, were
defined according to the four possible
combinations of genetic changes
at codons 763 and 774 (Table
2).
Due to the limited amount of
peripheral blood mononuclear cell
DNA available for our study, only 36 samples were analyzed with
regard to MCP alleles. The three alleles
MCP-A, MCP-B, and MCP-C
were found in 13 (36%), 3 (8%), and 20 (56%)
of the samples studied,
respectively. The putative allele MCP-D was not
found, which suggested
it was uncommon (<3%), if it existed. The same
picture was observed
for the putative gL allele B, which was not found
in any of the
50 HHV-7-positive samples studied. The codon TAA at
position 59
of the gL gene then appeared to be specific for RK and was
no
longer considered in our analysis. We concluded that the p100
and
MCP genes exhibited polymorphism markers which permitted classification
of the different HHV-7-positive samples into distinct groups as
done
previously with gB alleles.
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TABLE 2.
Distribution of putative alleles of the p100, MCP, and gL
genes among independent
HHV-7-positive samples
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|
Definition of two distinct HHV-7 prototype strains.
The
recognition of the putative alleles of the p100, gB, and MCP genes,
located at three distant positions of the HHV-7 genome, raised the
question of whether the association between these alleles occurred at
random. We then analyzed the frequency of allele combinations among the
36 samples for which the characterization of the three genes had been
carried out (Table 3). Theoretically,
when considering two, six, and three alleles for the p100, gB, and MCP
genes respectively, 36 different combinations were possible. Only seven
of these combinations (Co) were observed, and 75% of the samples
corresponded to two of them, designated provisionally Co1 and Co2. The
reference strain JI was defined as Co2, as were our reference isolate,
IM (17), and the original HHV-7-positive sample which was
the source of IM. The reference strain RK corresponded to an eighth
combination (Co8) which was not observed in the group of human samples
we investigated. In accordance with the nonrandom distribution of allele combinations, statistical analyses showed a significant association between the alleles gB-C, p100-A, and MCP-C on one hand,
and the alleles gB-F, p100-B, and MCP-A on the other (P < 0.001; chi-square test). These results supported the idea that Co1
and Co2 were the genetic signatures of two predominant prototype variants of HHV-7 and that less frequent allele combinations might correspond to HHV-7 strains which have been derived from prototype variants by point mutation and/or recombination events.
The genome of HHV-7 appears to be highly conserved, as reflected by the
high degree of nucleotide sequence homology between
the unrelated
strains JI and RK (
15,
16). This conservation
has been
previously shown for the gB gene with 99.8% nucleotide
sequence
homology (
8) and was confirmed here for three other
genes
(p100, MCP, and gL) distantly located on the viral genome.
However,
albeit limited, the polymorphism of the gB gene permitted
the
definition of alleles, this term designating stable associations
of
genetic markers within this gene. We have now extended this
concept to
the p100 and MCP genes: one critical codon in the former
gene and two
critical codons in the latter permitted the classification
of
HHV-7-positive samples into two distinct groups in the case
of the p100
gene and three distinct groups in the case of the
MCP gene. The two
groups based on the polymorphism of codon 721
of p100 differed from
each other by the amino acid residue (asparagine
or aspartic acid)
corresponding to this codon. In contrast, the
three groups based on the
polymorphism of codons 763 and 774 of
MCP did not differ from each
other with regard to the amino acid
sequence at the corresponding
position. This was also the case
for the gB gene alleles
(
8), and we have shown here that gB
gene polymorphism,
silent at the protein level, was apparently
not associated with
differences in either transcription or translation
processes. Given
that the effects of genetic polymorphism on virus
phenotypes are very
modest, if they exist, the different alleles
we have characterized may
be considered simply as stable genetic
entities transmitted without
apparent selection pressure. What
we presently know about HHV-7
epidemiology and pathogenicity fits
this scenario. HHV-7 is transmitted
early in life via saliva,
inducing an asymptomatic or poorly
symptomatic primary infection
which results in a lifelong chronic
infection with apparently
no major associated disease. This ubiquitous
virus is therefore
assumed to be serially propagated in human
communities with a
very high conservation of its genetic content
through generations.
This genetic stability implies that not only
individual alleles
but also allele combinations would be maintained
through serial
transmission. In agreement with this view, we have
described in
the present paper two preferential associations of p100,
gB, and
MCP alleles, representing 44 and 31% of the HHV-7-positive
samples
studied, which, in our opinion, may support the concept of
prototype
HHV-7 variants. These two allele combinations may represent
two
major prototype strains that have been propagated independently
of
each other. Of note, Co1 contains the allele gB-C, which has
been found
more frequently in African and Caribbean subjects,
while Co2 contains
the allele gB-F, which is more frequent in
European subjects
(
8). It is tempting to anticipate that the
two putative
variants are distributed as gB alleles, according
to the geographical
origin of the samples. This conclusion, which
requires a definite
demonstration by means of an ongoing larger
study, points out the
interest in HHV-7 variants as markers for
studying population movements
and genetics, as previously concluded
from the study of gB alleles. Two
other problems remain unsolved:
(i) the possibility of different
biological behaviors of the two
HHV-7 variants due to subtle genetic
differences and (ii) the
genetic mechanisms by which minor variants (at
least five allele
combinations representing 25% of the samples
studied) have been
derived from the two major prototype variants.
Recombination after
a mixed infection appears to be a likely
hypothesis, keeping in
mind that the infection with two distinct
strains of HHV-7 may
be observed in vivo (
8). However, the
occurrence of rare point
mutations at critical codons still fits the
very high degree of
conservation of the HHV-7 genome and cannot be
ruled out as a
factor in genetic
evolution.
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ACKNOWLEDGMENTS |
This work was supported in part by the Association Claude Bernard,
the Action Concertée Coordonnée des Sciences du Vivant of
the French Ministry of Research, and MRTC research grant no. 97-5-12172 to M.F.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Laboratoire de
Virologie, C.E.R.V.I., UPRES EA 2387, Hôpital
Pitié-Salpétrière, 83 bd de l'Hôpital, 75651 Paris Cedex 13, France. Phone: 33.1.42.17.74.01. Fax: 33.1.42.17.74.11. E-mail: henri.agut{at}psl.ap-hop-paris.fr.
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Journal of Virology, November 1999, p. 9655-9658, Vol. 73, No. 11
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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