- Open Access
Use of a multi-virus array for the study of human viral and retroviral pathogens: gene expression studies and ChIP-chip analysis
- Elodie Ghedin†1, 2,
- Anne Pumfery†3,
- Cynthia de la Fuente3,
- Karen Yao4,
- Naomi Miller1,
- Vincent Lacoste3,
- John Quackenbush1,
- Steven Jacobson4 and
- Fatah Kashanchi1, 3Email author
© Ghedin et al; licensee BioMed Central Ltd. 2004
- Received: 16 April 2004
- Accepted: 25 May 2004
- Published: 25 May 2004
Since the discovery of human immunodeficiency virus (HIV-1) twenty years ago, AIDS has become one of the most studied diseases. A number of viruses have subsequently been identified to contribute to the pathogenesis of HIV and its opportunistic infections and cancers. Therefore, a multi-virus array containing eight human viruses implicated in AIDS pathogenesis was developed and its efficacy in various applications was characterized.
The amplified open reading frames (ORFs) of human immunodeficiency virus type 1, human T cell leukemia virus types 1 and 2, hepatitis C virus, Epstein-Barr virus, human herpesvirus 6A and 6B, and Kaposi's sarcoma-associated herpesvirus were spotted on glass slides and hybridized to DNA and RNA samples. Using a random priming method for labeling genomic DNA or cDNA probes, we show specific detection of genomic viral DNA from cells infected with the human herpesviruses, and effectively demonstrate the inhibitory effects of a cellular cyclin dependent kinase inhibitor on viral gene expression in HIV-1 and KSHV latently infected cells. In addition, we coupled chromatin immunoprecipitation with the virus chip (ChIP-chip) to study cellular protein and DNA binding.
An amplicon based virus chip representing eight human viruses was successfully used to identify each virus with little cross hybridization. Furthermore, the identity of both viruses was correctly determined in co-infected cells. The utility of the virus chip was demonstrated by a variety of expression studies. Additionally, this is the first demonstrated use of ChIP-chip analysis to show specific binding of proteins to viral DNA, which, importantly, did not require further amplification for detection.
- Infected Cell
- Viral Gene Expression
- Infected Cell Line
- Cell Leukemia Virus Type
- KSHV Infected Cell
Microarray technology, developed in the wake of various genome projects, has increasingly become one of the most widely used functional genomic tools. Global gene expression arrays, in a single co-hybridization assay, can query the differing expression patterns of thousands of genes [1, 2]. Differential gene expression can be measured by treating identical cells with different stimuli, such as drug treatment or cellular stress, or by analyzing related but distinct cells/tissues, for example normal tissue versus malignant tumors [1, 2]. More recently, microarrays have shown great potential for clinical applications as well, including diagnosis of disease states, viral or bacterial subtyping, and even virus discovery [3–5].
Moreover, increasing attention is also being paid to the identification of viral genes expressed during latent and/or lytic infection as targets for the development of antiviral treatments, an approach that has proven successful for the herpesviruses [6–9]. A viral microarray comprised of spotted amplicons from the open reading frames (ORFs) of a number of different viruses would have distinct advantages over conventional technologies, including Enzyme Linked Immunosorbent Assay (ELISA) and Northern blot analysis that do not allow for simultaneous multi-target analysis.
To determine whether a multi-virus microarray had potential in specific applications, for instance viral gene expression inhibition studies and protein-DNA binding experiments, we developed and characterized an array containing the amplified ORFs of eight human viruses. These include human immunodeficiency virus type 1 (HIV-1), human T cell leukemia virus types 1 and 2 (HTLV-I and -II), hepatitis C virus (HCV), Epstein-Barr virus/human herpesvirus 4 (EBV/HHV-4), human herpesvirus 6A and 6B (HHV-6A and -6B), and Kaposi's sarcoma-associated herpesvirus/human herpesvirus 8 (KSHV/HHV-8). These viruses were chosen for their importance in AIDS and AIDS-associated diseases. In addition, these viruses vary widely in their pathogenesis, including high (EBV, HHV-6A, HHV-6B, and KSHV) and low copy number (HIV-1, HTLV-I, and HTLV-II); differing viral expression during latent and lytic infection (herpesviruses); low (HCV) versus high (HTLV-I) replication rate; and representing both DNA (herpesviruses) and RNA (HTLV-I, HTLV-II, HIV-1 and HCV) genomes. Our hope was that by demonstrating the feasibility and applicability for a single microarray representing diverse, pathogenically important viruses, we would set the stage for a wide range of applications ranging from multiple virus detection to functional studies. In addition, larger arrays consisting of more viruses could easily be developed based on the data generated from this first-generation virus array.
