Department of Experimental and Diagnostic Medicine, Section of Microbiology, University of Ferrara, Via L. Borsari 46, 44100 Ferrara , Italy1
Author for correspondence: Dario Di Luca.Fax +39 0532 247618. e-mail dil{at}dns.unife.it
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Abstract |
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Introduction |
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Primary infection is associated with exanthem subitum (Tanaka et al., 1994 ) and benign febrile disease (Portolani et al., 1995
). The majority of young children, by three years of age, are positive for HHV-7-specific antibodies (Clark et al., 1993
; Wyatt et al., 1991
) and over 80 % of adults harbour viral sequences in their PBMCs (Di Luca et al., 1995a
) at low copy number (Kidd et al., 1996
). Viral DNA is found in salivary glands (Di Luca et al., 1995b
) and virus is shed in saliva, suggesting that active replication may take place in this region of the body.
The determination of the HHV-7 DNA sequence (Nicholas, 1996 ) has not reflected a corresponding increase in knowledge of its molecular biology, mostly because its low pathogenic potential does not encourage investments in time and resources towards its study. Nevertheless, HHV- 7 is an interesting subject of study for three main reasons: (i) HHV-7 can reactivate HHV-6 from latency (Katsafanas et al., 1996
), and thereby it can indirectly contribute to life- threatening infections supported by HHV-6 in immunocompromised individuals; (ii) HHV-7 has an antagonistic effect with human immunodeficiency virus, interfering at the receptor level (Lusso et al., 1994
); and (iii) the tropism of HHV-7 for CD4 T lymphocytes and its low pathogenic potential suggest that the virus may be a good candidate as a vector for gene therapy, delivering therapeutic genes specifically in CD4+ cells.
To gain insights into HHV-7 molecular biology, we have studied the temporal regulation of selected genes and characterized splicing patterns. Gene transcription of herpesviruses is tightly and coordinately regulated during the infectious cycle. Immediate-early (IE) functions are encoded by genes and do not require prior protein synthesis. They activate ß genes (early) which switch on the production of late proteins encoded by
genes. ß genes can be further differentiated into two groups (ß1 and ß2), according to their temporal appearance. The
genes also form two groups 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 and human cytomegalovirus. Recently, we reported that HHV- 6 transcription is also regulated by the typical herpesvirus cascade mechanism (Mirandola et al., 1998
). It is reasonable to assume that HHV-7 genes are coordinately transcribed in a cascade fashion, but experimental evidence is not available and specific studies have yet to be reported. The purpose of this study was to determine the kinetics of HHV-7 transcription in productively and abortively infected cells maintained in the presence of cycloheximide (CEX) or emetine (EME), inhibitors of protein synthesis, or phosphonoacetic acid (PAA), an inhibitor of viral DNA replication.
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Methods |
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To identify genes, the cells were infected in the presence of 200 µg/ml CEX (Sigma) or 50 µg/ml EME (Sigma). Aliquots of 5x106 cells were collected 3, 6 and 8 h after infection for RNA extraction. To identify ß genes, cells were infected in the presence of 500 µg/ml PAA (Sigma), an inhibitor of viral DNA replication. 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. Cells were washed in PBS and immediately frozen at -80 °C until RNA extraction.
RNA purification and reverse transcription.
Total RNA was extracted from cells harvested at each time-point with RNazol B (Tel-Test). DNA contamination was eliminated by three cycles of digestion with 20 units RNase-free DNase (Boehringer Mannheim) at room temperature for 30 min in 5 mM MgSO4 and 100 mM sodium acetate buffer, pH 5·0. RNA was purified with two phenol/chloroform extractions and recovered by ethanol precipitation. After a 75% ethanol rinse, the RNA pellet was resuspended in water treated with diethyl pyrocarbonate (0·1%) and stored at -80 °C with the addition of 40 U RNase inhibitor (Amersham). The complete absence of DNA contaminants was checked by PCR, amplifying 200 ng total RNA with human ß- actin primers (Walther et al., 1994 ) and two different sets of primers designed to amplify HHV-7 U14 and U42 genes ( Table 1
).
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PCR analysis.
