Dipartimento di Medicina Sperimentale e
Diagnostica, Sezione di Microbiologia, Università di Ferrara,
Ferrara, Italy
To define the molecular features characteristic of the early stages
of infection of lymphocytes with human herpesvirus 6 (HHV-6) variant A
or B, we studied the temporal regulation of expression of selected sets
of viral genes. Thus, U42, U94, U89-U90, U73, and U39 are 
genes
since their transcripts (i) were made in the presence of inhibitors of
protein synthesis and (ii) were detected 3 h after infection of
untreated cells. U41, U53, U31, and U19 are 
genes since their
expression is inhibited by cycloheximide but not by phosphonoacetate,
an inhibitor of DNA synthesis. U100 is a 
gene since its spliced
transcript encoding the structural glycoprotein gp82/105 was first
detected 16 h after infection of untreated cells but could not be
detected in cells treated with phosphonoacetate. HHV-6 variants differ
in the transcription patterns of their genes. U16-U17 originates a
splice transcript and is regulated as in HHV-6B and as in
HHV-6A. U91 generates two transcripts, amplified as 476- and 374-bp PCR
fragments. The 476-bp fragment is in HHV-6A-infected cells but in HHV-6B-infected cells. Conversely, the 374-bp fragment is in
HHV-6A-infected cells and in HHV-6B-infected cells. Furthermore,
the spliced product of U18-U19-U20 (526 bp) is in HHV-6A-infected
cells, but only a partially spliced form (1.9 kb) was detected at late stages of infection in HHV-6B. HHV-6 transcription was also studied in
nonproductive lymphoid cells, and the same transcription pattern detected during lytic infection was observed. Also, HHV-6 variants maintain the differences in U91, U16-17, and U18-U19-U20. We conclude that, as expected from the sequencing data, gene expression is generally similar in HHV-6 variants. However, transcription of selected
genes in HHV-6A and HHV-6B differs with respect to temporal regulation
and splicing pattern. Furthermore, the identification of viral
functions expressed during the different stages of lytic replication
suggests that reverse transcription-PCR for HHV-6 genes is a useful
diagnostic approach to differentiate between latent and productive
HHV-6 infection.
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INTRODUCTION |
Human herpesvirus 6 (HHV-6) is
widespread in the human population. Primary infection usually takes
place in the first years of life, causing exanthem subitum (ES)
(37). Severe or fatal involvement of HHV-6 in pneumonitis,
hepatitis, and encephalitis has also been described, although this is
less common (3, 4, 18, 28).
The virus establishes a latent infection (20), and
spontaneous reactivation of HHV-6 is a rare event in the healthy adult population. In contrast, the virus is frequently isolated from patients
with acquired or drug-induced immunodeficiency, such as human
immunodeficiency virus-infected individuals, transplant recipients, and
patients with autoimmune diseases. In these cases, viral reactivation
has been associated with significant pathologic findings (8, 9,
12, 17, 26).
HHV-6 isolates form two groups, HHV-6A and HHV-6B. These groups are
genetically related but show consistent differences in biological,
immunological, and molecular properties (reviewed in reference
14). For example, (i) specific T-cell lines support the growth of HHV-6A but not of HHV-6B, and vice versa; (ii) monoclonal antibodies can recognize variant specific epitopes; and (iii) each
variant displays a characteristic DNA restriction pattern. These
differences are highly conserved, and until now no chimeric form
between HHV-6A and HHV-6B has been described. HHV-6 variants also
differ with respect to the pattern of disease with which they are
associated. Thus, HHV-6B, but not HHV-6A, is frequently isolated from
children with ES or febrile disease (29). It has been
suggested that HHV-6 is more virulent than HHV-6B in that it has a more
pronounced effect on suppression of bone marrow function
(10). The molecular mechanisms responsible for the biological differences between HHV-6A and HHV-6B have yet to be identified.
Herpesvirus gene transcription and expression are tightly and
coordinately regulated during the infectious cycle:
immediate-early (IE) proteins, encoded by -genes, activate
-genes, which in turn switch on the synthesis of late products
encoded by -genes. -Genes can be further differentiated into two
groups (1 and 2), according to their temporal appearance.