Most DNA arrays are designed for the analysis of thousands of genes representing a single species. We chose to construct a virus chip for the detection of various human viruses and demonstrate that this technique can address complex problems in virology. We demonstrate that we can detect, with great specificity, genomic viral DNA from cells infected with the human herpesviruses, EBV, KSHV, HHV-6A, and HHV-6B. We further demonstrate that we can induce and observe viral gene expression in HIV-1 and KSHV latently infected cells, and that we can use this chip to detect inhibition of HIV and KSHV gene expression when subjecting infected cells to a cyclin dependent kinase inhibitor. In addition, we used the virus chip for new applications such as chromatin immunoprecipitation (ChIP) followed by hybridization to the virus chip (ChIP-chip) and observed specific hybridization of KSHV DNA. Importantly, we were able to detect immunoprecipitated DNA from latently infected as well as induced cells by direct labeling of immunoprecipitated DNA without further PCR amplification.
Design of the virus chip
Microarray technology allows the analysis of thousands of genes in a single assay. Although most DNA arrays are designed to represent a single species, we chose to construct a comprehensive array representing the annotated open reading frames of eight human viruses that are often associated with AIDS: HIV-1, HTLV-I, HTLV-II, HHV-6A, HHV-6B, EBV, KSHV, and HCV. In doing so, we hoped to be able to simultaneously detect the presence of these viruses while monitoring their patterns of gene expression. As a means of quality control, we also included thirty-one human sequences; 29 control cDNA clones obtained from a human EST library and glyceraldehyde-3-phosphate dehydrogenase exon 7 as well as β-actin exon 3.
Although there are advantages in using oligonucleotides (oligos) over PCR products (amplicons) for the construction of DNA microarrays [10, 11], we chose to use amplicons for a number of reasons. First, viral genomes are compact enough that it is comparatively easy to cover the entire genome, including both coding and non-coding regions, with a small number of PCR amplification reactions. This has a significant cost advantage compared to oligo synthesis. Second, although oligos offer the possibility of greater specificity, the ability of longer products to hybridize effectively despite some number of mismatches can be an advantage for viruses that exhibit a relatively high mutation rate and for the detection of virus strains from related families. One potential drawback of PCR is that, in certain cases, due to either the size of the gene to be amplified, its nucleotide composition, or other factors, amplification may be unsuccessful. For example, in our virus chip, primers could not be designed for 15 of the 86 EBV ORFs due to their repetitive nature. However, these genes may also prove difficult for long oligo designs as the repeats may pose similar problems. Overall, we were able to successfully amplify and array probes representing 254 to 264 of the 329 (>75%) ORFs within our eight target viruses.
Specificity of DNA hybridizations
As can be seen in Figure 2A, we were able to accurately detect each virus with little cross hybridization to the others represented on the array. Hybridizations were performed with three different EBV infected cell lines, MM2, B-95A, and Jijoye. Representative data from the B-95A hybridization are shown in Figure 2A. The three cell lines exhibited similar hybridization patterns, although we did observe a slightly lower hybridization signal from the Jijoye cell line than from the others (data not shown).
