1 Laboratory for Clinical and Molecular Virology, The University of Edinburgh, Summerhall Square, Edinburgh EH9 1QH, UK
2 Scottish Centre for Genomic Technology and Informatics, The University of Edinburgh, Summerhall Square, Edinburgh EH9 1QH, UK
3 MRC Centre for Inflammation Research, The University of Edinburgh, Summerhall Square, Edinburgh EH9 1QH, UK
Correspondence
Bahram Ebrahimi
ebrahimi{at}staffmail.ed.ac.uk
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ABSTRACT |
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Present address: Departamento de Bioquimica y Biología Molecular, Facultad de Medicina, 02071 Albacete, Spain.
Present address: Department of Medical Microbiology, The University of Liverpool, Liverpool L69 3GA, UK.
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Introduction |
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The MHV-68 genome contains 118 311 bases of unique sequence with variable numbers of internal repeats flanked by multiple copies of terminal repeats. The genome encodes eight tRNA-like sequences and 73 protein-coding open reading frames (ORF) and is broadly contiguous with other herpesviruses. Also present are genes unique to MHV-68 (M1M4), as well as homologues of cellular genes such as complement regulator protein, Bcl-2, cyclin-D and G-protein-coupled receptor, believed to play a role in establishment of infection and evasion of host immune responses (Bowden et al., 1997; Virgin et al., 1997
; Kapadia et al., 1999
; van Dyk et al., 1999
; Wang et al., 1999
; van Berkel et al., 1999
; Roy et al., 2000
; Parry et al., 2000
; Nash et al., 2001
).
Herpesvirus replication follows a regulated pattern of viral gene expression. Using either sensitivity to drug inhibitors and/or measurements of viral gene expression, it is feasible to classify these viral proteins into immediate-early (IE), early (E) and late (L). IE genes encode predominantly transcription factors, associated with transactivation, whereas Egenes include enzymes associated with DNA replication. L genes are mainly associated with structural components of viral particles.
DNA microarrays offer a powerful platform for parallel analysis on a genome-wide scale. DNA microarrays for viral pathogens can facilitate identification of transcription patterns during replication, persistence and latency. The parallel and comprehensive transcriptome analysis by DNA microarrays will be useful in exploring transitions between different stages of virus infection and disease pathogenesis.
Analysis of MHV-68 gene expression has been limited to a small set of genes. In this paper we sought to: (a) design and validate a DNA microarray specific for MHV-68, (b) develop new PCR assays for tRNA-like genes of MHV-68 and (c) provide an overview of the stage-specific kinetic class of all viral genes during permissive infection in vitro.
We show the simultaneous expression of MHV-68 genes by using classical drug inhibitors. Data obtained from the microarray platform and complemented with RT-PCR assays constitute a comprehensive transcriptome analysis of MHV-68.
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Methods |
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Synthesis, isolation and labelling of nucleic acids.
Cloned Bacillus subtilis genes obtained from the ATCC (pGIBS-TRP, accession #K01391; pGIBS-DAP, accession #L38424; pGIBS-PHE, accession #M24537; pGIBS-LYS, accession #X17013) were linearized and mRNA transcribed in vitro using the MEGAscript kit (Ambion) according to the manufacturer's protocol.
Viral DNA was extracted from purified MHV-68 virions as described previously (Efstathiou et al., 1990b). The purity and integrity of viral DNA were checked by digestion with EcoRI, BamHI and HindIII followed by gel electrophoresis (data not shown).
Purified viral DNA (1 µg) was digested with 5 mU DNase 1 (Life Technologies) at 15 °C for 15 min, and heat-inactivated at 65 °C for 20 min. To generate labelled DNA fragments, digested DNA was incubated with 10 U E. coli DNA polymerase 1 (Life Technologies), dATP, dGTP, dTTP (4 mM each) and 20 nM FluoroLink Cy3-dCTP (Amersham Pharmacia). Labelled DNA was mixed with 20 µg COT-1 human DNA (Life Technologies) and excess label was removed by YM-30 microfiltration (Amicon). This step was also used to reduce the final reaction volume of labelled DNA to 12 µl.