-Genes also form two groups differentiated on the basis of their
independence (1) or dependence (2) on viral DNA synthesis for
their expression. The regulatory cascade has been extensively studied
in cells infected with herpes simplex virus (HSV) and, more recently,
in those infected with human cytomegalovirus (HCMV). Both HHV-6 and
HCMV belong to the Betaherpesvirinae subfamily of the
herpesvirus family as determined by colinearity of gene clusters and
homology of their proteins (19, 21). It is reasonable to
assume that HHV-6 gene transcription is coordinately expressed in a
cascade fashion, but no experimental evidence is available and specific
studies have yet to be reported.
Elucidation of the pattern of HHV-6 gene regulation would be expected
to affect the current knowledge of HHV-6 molecular biology, provide the
basis for identification of viral gene expression during latent
infection and the various stages of productive infection, and provide
useful markers to diagnose the state of viral infection in clinical
infections with these viruses.
In this report, we describe the results of analyses of the temporal
patterns of regulation of transcription of HHV-6 in untreated infected
cells and in cells infected and maintained in the presence of
cycloheximide (CEX) or emetine, inhibitors of protein synthesis, or
phosphonoacetate (PAA), an inhibitor of viral DNA replication (13). In these studies, viral transcripts were detected by
reverse transcription-PCR (RT-PCR). The objectives of the studies were to determine the kinetic class of selected viral genes with the aim of
identifying the pattern of transcription of the HHV-6A and HHV-6B DNAs
in productive and latent infection.
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MATERIALS AND METHODS |
Virus and cells.
Strain U1102 of HHV-6A (16) was
grown and analyzed in the JJhan T-cell line. Strain CV of HHV-6B
(15) was grown and analyzed in the SupT1 T-cell line. Both
cell lines were grown in suspension at 37°C in RPMI 1640 supplemented
with 10% fetal calf serum. Cell-free viral inocula were obtained by
pelletting 1 liter of HHV-6-infected cell cultures exhibiting complete
cytopathic effects (CPE). Infected cells, resuspended in 2 ml of 100%
fetal calf serum supplemented with RNase (Boehringer; 50 µg/ml), were
disrupted by four cycles of freezing in liquid nitrogen and thawing at
37°C. The resulting inoculum was completely free of living cells, as
checked by microscopic observation and cultivation, and was also
analyzed by RT-PCR (both for -actin and for the panel of viral
mRNAs) to ensure that RNA was completely absent. Infection was
performed by adding the viral inoculum to 107 cells/ml.
After 1 h of adsorption at 37°C, the cells were diluted with
fresh medium to a final concentration of 5 × 105
cells/ml. For cultures which were treated with drugs, the cells were
mixed with the appropriate concentrations of the drug 1 h before
infection. Adsorption of virus to cells, dilution, and incubation took
place in the continuous presence of the drug.
To identify -genes, the cells were infected in the presence of CEX
at 200 µg/ml. The experiments were repeated with another inhibitor of
protein synthesis, emetine (Sigma), at 50 µg/ml. Aliquots of 5 × 106 cells were collected 3, 6, and 8 h after
infection for RNA extraction. To identify -genes, cells were
infected in the presence of PAA (Sigma), an inhibitor of viral DNA
replication, at 500 µg/ml. The cells were harvested 8, 16, 24, and
36 h after infection. Control cells were infected under similar
conditions, but no drug was added to the medium. The cells were washed
in phosphate-buffered saline and immediately frozen at 
80°C until
used for RNA extraction.
RNA purification and RT.
Total RNA was extracted from cells
harvested at each time point and purified with Tripure isolation
reagent (Boheringer); DNA contamination was eliminated by three cycles
of digestion with 20 U of RNase-free DNase (Boheringer) at room
temperature for 30 minutes in MgSO4-acetate buffer. RNA was
purified by three phenol-chloroform extractions and recovered by
ethanol precipitation. After a 75% ethanol rinse, the RNA pellet was
resuspended in water treated with diethylpyrocarbonate and stored at
80°C with the addition of 40 U of RNase inhibitor (Amersham). The
complete absence of DNA contaminants was confirmed by PCR, amplifying
200 ng of total RNA with human -actin primers (38) and
two different sets of primers designed to amplify the HHV-6 U31 and U94
genes (Table 1).
Random primer first-strand cDNA synthesis from 1 µg of total RNA was
carried out with the cDNA cycle kit (Invitrogen) as recommended by the
manufacturer, with random hexamer primers. The cDNAs were purified by
phenol-chloroform extractions and ethanol precipitated. After being
rinsed with 75% ethanol, the cDNAs were stored at 80°C. Due care
was taken to avoid sample-to-sample contamination: different rooms were
used for RNA extraction, PCR setup, and gel analyses, all pipette tips
had filters for aerosol protection, samples were intersped with blank
reaction mixtures, etc. To assay whether cDNAs from different samples
were retrotranscribed with similar efficiencies, 10,000-fold dilutions
of cDNAs were analyzed by PCR for the detection of the human -actin
gene.