HHV-6A and HHV-6B are two variants of HHV-6 that differ in epidemiology, in vitro growth properties, and nucleotide sequence [13, 14]. Although the majority of the ORFs in HHV-6A and HHV-6B have high sequence identity, there is a cluster of genes that exhibit less than 80% identity at the right end of the unique region of the viral genome spanning ORFs 86 to 100 [15, 16]. Therefore, all of the HHV-6A ORFs were amplified, whereas only those HHV-6B ORFs that exhibited less than 80% sequence identity with HHV-6A were printed. Hybridization with genomic DNA from HHV-6A (U1102) infected cells hybridized to the HHV-6A targets with little to no cross-hybridization with the HHV-6B amplicons (Figure 2A). As was observed with the EBV infected cells, there was very little hybridization to other viral ORFs. Furthermore, as expected, hybridization with genomic DNA from HHV-6B (Z29) infected cells detected HHV-6A and HHV-6B targets.
Hybridization using the KSHV infected cell line, BCBL-1, gave a good signal for the KSHV ORFs (Figure 2A). Although KSHV cross-hybridized to some viral probes, those that cross-hybridized were not consistent between the replicates (N = 3), with the exception of the HHV-6A U80 and HCV NS5 probes. These results indicate that we can accurately identify several closely related herpesviruses by their specific hybridization profile. The copy number for KSHV+ cell lines ranges from 50 to 2000 copies/cell. Therefore, the minimum copy number required to detect viral sequences from infected cells appears to be 70 copies per cell, as this is the lowest copy number determined from the KSHV infected cell line, BCBL-1 . However, subsequent analyses using the co-infected cell lines, Cra-BCBL and BBG1 (Figure 1C), indicated that the minimum detectable viral copy number is 20.
We were not able to detect HIV-1, HTLV-I, HTLV-II, or HCV DNA sequences from infected cell lines (data not shown). This is likely due to the low copy number of HIV, HTLV-I, and HTLV-II in the infected cells used. The HIV-1 latently infected cell lines, ACH2 and U1, contain one and two integrated copies [18, 19], respectively, while the HTLV-I and HTLV-II cell lines, MT-2 and C19, contain 5 to 8 integrated copies [20, 21]. HCV is a RNA virus and hybridization with genomic DNA from infected cells was not expected. As we were unable to detect HIV-1, HTLV-I, or HCV using genomic DNA from infected cells, we labeled infectious plasmid clones with Cy3-dCTP or Cy5-dCTP by nick translation. As can be seen in Figure 2B, specific hybridization was observed for all three viruses. Since the experiments were performed using plasmid DNA rather than DNA from infected cell lines, the same DNA was labeled with both Cy-dyes and "self-self" hybridizations were performed. The data is represented for each gene as the ratio of gene-specific hybridization intensity on the average intensity for all spots. The cut-off ratio for high hybridizations was set to five rather than ten. While no cross-hybridizations could be observed, only two of the 11 HIV probes represented on the array successfully hybridized with the labeled plasmid DNA.
We also wanted to determine if we could specifically detect multiple viruses in co-infected cells. Therefore, we used genomic DNA from two different KSHV/EBV co-infected cell lines, Cra-BCBL  and BBG1  (Figure 2C). Hybridization to the KSHV and EBV targets was observed. However, there was much less hybridization to the EBV targets than was observed using DNA from a singly infected cell line (Figure 2A, EBV probe). BBG1 cells contain approximately 2000 copies/cell of the KSHV genome and 20 copies/cell of the EBV genome . The Cra-BCBL cell line contains 130 copies/cell of the KSHV genome (unpublished data) but the copy number for EBV is not known. Results suggest that the effective limit of detection is approximately 20 genomic copies per cell, as we observed hybridization of some of the EBV amplicons despite the large difference in copy number relative to KSHV.
In principal, genomic DNA should hybridize uniformly to all of the genes of a specific array. There are several possibilities why we observed differences in intensity levels for the various amplicons, including the size of PCR products and the G/C composition of the amplicons. Larger ORFs may exhibit a higher fluorescent intensity as there is more incorporated label than in smaller ORFs, and amplicons with a higher A/T content may not hybridize as strongly due to their lower melting temperature. The PCR products spotted on our virus array are not uniform in size as they vary from 100 bp to 3.4 Kbp. The genes that failed to hybridize tended to be between 100–300 bp in size: 50% of all amplicons smaller than 300 bp did not hybridize, or hybridized weakly, while the failure rate was approximately 5% for the remaining amplicons. The G/C content of the viruses varies, as does the G/C content of some ORFs within viruses. However, there did not appear to be any correlation between weak hybridization and G/C composition of the amplicons.