Total RNA was harvested from cells using RNeasy columns (Qiagen) according to the manufacturer's recommendations. Briefly, adherent cells were trypsinized and the cell pellet was resuspended in RLT lysis buffer (Qiagen). Contaminating DNA was digested with 10 U AmpGrade DNase 1 (Life Technologies) for 15 min at room temperature. RNA was recovered and purified using RNeasy columns and the quality of RNA was checked by spectrophotometry and gel electrophoresis. Purified RNA (25 µg in 11 µl) was mixed with 4 µl (2 µg) of poly(dT)21, heated to 70 °C for 10 min and snap-cooled on ice for 5 min. Direct labelling was carried out in a 30 µl reaction volume containing 400 U Superscript II reverse transcriptase (Life Technologies), 6 µl of 5x times; first-strand buffer, 3 µl of DTT (10 mM), 0·6 µl of dNTP mix (dATP, dGTP and dTTP at 0·5 mM; dCTP at 0·3 mM) and 3 µl of Cy3-dCTP (0·1 mM) at 42 °C for 2 h. This was followed by addition of 15 µl 0·1 M NaOH at 70 °C for 10 min, and then neutralized by addition of 15 µl 0·1N HCl. From this stage onwards, labelled cDNA was treated in an identical manner to labelled viral genomic DNA.
Probe design and array fabrication.
Probes were designed using the Oligo6 primer design software (Molecular Biology Insights: www.oligo.net/contact.htm). ORF- and strand-specific viral probe sequences were based on the g2.4 strain of MHV-68 (accession #AF105037) (Table 1). Design parameters for probe selection included: lack of homo-oligomers or sequence repeats, 4060 % GC-rich and a melting temperature range of 8595 °C. Control elements on the array included 54 murine cellular genes, five negative control genes (plant and bacterial), four B. subtilis genes for spike RNA and printing buffer without nucleic acids (Table 2
). Probes were synthesized by MWG-biotech, resuspended at 0·4 µg ml-1 in printing buffer (3x times; SSC) and printed in triplicate using an Affymetrix 417 arrayer onto poly-L-lysine-coated glass slides (Eisen & Brown, 1999
). Arrays were stored in desiccators at room temperature prior to use.
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Labelled target DNA was resuspended in 15 µl of hybridization solution (3·5x times; SSC/0·3 % SDS), and incubated on the array at 65 °C overnight. Following this, arrays were rinsed in 1x times; SSC/0·2 % SDS, followed by 0·1x times; SSC/0·2 % SDS, and 0·1x times; SSC. All stringency washes were done at room temperature.
Primer design and RT-PCR for tRNA-like sequences.
MHV-68 encodes eight tRNA-like sequences which lack poly(A) tracts and are therefore unsuitable for oligo(dT)-directed reverse transcription. We therefore designed PCR primers for tRNA1tRNA8 to complement the array data (Table 3). The design parameters included: 4060 % GC content; 3'-end stability; and a melting temperature of 8490 °C. RNA was isolated and treated with DNase 1 as for microarray experiments and was used in RT-PCR assays. PCR included 1 U per reaction of Taq DNA polymerase (Life Technologies), 100 nM dNTP and 100 pmol of each primer. Cycling parameters were: hot-start at 95 °C for 5 min followed by addition of Taq DNA polymerase; 94 °C for 60 s; appropriate annealing temperature for 60 s; 72 °C for 60 s; followed by 35 cycles with a final extension at 72 °C for 5 min. PCR products were resolved by agarose gel electrophoresis and DNA visualized by UV transillumination.