PCR analysis.
We analyzed 13 HHV-6 genes. All the primers
were derived by us from the published HHV-6 sequence (19),
with the exception of the primers for U31, which were described by
Aubin et al. (5), and the primers for U73 and U41, which
were developed by Rapp and Pellett (29a). The primer
sequences, as well as the expected size of amplified fragments
resulting from DNA and cDNA, are shown in Table 1. All primer sets
amplified the DNA of both HHV-6 variants, with the exception of
U89-U90, for which separate sets of primers had to be developed.
PCRs were done in the presence of 400 nM primers (500 nM for -actin
and U31), 1.5 mM MgCl2 (3 mM for U41, U39, and U16-U17; 2.5 mM for U31), 200 mM deoxynucleoside triphosphates, and 1 U of AmpliTaq
DNA polymerase (Perkin Elmer) in the buffer supplied by the
manufacturer.
After an initial denaturation of 5 min at 94°C, a thermal cycle of 1 min at 94°C, 1 min at 55°C (63°C for U91 and U42; 68°C for
U100; 58°C for U89-U90; 60°C for U31), and 1 min at 72°C (with a
3-s increase at each new cycle) was repeated 35 times. The
amplification products were run on an agarose gel (1 to 2%, according
to the expected fragment size) and visualized under UV illumination
after ethidium bromide staining.
Sequencing.
After electrophoresis, the amplified fragments
were purified with Gene Clean II (Bio 101) and were sequenced with the
FMOL sequencing system (Promega). Reaction products were separated on a
6% polyacrylamide gel, with 8 M urea and Tris-borate buffer. The gels
were dried on filter paper and exposed to autoradiographic film. The
sequences, obtained with both sense and antisense primers, were
manually determined. Open reading frames were determined with the DNA
Strider 1.0 software.
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RESULTS |
Temporal regulation of HHV-6 transcription.
In this series of
experiments, cells were infected with HHV-6A or HHV-6B and maintained
in the presence or absence of CEX or emetine (inhibitors of protein
synthesis) and PAA (inhibitor of viral DNA replication) to identify the
temporal class of viral transcripts. The concentrations of drugs
compatible with cell viability but effective in inhibiting viral
protein or DNA synthesis, respectively, had been determined in
preliminary experiments. All the results were verified in at least two
independent experiments. To minimize experimental variations, RNA
extracted from infected cells was retrotranscribed after priming with
random hexamers, and therefore the resulting cDNAs could be analyzed
for the whole panel of HHV-6 genes. In addition, the same samples were
retrotranscribed in different experiments, and equivalent results were
always obtained. Moreover, aliquots of the viral inoculum were
carefully analyzed by rtPCR, to ensure that the cDNAs detected
originated from newly synthesized mRNAs and not from transcripts
present in the inoculum. The samples were also checked for the absence
of viral DNA: samples from all time points were analyzed by PCR,
without retrotranscription, for the presence of residual HHV-6 DNA, and
were retrotranscribed only when found negative. No contaminant DNA was
present in the samples, as shown in Fig.
1 (U41, 8 h postinfection [p.i.]).
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FIG. 1.
Agarose gels stained with ethidium bromide showing the
results of RT-PCR for human -actin and HHV-6 U42 (an -gene), U41
(a -gene), and U100 (a -gene). RNA was extracted at the indicated
time (hours) after infection from cells infected with HHV-6 in the
presence or absence of CEX and PAA. D, PCR on DNA from HHV-6 infected
cells, extracted 4 days p.i., at complete CPE; R, RT-PCR on RNA
extracted from HHV-6-infected cells at complete CPE; M, human DNA
extracted from mock-infected cells. The asterisk marks a sample
yielding a faint band, difficult to reproduce in print. The sizes of
amplified fragments are shown. No contaminant DNA was detected in the
samples (U41, 8 h after infection).
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The efficiency of retrotranscription was checked by amplification of
the human -actin cDNA, as shown in Fig. 1, after a 1:10,000 dilution. Samples from all time points were amplified with similar efficiencies, excluding the possibility of marked differences in
retrotranscription for different samples. Furthermore, the presence of
CEX and PAA did not affect the mRNA levels of a housekeeping gene such
as the -actin gene.