All primers were derived by us from the published HHV-7 sequence (Nicholas, 1996 ). The primer sequences, as well as the expected sizes of amplified fragments resulting from DNA and cDNA, are shown in Table 1
.
PCR reactions were done in the presence of 400 nM primers, 1·5 mM MgCl2 (2·5 mM for U16/17 and U98/100; 2·0 mM for U31, U39, U53 and U66), 200 µM dNTPs and 1·25 U 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 58 °C (54 °C for U19 and U66; 55 °C for U60/66; 56 °C for U10, U18/20, U39 and U98/100; 57 °C for U18 and U99/100) and 1 min at 72 °C (with 3 s increase at each new cycle) was repeated 35 times. Products of amplification were run on agarose gel (12%) or polyacrylamide gel (4%), according to the expected fragment size, and UV-visualized after ethidium bromide staining.
Due care was taken to avoid sample-to-sample contamination. Different rooms and dedicated equipment were used for mRNA extraction and processing, PCR set-up and gel analyses; all pipette tips had filters for aerosol protection and all experimental samples were interspersed with blank reactions. All PCR reactions for HHV-7 transcripts had similar sensitivities; tenfold dilutions of DNA extracted from HHV-7 infected cells were similarly amplified by all primers for nested PCR.
Sequencing.
After electrophoresis, the amplified fragments were purified by GeneClean II (Bio 101) and were sequenced by the fmol sequencing system (Promega). Reaction products were separated on a 6% polyacrylamide gel with 8 M urea and Trisborate buffer. Gels were dried on filter paper and exposed to autoradiographic film. The sequences, obtained using both sense and antisense primers, were manually determined. Open reading frames were determined with the DNA Strider 1.0 software. Consensus sequences for initiation of translation were determined according to Kozak (1987 , 1996
).
Analysis of PBMCs.
Buffy coats were collected from 36 healthy blood donors and PBMCs were purified on Ficoll gradients. Aliquots corresponding to 5x10 6 cells were frozen at -80 °C. Nucleic acid extraction and retrotranscription of mRNA was performed as described recently (Rotola et al., 1998 ). Nested PCR was performed by amplifying 5 ml 1:100 dilution of template from the primary reaction. Sequences of nested primers were: TAACACCGAAGAGGCTATGC and CTGGAAGACCATTCTCATGC for U14; GCTGCTGTTGTTACGTCGTT and AACCGAGCTGCAAGACCTAT for U16/17; CGCGTATGAACTGAGGTTGT and CCAGCTCATAGGATTCGAGA for U42; AAGAAGGAGCTTCCTCGGAT and TGCAGGCACTAATGGACTGA for U89/90.
PCR reactions were done in the presence of 400 nM primers, 1·0 mM MgCl2 (1·25 mM for U14; 2 mM for U89/90), 200 µM dNTPs and 1·25 U Ampli Taq 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 58 °C and 1 min at 72 °C (with 3 s increase at each new cycle) was repeated 35 times.
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Results |
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All PCR reactions had similar efficiencies, as shown by amplification of 10-fold dilutions of DNA extracted from HHV-7 infected cells (data not shown).
To analyse the temporal regulation of HHV-7 transcription, SupT1 cells were infected with HHV-7 in the presence of inhibitors of protein synthesis (CEX or EME) or an inhibitor of viral DNA replication (PAA). The concentrations of drugs compatible with cell viability that are effective in inhibiting viral protein or DNA synthesis were determined in two independent preliminary experiments. To ensure reproducibility, RNA extracted from infected cells was retrotranscribed after priming with random hexamers, and the resulting cDNAs were analysed for the whole panel of HHV-7 genes. The same samples were retrotranscribed in different experiments and equivalent results were always obtained. Aliquots of the viral inoculum were carefully analysed by RTPCR to be sure 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; all time-points were analysed by PCR, without retrotranscription, for the presence of residual HHV-7 DNA. No contaminant DNA was present in the samples.