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To define the linear dynamic range of the fluorescence dye and to control for scanning parameters (laser settings and gain), arrays were scanned at five combinations of increased laser/gain settings; therefore generating five datasets. These datasets were then analysed against each other by scatter plots. Scan settings that gave the optimum scatter without signal saturation (assessed by least-squares fit analysis) were chosen for subsequent analysis. Normalization (scaling) between arrays was based on four B. subtilis genes transcribed in vitro. The in vitro-transcribed spike mRNA was added to the test RNA prior to reverse transcription. This approach normalizes for intensity differences between arrays because of scan settings and hybridization efficiencies and is also independent of potential changes in cellular transcripts because of virus infection. Signal intensities of viral and cellular genes above the arithmetic mean plus 2 SD of signal intensities for negative control genes were used for further analyses. Each data point was obtained from three experiments (n=3).
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Results |
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Immediate-early transcripts of MHV-68
Herpesviruses encode a number of IE (or ) genes transcribed immediately after infection and independently of de novo protein synthesis. In this study, viral transcripts whose median fluorescence signals were increased at least twofold in the presence of inhibitor (CHX) when compared with CHX-treated but uninfected cultures were grouped as IE transcripts. No viral transcripts were detected in the presence of CHX at 2 and 4 h post-infection. In contrast, six transcripts were detected 8 h post-infection in the presence of CHX. These IE transcripts were M4, ORF12 (K3), ORF38, ORF50, ORF57 and ORF73 (Fig. 1
).
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Early, early-late and late transcripts of MHV-68
We used PAA to generate stage-specific RNA to identify the kinetic class of E and L genes. Most E gene products are involved in viral nucleic acid metabolism. Classically, E genes show increased expression in the presence of PAA (compared to virus-infected cells without PAA) and are sensitive to CHX treatment. These E transcripts included ORF24, ORF29a (packaging), ORF36 (kinase), ORF54 (dUTPase), ORF63 (tegument), ORF64 (tegument), ORF68, ORF75b and M12 (Fig. 2).
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Based on sequence comparisons with other herpesviruses, ORF75a, ORF75b and ORF75c may encode tegument proteins (Virgin et al., 1997). Interestingly, the array analysis shows a hierarchy of expression for these genes, with abundant expression of ORF75c transcript compared to ORF75a and ORF75b (Fig. 2
).
Development of PCR primers and kinetics of viral tRNA genes
The MHV-68 genome encodes eight tRNA-like sequences which lack poly(A) tracts and are therefore unsuitable for analysis by our microarray approach. Therefore, we designed and validated primers specific for these viral sequences and identified the expression kinetics of these unique MHV-68 genes in C127 cells in the presence and absence of inhibitors. All eight tRNA-like transcripts were detected in RNA representing the three kinetic stages of MHV-68 transcription (Fig. 3).
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Discussion |
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Drug inhibitors were used to map the expression kinetics of MHV-68 genes during the lytic cycle of infection in vitro. Viral transcripts were not detected at 2 and 4 h post-infection with MHV-68 in the presence of CHX by DNA microarrays. However, six transcripts were detected in the presence of CHX at 8 h post-infection. These included previously identified IE genes K3, ORF50 and ORF73 (Liu et al., 2000; Rochford et al., 2001
; Virgin et al., 1999
), as well as M4, ORF38 and ORF 57. ORF50 is homologous to Rta genes of other gammaherpesviruses and encodes the major transactivator of MHV-68 lytic cycle antigens as well as playing a role in reactivation from latency (Wu et al., 2000
, 2001
). The M4 ORF of MHV-68 contains a heparan sulphate-binding domain, which may have a role in initial virion interaction with the cell membrane via proteoglycan heparan sulphate. In BC-3 cells, the KSHV K8.1A gene, which also contains a heparan sulphate-binding domain, showed an early expression profile after induction (Jenner et al., 2001
). However, expression of this gene in the presence of CHX may indicate other roles for this protein during the infection cycle.