Since the primers for PCR amplification were derived from HHV-6A
sequence, we checked whether they could also amplify HHV-6B DNA.
Similar efficiencies were obtained, with the exception of U89-U90, for
which an HHV-6B-specific set of primers had to be developed (Table 1).
The genes analyzed in our study are shown in Table
2; they include genes with predicted
regulatory functions, genes encoding proteins which orchestrate viral
replication, and genes encoding structural proteins. The -genes
which included U42, U89-U90, U94, U39, and U73, were expressed as early
as 3 h after infection even in the presence of CEX (Fig.
2). The results obtained with CEX and
emetine are identical (data not shown). A typical example of an
-gene whose expression is not affected by CEX or PAA is U42 (Fig.
1).
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FIG. 2.
Agarose gel stained with ethidium bromide showing the
results of RT-PCR for HHV-6 -genes (U42, U73, U89-U90, and U94). RNA
was extracted 3, 6, or 8 h after infection from cells infected in
the presence of absence of CEX. D, PCR on DNA from HHV-6 infected
cells, extracted 4 days p.i., at complete CPE. The sizes of the
amplified fragments are shown.
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U19, U31, U41, and U53 were regulated as -genes. Transcripts of
these genes were detected at 8 h after infection. They were inhibited by CEX but unaffected by the addition of PAA. The example of
U41 is shown in Fig. 1.
Only one gene in our panel, U100, encoding gp82/105, was regulated as a
-gene. The cDNA of HHV-6A U100 was identified by Pfeiffer et al.
(27) as a multiply spliced product of a gene containing 12 exons. The primers used by us amplified a 1,045-bp fragment of the DNA,
encompassing part of exon 2, exon 3, and part of exon 4, and detected
both unspliced and spliced mRNAs (Fig.
3). Low levels of unspliced mRNA were
detected at 8 h after infection but could not be detected in cells
infected and maintained in the presence of CEX (Fig. 1). Spliced RNA
was detected at late times after infection and was detected 24 h
after infection. The unspliced form gradually disappeared, and at the
time the cells exhibited complete CPE, 4 days p.i., only spliced RNAs
were detected. In the presence of PAA, no spliced RNAs could be
detected, suggesting that splicing of U100 RNA was regulated as or by a
-gene function.
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FIG. 3.
Schematic representation of part of U100 (exons 2 to 4)
from HHV-6A (27), containing two introns of 241 and 101 bases. The PCR amplified both a genomic fragment of 1,045 bp,
highlighted in the shaded area, and a multiply spliced cDNA of 703 bp.
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Transcription of HHV-6A and HHV-6B IE-A region.
The patterns
of expression of the genes listed above are similar in HHV-6A- and
HHV-6B-infected cells. However, other genes exhibited temporal patterns
specific for HHV-6A or HHV-6B. U91 and U89-U90 are encoded by the HHV-6
IE region homologous to the major IE locus of HCMV. The domain of this
region relevant to our studies is represented in Fig.
4. Schiewe et al. (31)
described two spliced transcripts derived from U89-U90 in the published HHV-6 sequence and identified them as -genes on the basis of the
observation that they are expressed in the presence of CEX. In
addition, they reported the presence of another spliced mRNA, derived
from U91 mapping on the complementary strand. Our results confirm that
U89-U90 of both HHV-6A and HHV-6B are -genes and that similar
splicing events take place in the homologous regions of HHV-6A and
HHV-6B. However, the pattern of transcription of U91 differed in cells
infected with these two viruses. Our assay amplified part of exons 1 and 2 and detected both unspliced and spliced transcripts (Fig. 4). The
results, shown in Fig. 5, indicate that
the unspliced sequence (476 bases) of HHV-6A was detected in cells
infected and maintained in the presence of CEX whereas the spliced form
(374 bases) was detected only in untreated, infected cells, suggesting
that the splicing requires the intervention of a function expressed
earlier in infection. Interestingly, the same transcripts exhibited a
different pattern of synthesis in cells infected with HHV-6B. In fact,
the spliced form was maintained in the presence of CEX whereas the
unspliced transcript (484 bases) was not detected (Fig. 5). Therefore,
in HHV-6B the shorter transcript is and the longer is transcribed
later in infection.
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FIG. 4.