In the case of hybridizations to plasmid DNA, as seen in Figure 2B, there is less cross-hybridization and better representation of all targeted genes for HTLV-I and HCV. For HIV, while there was no cross-hybridization to amplicons of other viruses, only two (proviral LTR and nef) of the 11 spotted regions of the HIV-1 genome hybridized to the DNA sample. The G/C content of the entire coding region of HIV is 42%. However, the G/C content for Nef and the LTR is 49% and 53%, respectively. As the HIV proviral DNA was labeled with Cy-dCTP, the higher G/C content of Nef and LTR may explain the better hybridization observed for these amplicons.
Sensitivity of DNA hybridizations
DNA hybridizations allowed us to validate hybridization for each target and determine the specificity and sensitivity for each virus in the array. However, many of the applications we envision will require determination of RNA expression levels. To demonstrate this capability, we focused on the expression of HHV-6B, HIV-1, and KSHV genes from virally infected cells.
To further demonstrate the utility of the virus chip, we analyzed the effect of drug treatment on viral gene expression in HIV-1 and KSHV-infected cells. In the first experiment, we compared HIV gene expression before and after drug treatment. Cellular cyclins and cyclin dependent kinases (CDK) have been shown to be critical for the expression and replication of a number of viruses, including HIV and several herpesviruses [29–35]. We and others have previously shown that inhibiting CDKs with ATP analogs, such as CYC202 (r-CYC202; Cyclacel Ltd; http://www.cyclacel.com), can suppress HIV-1 expression and replication in vitro and in vivo [35, 36]. However, we were previously unable to specifically determine which ORFs (from doubly spliced or singly spliced messages) was inhibited in these drug treated cells. To determine which viral transcripts were suppressed by Cyc202, HIV-1 expression was induced in ACH2 cells, a latently infected HIV-1+ cell line, by incubation with TNF-α for two hrs. Cells were subsequently washed and fresh media was added, with or without CYC202 (5 μM). Similar treatments were performed in uninfected CEM parental cells. Nine hours after induction, total RNA from uninfected, infected and CYC202 treated cells were isolated, labeled, and hybridized to the virus chip (Figure 4B). Without any induction, both the LTR and Gag genes showed detectable levels of expression, while low expression was observed for Rev (red bars). This is consistent with previously published reports showing that latent cells exhibit low basal transcription with a mostly non-processive RNA polymerase II . When cells were treated with TNF-α, all of the expected HIV-1 RNAs were transcribed and detected on the virus chip (green bars). This is also consistent with previously published reports, where the mRNA species producing Tat, Rev, and Nef are coordinately regulated by Rev and therefore these transcript levels accumulate in the absence of Rev protein and are down regulated in the presence of Rev .
When cells were treated with CYC202, we observed the down-regulation of a number of transcripts. Infected cells treated with CYC202 should have expression levels similar to infected cells not induced with TNF. We can in fact observe this in Figure 4B, where hybridizations from CYC202 treated cells, expressed as a Log2 (ratio) with untreated cells (yellow bars), were comparable to the Log2 ratios of uninduced cells. If CYC202 did not have an effect, the Log2 ratio values would have been closer to 0 (Log2 of a ratio of 1 is zero). Log2 intensity ratios less than one indicate that the drug had little or no effect on the expression of that gene. CYC202 mainly inhibited expression of the LTR and Pol and Nef genes. This is consistent with the mechanism of CYC202 inhibiting at the promoter (LTR) and the first ORF (Gag). The down-regulation of Nef, which is the most abundant doubly spliced transcript in infected cells, also implies that these transcripts are susceptible to regulation by CDK inhibitors. This novel finding was unexpected; however, it further characterizes which of the doubly or singly spliced messages are regulated by cyclin/cdk complexes in HIV-1 infected cells. Although these experiments indicate that CYC202 inhibits activated transcription at the HIV-1 LTR by inhibiting cdk2 and cdk9 , it does not address the changes in the half-lives of the other HIV-1 ORFs. However, in the absence of Gag or Nef, no HIV-1 particles were made, as demonstrated by the lack of the p24/gag antigen in the supernatants [35, 36].