The efficiency of retrotranscription was monitored by amplification of the human ß-actin cDNA, after a 1:10000 dilution. All samples taken at different intervals were amplified with similar efficiencies, which excluded the possibility of marked differences in retrotranscription. Furthermore, the presence of drugs inhibiting protein synthesis did not affect the mRNA levels of a `housekeeping' gene such as ß-actin.
The results of temporal analysis of HHV-7 transcripts are shown in Table 2. The
genes, which included U10, U14, U18, U31, U39, U41, U42, U53, U73 and U89/90, were transcribed as early as 3 h after infection even when protein synthesis was inhibited. The results obtained with CEX and EME were identical (data not shown). A typical example of an
gene is U42 (Fig. 1a
). U19 and U18/20 were regulated as ß genes. Transcripts of these genes were detected 8 h after infection; they were inhibited by EME but were unaffected by the addition of PAA. The example of U19 is shown in Fig. 1(b)
. Two genes in the panel, U60/66 and U98/100, had spliced products regulated as
functions and are described below.
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U16/17. Two mRNAs were detected. Sequence analysis showed that both are transcribed from the same coding strand (Fig. 2a ). The unspliced mRNA was
, as it was synthesized 3 h after infection in the presence of protein inhibitors (Fig. 2b
). The spliced form was transcribed at 16 h post- infection (p.i.). It was inhibited by EME but not by PAA and therefore it belongs to the ß transcriptional class. The unspliced mRNA encoded two ORFs (Fig. 2b
). U17 had canonical start and stop signals and could be translated. In comparison, U16 had no start signal and the 3' end lacked Kozak consensus sequences, and therefore translation could not take place. The splice event (donor site, ACCTAT/GGTAAGT; acceptor site, TCATAG/GTTGCT) excised 72 bases, including the stop codon for U17. The fusion product acquired the start signals from U17 and the stop codon from U16 and has the potential to be translated (Fig. 2
).
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Detection of transcripts associated with in vivo persistence of HHV-7 |
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Discussion |
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Transcription of HHV-7 was regulated with the typical herpesvirus cascade pattern, in which , ß and
genes are sequentially transcribed (Table 2
). As we recently discussed for HHV-6 (Mirandola et al., 1998
), several factors complicate the analysis of transcriptional classes. The presence of a specific mRNA does not necessarily imply protein expression and transcript may accumulate before protein synthesis is observed. Furthermore, the levels of mRNA increased significantly as infection progressed, suggesting that transcription begins early p.i. and that specific steps in the replication cycle might enhance promoter activity and transcription. In these instances, RTPCR will result in an enrichment of transcripts scarcely transcribed. Furthermore, the block of protein synthesis is not absolute, and RTPCR is sensitive enough to detect even low levels of transcription from ß genes with strong promoters. For this reason, the experiments were performed independently with two different inhibitors of protein synthesis (CEX and EME). EME exerts a stronger inhibition on protein synthesis, but it has a more pronounced toxic effect. The same results were obtained with both drugs, showing that the effects on virus transcription could not be ascribed to toxicity.
The kinetic classes of HHV-7 genes correspond to those recently described for HHV-6 (Mirandola et al., 1998 ). Exceptions are represented by U41 and U53 which are transcribed in the IE phase of HHV-7, but are ß genes in HHV-6. Other differences between HHV-7 and HHV-6 transcription are observed in U18/20, which encodes transcriptional activators. The splice pattern of this region in HHV-6 is variant-specific, due to different recognition of splice donor and acceptor sites, and consequently each variant originates transcripts expressing partially different proteins (Mirandola et al., 1998
). The PCR reaction for this region of HHV-7 was designed to detect the potentially occurring splice mechanisms, but no splice was observed. HHV-6 variants and HHV-7 are closely related viruses and yet possess distinct biological properties, mainly reflected by specific in vitro cell tropism and by different pathogenic potential. It will be interesting to determine whether the distinct biological behaviours of these viruses are associated with the different patterns of transcription of U18/20.