Schematic representation of the portion of U89-U90 and
U91 from HHV-6A (31) amplified by PCR. The amplified DNA
fragment is shown within the shaded area. U89-U90 amplified cDNAs were
789 bp (unspliced) and 682 bp (spliced); the U91 amplified cDNAs were
476 and 374 bp, respectively.
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FIG. 5.
Agarose gels stained with ethidium bromide showing the
cDNA amplified by RT-PCR for U91 and U18-U19-U20. RNA was extracted
8 h p.i. from cells infected with HHV-6A (U1102) or HHV-6B (CV) in
the presence or absence of CEX. D, PCR on DNA from HHV-6-infected
cells, extracted 4 days p.i., at complete CPE. R, RT-PCR on RNA
extracted from HHV-6-infected cells at complete CPE; M, 123-bp ladder
molecular size marker; M1, lambda HindIII molecular size
marker, showing the 2,322-, 2,027-, and 564-bp fragments. The sizes of
the amplified fragments are shown.
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Transcription of the HHV-6A and HHV-6B IE-B region.
The IE-B
region, homologous to HCMV IE, encodes two sets of spliced genes. U16
and U17 correspond to the EFLF-2 and EFLF-1 open reading frames (ORFs)
originally described by Nicholas and Martin (25). Our
analysis confirms that this region yields a spliced RNA transcript
hypothesized earlier (25). In HHV-6A-infected cells, the
transcript was regulated as a -gene function: it appeared early in
the course of infection, being detected by 3 h p.i., but was
absent in cells treated with inhibitors of protein synthesis (Fig.
6). However, this transcript has a
different regulation in HHV-6B: the spliced product is unaffected by
the presence of either CEX or emetine and therefore is an -gene
function (Fig. 6).
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FIG. 6.
Agarose gel stained with ethidium bromide showing the
results of RT-PCR for HHV-6 U16-U17. RNA was extracted 3, 6, or 8 h p.i. from cells infected in the presence or absence of CEX. D, PCR on
DNA from HHV-6 infected cells, extracted 4 days p.i. at the time the
infected cells exhibited complete CPE.
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The other group of spliced genes is U18, U19, and U20. We detected a
transcript originating from U19, presumably regulated as a -gene
function in both variants (Table 2). However, this region also encodes
multiply spliced transcripts, and some spliced forms of U18-U19-U20
differ in cells infected with HHV-6A or HHV-6B. Nicholas and Martin
(25) suggested that the EJFL6, EJFL4, and EJFL3 ORFs,
renamed U18, U19, and U20 in the published HHV-6 sequence (19), were joined as result of multiple splice events, and
mRNA species were proposed on the basis of putative splice donor and acceptor sites. We designed PCR primers that permitted the
amplification of a 2,064-bp DNA sequence comprising part of U18, all of
U19, and part of U20 (Fig. 7). Analyses
of HHV-6A-infected cells showed that a spliced mRNA was detected 8 h after infection. This mRNA was detected in infected cells maintained
in the presence of PAA (data not shown) but not in cultures infected
and maintained in the presence of CEX (Fig. 5). The amplified product
of HHV-6A cDNA was completely sequenced and showed a perfect
correspondence to the published sequence (19). The amplified
fragment from cDNA was 526 bp long, and the splice pattern is shown in
Fig. 7. Splice donor and splice acceptor sequences were identified at
the intron boundaries (Table 3). Two
splicing sites were present (donor sites D1 and D2 and acceptor sites
A1 and A2), originating two introns of 687 and 851 bases. According to
the analysis of the coding regions, this transcript has the potential
of synthesizing two proteins, one corresponding to the complete U18 and
the other corresponding to U20 partially deleted in the carboxy
terminus (Fig. 7).
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FIG. 7.
Schematic representation of the portion of U18-U19-U20
amplified by our PCR assay. The amplified DNA fragment is shown within
the shaded area. The sequenced cDNAs of HHV-6A (U1102) and HHV-6B (CV)
are indicated by thick lines, and the white line indicates the
undetermined sequence. Spliced regions and their sizes are indicated.
The size of exon D1/A2 is 170 bp. The locations of splice acceptor (A)
and splice donor (D) sites are shown. The corresponding ORFs are shown.
Full bars indicate stop codons, and small bars indicate start codons.