In a second experiment, we evaluated viral gene expression in the KSHV infected cell line, BCBL-1. BCBL-1 is a primary effusion lymphoma cell line established from a HIV+ patient . The KSHV genome remains latent with very little viral gene expression in infected cells cultured in vitro. After treatment with TPA, viral gene expression is activated . As can be seen in Figure 4C, following nine hours of TPA treatment, low but significant expression (p < 0.05) was detected for a number of KSHV ORFs, including K5, K7, ORF72, and ORF73. The expression of these genes was previously shown to peak later in the viral life cycle . We also detected hybridization to ORF50/Rta, which was previously shown to exhibit significant expression by 10 hours post-induction . CYC202 has been shown to inhibit the gene expression and replication of a number of herpesviruses including herpes simplex virus (HSV) and cytomegalovirus (CMV) [29, 32, 36]. Therefore, we attempted to determine if CYC202 could inhibit KSHV gene expression. At the time of induction with TPA, CYC202 (5 μM) was added and nine hours after induction total RNA was isolated, labeled, and hybridized to the virus chip (Figure 4C). Values were determined as the mean log2 ratios of four experiments. Evidence of suppression by CYC202 was demonstrated by the fact that the sample treated with CYC202 behaved like the sample with no TPA induction. The data indicate that CYC202 suppressed the expression of the lytically induced gene, K7. These results are similar to what has been observed with HSV .
These results demonstrate the utility of using a virus chip for gene expression studies of virally infected cells. The advantage of using a chip containing all ORFs of a virus is that it allows for analysis of global changes in gene expression. Furthermore, the ability of longer products to hybridize effectively despite some number of mismatches can be an advantage for viruses that exhibit a relatively high mutation rate and for the detection of virus strains from related families.
Use of ChIP-chip analysis to identify proteins bound to a viral genome in vivo
We have developed an amplicon based virus chip representing the genomic sequences from eight human viruses. Using labeled DNA from infected cells or infectious plasmids we were able to specifically identify each virus, with minimal cross-hybridization between the various viral species. Furthermore, we were able to accurately identify both viruses from co-infected cells. We further demonstrated the utility of the virus chip with a variety of expression studies as well as ChIP-chip analysis. Significantly, we were able to detect specific viral sequences immunoprecipitated from infected cells without further amplification. Given the above positive results, we plan to further develop this chip by including the open reading frames of other viruses that are known human pathogens. The potential applications of a viral microarray representing pathogenic viruses extends beyond profiling expression of viral genes to the discovery of novel viruses, drug target identification and drug development, and widespread screening of blood or organs for viral contamination prior to transplantation [11, 64, 65].
All cell lines were grown in RPMI 1640, 10% FBS, 1% streptomycin/penicillin, and 1% L-glutamine (Invitrogen). Uninfected control T and B cell lines included CEM, SupT1, and BJAB. The following virally infected cell lines, MT-2 (HTLV-I); C19 (HTLV-II); B-95A, MM2 and Jijoye (EBV); BCBL-1 (KSHV) , Cra-BCBL  and BBG1  (dually infected with KSHV and EBV); and ACH2 (HIV-1) were also used. Genomic DNA from EBV strain Jijoye was a generous gift from Dr. Charles Wood (University of Nebraska). Uninfected SupT1 cells were co-cultivated with SupT1 infected with HHV-6A (U1102) and HHV-6B (Z29). Seven days post-infection, RNA and DNA were isolated. Viral replication was induced in HTLV and HIV infected cell lines with TNF-α (10 ng/ml) for two hrs. Viral replication was induced in KSHV-infected cells with TPA (20 ng/ml) or sodium butyrate (0.3 mM). For the CYC202 (r-CYC202) studies, induced cell lines were treated with CYC202 (5 μM) at the time of induction.