It is difficult to predict if the primary transcript is actually translated. Indicators predictive of whether an mRNA can be translated include the presence of an upstream terminator codon, an ATG codon and Kozak consensus signals for the start of translation (Kozak, 1996 ). For example, U16 lacks both the ATG and the Kozak consensus, and therefore it is extremely unlikely that the primary transcript can be translated. Instead, the mature mRNA acquires the potential for translating U16 as a fusion product with U17 ( Fig. 2
). Therefore, the splice product might be reasonably considered to be the functional messenger. Also, the expression of U98/100 requires post-transcriptional processing. The pre- mRNA is tricistronic, but can possibly translate only U100. In fact, the three ORFs are not in-frame and only U100 has both a start codon and Kozak consensus sequences. The singly spliced mRNA is bicistronic and could express a U100/99 fusion product. Only the doubly spliced mRNA can express all three ORFs, as a U100/98 product. The study of the splice pattern suggests that post-transcriptional processing is essential for HHV-7 expression.
Interestingly, in addition to the synthesis of the primary transcript, the splice mechanisms are also temporally regulated. In fact, in several cases the pre-mRNA and the spliced product have a different kinetic class. For example, the primary transcript of U16/17 is synthesized with a typical pattern, and splicing takes place only during the ß phase of infection.
U60/66 is another example of the temporal regulation of splice mechanisms. The primary transcript is abundant and belongs to the kinetic class. The spliced transcript was detectable in small amounts as early as 3 h after infection and levels of synthesis increased over time. Addition of PAA prevented accumulation of the mature mRNA, suggesting that a virus function associated with virus replication might enhance the splicing activity and increase the rate of conversion from the pre-mRNA (
) into the final messenger (
1).
Also, the splicing of U98/100 is temporally regulated. RTPCR showed the presence of three mRNAs of different sizes. The primary transcript is a ß function (Fig. 5). The singly spliced transcript (splice sites A2/D2) is synthesized in low abundance, but with typical
kinetics. The observation that the singly spliced transcript is detected earlier than primary mRNA could reflect a high rate of splicing activity specific for A2/D2 allowing accumulation of the pre-mRNA only at later stages of infection. The doubly spliced mRNA is produced at 8 h p.i. in limited amounts even in the absence of protein synthesis, but subsequently it is drastically reduced until 40 h p.i., when the product accumulates. PAA inhibits the accumulation at late times, showing that it is (or is dependent on) a
1 function (Fig. 5
). The observation that a singly spliced transcript A1/D1 was not detected suggests that the A2/D2 junction might be a requirement for efficient A1/D1 splicing, regulated as a
event.
These results show that the splicing machinery can recognize and select targets and is dependent upon factors synthesized in different moments of the infectious cycle. Furthermore, it is possible that the splicing pattern is regulated by virus-induced functions, possibly affecting the formation and/or the specificity of cell spliceosomes.
In conclusion, HHV-7 transcription is regulated by complex mechanisms, involving both pre-transcriptional factors, such as the temporal coordinated activation of specific viral promoters, and post- transcriptional processing, mediated by virus-induced factors affecting the specificity of splicing.
To examine whether HHV-7 establishes a true virus latency, we analysed PBMCs from healthy blood donors for the presence of viral RNA transcripts. We focused our attention on transcription of four IE genes, transcribed also during abortive infection, reasoning that their presence is a necessary requisite for productive virus replication. The lack of detection of viral transcripts, even in samples harbouring fairly large amounts of viral DNA (1 copy in 150 cells) supports the notion that HHV-7 establishes a true latency in PBMCs.
The pathogenic role of HHV-7 is not clear and current information is derived from few case reports. Exanthem subitum (Tanaka et al. , 1994 ), childhood febrile syndrome (Portolani et al. , 1995
) and pityriasis rosea (Drago et al., 1997
) have been associated with productive HHV-7 infection. Pathogenic studies are complicated by the fact that infection with HHV- 7 is widespread in the human population and serological analysis is of little value. PCR is often used to detect HHV-7 DNA, but viral sequences are detected in the majority of healthy adults (Di Luca et al., 1995a
) and PCR cannot discriminate the state of virus replication. RTPCR instead might allow a relatively simple differentiation between latent and active infection and will be an useful approach to study pathogenic associations of HHV-7.
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Acknowledgments |
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References |
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Received 13 May 1999;
accepted 7 July 1999.