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The cDNA from HHV-6B-infected cells yielded a significantly larger
amplimer, which differed from that of HHV-6A-infected cells both for
size, approximately 1.9 kb, and for the time of its appearance, being
detected only 24 h p.i. (Fig. 5). We should stress that preliminary experiments had shown that the PCR had similar efficiencies in amplifying the transcripts of both HHV-6A and HHV-6B and therefore the disparity of results cannot be attributed to the amplification procedure. HHV-6B cDNA, as well as DNA, were partially sequenced, and
only one intron was detected. The splice donor and acceptor sites are
given in Table 3. The splice acceptor site A1, also present in HHV-6A,
was maintained. However, the corresponding donor site (D3) was
different from the site in HHV-6A, being located only 214 bp upstream
on the HHV-6B DNA sequence (Fig. 7). Analysis of the coding regions
from HHV-6B DNA showed that this transcript has the potential of
synthesizing three complete ORFs, corresponding to U18, U19, and U20
(Fig. 7). Therefore, U19, spliced out in the final transcript of
HHV-6A, is instead transcribed in HHV-6B.
Interestingly, HHV-6A DNA contains an identical splice donor site
(GCTGAG/GTAGGT) in a position corresponding to the D3 site in HHV-6B (19), but it does not give rise to introns.
Likewise, HHV-6B DNA also contains typical splice donor sites
(GGGACG/GTGTGT and
CAAAAC/GTCCGT) in the same position as D1 and
D2, respectively, with some point mutations (underlined) not affecting
the consensus sequence, which do not originate introns.
HHV-6 transcription in nonproductive cells.
To ensure that the
differences detected between HHV-6A and HHV-6B were not due to the
different cell lines used, the experiments were repeated but SupT1
cells were infected with HHV-6A (U1102) and JJhan cells were infected
with HHV-6B (CV). As expected (14), viral growth did not
occur. Nevertheless, transcription took place with the same pattern as
in lytically infected cells (data not shown). The results reported in
Fig. 8 show that for nonproductive infection, HHV-6 variants also maintain the three main differences already described for U91, for U16-U17, and for the multiply spliced transcript of U18-U19-U20. The larger transcript from U91 is temporally regulated as in HHV-6A and as in HHV-6B, while the supposedly spliced form is in HHV-6A and in HHV-6B. The U16-U17 splice is
regulated as in HHV-6B and as in HHV-6A. Finally, the spliced transcript from U18-U20, revealed as a 526-bp fragment by the
RT-PCR assay, was present only in HHV-6A, whereas HHV-6B showed only
the larger, 1.9-kb fragment (Fig. 8).
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FIG. 8.
Agarose gels stained with ethidium bromide showing
RT-PCR of mRNA transcribed during nonproductive HHV-6 infection of
lymphoid cells. SupT1 cells were infected with HHV-6A (U1102), and
JJhan cells were infected with HHV-6B (CV), in the presence or absence
of CEX. The PCR products of amplification upon DNA are shown (D). Sizes
are shown in base pairs. The sizes shown for U91 are derived from
HHV-6A; the respective sizes in HHV-6B are 484 and 388 bp. Molecular
weight markers are indicated by 1 (123-bp ladder) and 2 ( DNA
digested with BstE).
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DISCUSSION |
HHV-6 was discovered in 1986 (30), the existence of two
distinct groups (HHV-6A and HHV-6B) was described in 1991 (32), but the knowledge of its molecular biology has
experienced slow progresses and is still limited. The nucleotide
sequence of HHV-6A was published only in 1995 (19), the
complete sequence of HHV-6B is still not available, and studies on the
transcription of viral genes have been restricted to ORFs homologous to
IE genes of HCMV. We describe here for the first time the temporal
regulation of transcription of a selected set of HHV-6 genes. These
studies were undertaken for two practical reasons: (i) the temporal
mapping of HHV-6 transcripts is a necessary step to identify viral
genes which may be expressed during latency, following reactivation, and during the early stages of infection; and (ii) the different biological behaviors of HHV-6A and HHV-6B could reflect differences in
gene expression, potentially revealed by a comparative analysis of
their transcription.