A subset of MHV-68 genes showed L expression kinetics and includes previously identified L genes ORF8 (gB), gp150, ORF65 (M9) and ORF46 (DNA Gly). In total, 28 transcripts showed L kinetics. These data are summarized in Table 4.
A recent sequence re-evaluation of the MHV-68 genome suggested a number of viral genes unlikely to encode proteins (Nash et al., 2001). These include M5, M6, M8, M10ac, M12, M13 and M14. We were able to detect transcripts for M5, M6, M8, M10ac and M12 by a combination of microarray and PCR assays. However, it is not clear whether these transcripts encode proteins.
Our analysis by an oligonucleotide-based microarray compares favourably with a recently published PCR-based microarray (Ahn et al., 2002). For example, 22 transcripts which showed L expression kinetics were also grouped as
transcripts by the PCR microarray. Similarly, 15 transcripts with
expression kinetics as determined by the PCR microarray also showed E or E-L expression kinetics by the oligonucleotide microarray. Moreover, the simultaneous analysis of the MHV-68 transcriptome by both microarray studies supports previous observations of highly expressed genes such as M3 and M9 and genes expressed at a low level, for example M11 and ORF74 (Virgin et al., 1999
; Parry et al., 2000
; Rochford et al., 2001
; Bridgeman et al., 2001
; Roy et al., 2000
; Ahn et al., 2002
).
Overall, the array data parallel previously published data obtained by other methodologies in terms of specificity and sensitivity of the assay (Table 4). There are, however, some discrepancies. These are very likely a consequence of different experimental parameters for example, use of double-stranded PCR probes, as well as the cell types and the criteria used in the classification of virus transcripts, i.e. whether based on temporal expression rather than sensitivity to drug inhibitors only or a combination of both.
MHV-68 encodes eight tRNA-like sequences for which we developed gene-specific PCR primers. Using these assays, we were able to detect all eight tRNA-like transcripts during the three stages (IE, E, L) of MHV-68 infection in C127 cells.
Small RNA sequences that have been identified in HSV-1, EBV and adenoviruses may have a role in virus evasion of host responses and/or pathogenesis (Albrecht & Fleckenstein, 1992; Howe & Shu, 1989
; Mathews, 1995
). In EBV, two EpsteinBarr early RNAs (EBERs), which reside adjacent to the OriP origin of replication, are expressed during the latent but not lytic phase of infection (Albrecht & Fleckenstein, 1992
). This expression pattern is in contrast to tRNA14 transcripts, detected during lytic as well as latent phases of MHV-68 infection (Simas et al., 1998
).
Alphaherpesviruses encode homologues of host shut-off proteins associated with degradation of cellular RNA. In HSV-1, UL41, which encodes the host shut-off protein, was implicated in the decline of cellular transcripts (Schek & Bachenheimer, 1985). More recently, DNA microarray analysis showed a global reduction in cellular transcripts following HSV-1 infection (Stingley et al., 2000
). UL41 is an endonuclease and interacts with translation initiation factor eIF4H. No homologue of UL41 exists in the MHV-68 genome. This would suggest that either MHV-68 encodes a cryptic host shut-off gene product or that the loss of cellular transcripts is independent of viral gene products.
The present study is a global analysis of the MHV-68 genome using DNA microarrays and adds to the growing kinetic analysis of herpesvirus transcriptomes (Chambers et al., 1999; Stingley et al., 2000
; Jenner et al., 2001
; Paulose-Murphy et al., 2001
; Sarid et al., 1998
; Ahn et al., 2002
). Although not a substitute for other established methodologies, DNA microarrays provide a useful first-step platform for simultaneous and parallel analysis of large numbers of genes. This approach will help identify global transcription patterns (viral and cellular) associated with virus latency, reactivation and disease pathogenesis.
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ACKNOWLEDGEMENTS |
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Received 6 June 2002;
accepted 18 September 2002.