Our results show that HHV-6 transcription follows a typical herpesvirus
cascade pattern in which -, -, and -genes are coordinately and
sequentially transcribed. The kinetic classes of genes such as U42,
U89-U90, and U41 correspond to those of their homologous counterparts
in the other HHVs (Table 2), but other HHV-6 genes are transcribed
earlier than in other members of the family; this is the case for U31,
U39, U53, and U73. It should be noted that several factors may
complicate the temporal analysis of transcriptional classes. The
presence of a specific mRNA does not necessarily imply protein
expression, and transcripts may accumulate before protein synthesis is
observed. Furthermore, regardless of when it begins, transcription
continues throughout the infection and mRNA levels are significantly
higher at late times p.i. For example, mRNA from U39 is unaffected by
the presence or absence of CEX, but its levels are lower at 8 h
p.i. than at later times (data not shown), suggesting that a promoter
may be active at a low level throughout the reproductive cycle or that
its activity could be enhanced at specific times during infection. The
sensitive approach we used, RT-PCR, does not allow us to determine the
relative abundance of viral transcripts and could result in an
overestimate of the amounts of mRNAs from scarcely transcribed genes.
Furthermore, different members of the herpesvirus family express
homologous genes at different times during the lytic cycle. Such is the
case, for example, for a conserved herpesvirus transactivator gene: in
HSV, ICP27 is clearly an -gene product, but the counterpart of this
gene in HCMV, UL69, is a -gene, expressed at 7 h after infection and not detected under IE conditions (39). The
HHV-6 homolog U42 is regulated as an -gene and in this respect
resembles the HSV counterpart more closely than the HCMV counterpart.
Several genes were transcribed under IE conditions (Table 2) in that
their mRNA was present at 3 h after infection, CEX and emetine did
not inhibit their synthesis, and the mRNA was also detected at later
times p.i.
In this report, we show that U94, which does not have counterparts in
the other herpesviruses, is regulated as an -gene. It has been
reported that the product of U94, a homolog of the Rep protein of
adeno-associated parvovirus type 2, has transactivating potential and
can negatively regulate several heterologous promoters, such as the HIV
long terminal repeat and the ras promoter (2). The finding that U94 was expressed under IE conditions supports the
notion that it is a regulatory gene which may play an important role in
the viral replicative cycle, either by regulating viral activity or by
turning off host cell functions.
The only -gene revealed by our study is U100: the mRNA was
transcribed in its unspliced form under -gene conditions, but the
spliced product was detected only 16 h p.i. and was inhibited by
PAA (Fig. 1), showing that HHV-6 controls gp82/105 production by
temporally regulated splicing mechanisms. An indirect observation is
that the HHV-6 replicative cycle is longer than 16 h, since at
earlier times the viral envelope would not be complete.
Transcription from the IE-B region shows a distinctive pattern for each
variant. One difference resides in U16-U17. The final splice product is
present in both variants, but it is in HHV-6B and in HHV-6A
(Fig. 6). Therefore, HHV-6 variants modulate transcription from this
region differently. Other variant-specific differences were observed in
the remaining portion of IE-B. Nicholas and Martin proposed a
speculative mRNA structure for the U18-U19-U20 region (25),
on the basis of DNA sequence and by comparison with the splicing
pattern in the HCMV homologous region. Our results demonstrate that
transcription from this region is complex. In fact, both variants show
the presence of a distinct -gene transcript originating from U19,
but a multiply spliced transcript is also found. In HHV-6A the cDNA
amplified in our assay is larger than expected (25), since a
putative 118-bp intron is not spliced out and a splice junction is
present within an intron, resulting in an additional 170-bp exon (Fig.
7). Interestingly, HHV-6B shows a different cDNA structure. The size of
the amplified cDNA in HHV-6B is about 1.9 kb; sequence analysis showed
that one splice donor/acceptor site is missing (A2/D2, Fig. 7) and that
the D1 site, still present in HHV-6B, is not recognized and instead is
substituted by a donor site (D3) closer to A1. This pattern reflects a
different coding potential in the spliced transcript detected for each
variant (Fig. 7). Theoretically, the HHV-6A mRNA has the ability to
transcribe a complete product from U18 and a partially deleted protein
from U20. Instead, the HHV-6B transcript can perform synthesis for all
three ORFs, including U19. Furthermore, the cDNA in HHV-6B appeared
later during infection than did that in HHV-6A (24 and 8 h p.i.,
respectively). Considering the late appearance and large size, it is
possible that this 1.9-kb cDNA represents a low-abundance intermediate
species, yielding a final transcript(s) not detected by the PCR
primers, possibly due to a different splice pattern between HHV-6
variants. By analogy to the homologous UL36-38 region in HCMV, it is
also possible to propose that these transcripts function as
polycistronic messengers, encoding distinct proteins from
nonoverlapping ORFs (35). Each variant showed its
characteristic splice pattern in both cell lines used (Fig. 8),
strengthening the idea that the observed differences are variant
specific. The different splice patterns detected within U18-U19-U20 are
not due to differences in the sequence of HHV-6 variants, because splice sites D1 and D3 are present both in HHV-6A and HHV-6B. Therefore, it is possible that differences reside in spliceosome formation or in the splicing machinery.
Another difference in transcription between HHV-6 variants was observed
for U91. The 476- and 374-base transcripts generated by U91 show
opposite temporal regulations in HHV-6 variants (Table 2; Fig. 5).
Schiewe et al. (31), on the basis of sequence data on cDNA,
suggested that the transcript from this region is spliced. However, in
our experiments with HHV-6B, the 374-bp fragment corresponding to the
spliced mRNA appeared earlier than the 476-bp amplimer corresponding to
the full-length molecule, from which it should originate. The results
are compatible with the hypothesis that U91 transcription is regulated
by two promoters, temporally regulated as different functions in HHV-6
variants: the promoter yielding the 476-base transcript is in
HHV-6A and in HHV-6B, while the 374-base mRNA is transcribed from a
promoter in HHV-6A and from an promoter in HHV-6B. It is also
possible that, similarly to the situation already described for
U16-U17, different splicing mechanisms take place in HHV-6 variants.
The observation that transcription of HHV-6 variants differ in this
region is supported by sequence data, showing that the homology between
IE regions is approximately 75 to 85% for the DNA sequence and 62 to
70% for the amino acid sequence, whereas in other genomic regions the
sequence homology is often above 90% (11, 36).
The differences observed were not due to the cell lines, since all
three variant-specific transcription patterns were also detected when
HHV-6 strains infected cell lines that did not support viral growth
(Fig. 8). Incidentally, these results show that nonproductive lymphoid
cells are susceptible to infection and support active transcription;
however, the lack of viral growth is indicative of restricted or
abortive infection.
Although both HHV-6 variants have a predominant tropism for
CD4+ cells, HHV-6B requires cell maturation associated with
the expression of the CD3 antigen (34) but HHV-6A seems to
prefer less differentiated CD4+ T cells (23).
The two variants show distinct characteristics of growth in continuous
cell lines. Variant B, unlike HHV-6A, does not propagate in JJhan and
HSB-2 cell lines (1, 40) but grows in other T cells, such as
SupT1, Molt-3, and MT-4, which are not susceptible to HHV-6A productive
infection (1, 6). NK cells are more permissive to HHV-6A
than to HHV-6B infection and replication (22). Finally, both
variants can infect the monkey Macaca nemestrina, but only
HHV-6B, and not HHV-6A, infects and replicates in peripheral blood
mononuclear cells of Macaca mulatta (24). It has
also been suggested that HHV-6 variants may have a different disease
association or a different pathogenic potential, and, indeed, HHV-6B is
frequently isolated from children with ES whereas HHV-6A is isolated
mostly from immunosuppressed individuals. The biological and
pathological implications of these differences between HHV-6
variants are still controversial, but the present results, showing that
at least three viral genes expressed during the early phases of
replication have distinct features in each variant, provide a molecular
basis for the different biological behaviors of HHV-6 variants.
Several observations support the notion that HHV-6 can be an
opportunistic pathogen in immunosuppressed patients (33),
and viral infection or reactivation in transplant recipients may result in graft-versus-host disease, interstitial pneumonitis, and
encephalitis. In these cases, an early diagnosis of viral infection is
important to permit prompt antiviral treatment, and PCR for HHV-6 DNA
is often an obligate diagnostic choice. However, the mere detection of
viral genomes is not sufficient to discriminate between latent and
acute infection, and even quantitative PCR is not suitable to
distinguish between a low-level productive infection and an increased
load of latently infected cells in immunocompromised patients
(7). Such a distinction might be important, since even low
levels of productive infection by HHV-6 may cause clinical disease
(7, 10). Our results, identifying the viral functions associated with the different phases of lytic infection, provide the
necessary conditions for proposing RT-PCR as a useful diagnostic approach to the differentiation between latent and productive infection
by HHV-6.
This work was supported by grants from Ministero della
Sanità (Istituto Superiore Sanità, AIDS Project), from
BiomedII (European Community), from Associazione Italiana per la
Ricerca sul Cancro (AIRC), and from MURST.
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Abstract
Introduction