PCR primer sets (see Additional file 1) were generated for all specific viral open reading frames from the eight target viral species. Coding sequence coordinates of the ORFs were adjusted to prevent overlaps between amplified regions and to limit the amplicon size to less than 3.5 kb. PCR primer sets were designed using Primer 3.0  with optimized design parameters. Larger ORFs were represented by up to four amplicons. The largest PCR product obtained was 3.2 kb in length and the shortest 100 bp. We were able to successfully design primer sets for 302 of the 329 ORFs identified in the eight viruses (see details and primer sequences in Additional file 1). Coding regions for which primers could not be designed corresponded to very small target sequences or highly repetitive regions. Genomic DNA (1 ng) from infected cells was used as the template and was amplified with 800 nM primers and Taq DNA polymerase using the supplied buffer (Applied Biosystems). Conditions for PCR were 94°C, 30 sec; 60°C, for 45 sec; and 72°C for 2 min 35 sec; for 30 cycles, with a final extension at 72°C for 10 min. Following amplification, PCR products were purified with the Millipore 96-well filtration system, according to manufacturer's directions. Five microliters of purified product were separated through a 1% agarose gel and analyzed for the presence and correct size of each amplicon. Each product was graded as strong, acceptable, questionable, smear, mis-primed, or failed and scores are uploaded into the TIGR database. A score indicating an unsuccessful amplification will automatically give a null value for the particular spot corresponding to that amplicon. We obtained amplicons for 254 genes in the first generation of the chip and 264 in the second generation, with failed amplifications giving either no products or multiple bands. Figure 1 provides an overview of the genomic location of the ORFs that were successfully amplified and spotted for each virus. Purified amplicons were spotted in twelve replicate copies on aminosaline-coated microscope slides (Corning) using the Molecular Dynamics generation III arrayer (Sunnyvale, California). Printing was performed at room temperature and at a humidity level of 40–52%. Printed slides were cross-linked by drying for 2 hours at 80°C.
Sample labeling, hybridization, and scanning
Total RNA was isolated from control and infected cell lines with RNA-BEE (Tel-Test, Inc.) according to the manufacturer's directions. Residual genomic DNA was removed by DNase I digestion (RNase-free, Amersham Biosciences) and phenol-chloroform extraction. Total genomic DNA was isolated as described .
Genomic DNA (3 μg) was labeled using 15 μg random hexamers (Invitrogen), 3 mM Cy3-dCTP or 3 mM Cy5-dCTP (Amersham Pharmacia), and 15 units exo-Klenow large fragment (New England Biolabs), at room temperature for 3 hr. Labeled DNA probes were purified using a GFX column (Amersham Pharmacia).
Infectious plasmid clones were labeled by nick translation as described . Cy3-dCTP or Cy5-dCTP (1 mM) was included with 1 mM each dTTP, dATP, and dGTP. Following incubation at 15°C, the labeled products were purified using a GFX column (Amersham Pharmacia).
For expression profiling experiments, complimentary DNA was synthesized by reverse transcription in the presence of aminoallyl-tagged dUTP (aa-dUTP, Ambion). Total RNA (2 μg) and 6 μg of random hexamers (Life Technologies) were incubated at 70°C for 10 min and snap frozen. cDNA was synthesized overnight at 42°C by reverse transcription (SuperscriptII, Invitrogen) in the presence of 25 mM each dATP, dCTP, dGTP, 10 mM dTTP, and 15 mM aa-dUTP (Ambion). The RNA template was hydrolyzed with 1 M NaOH at 65°C. The resulting cDNA was filtered and concentrated with a Microcon-30 spin column (Millipore) and dried under vacuum. The sample was resuspended in 0.1 M carbonate (Na2CO3, pH 9.0) buffer and NHS-Cy3 or -Cy5 (Amersham Pharmacia) to fluorescently label the cDNA probe (at room temperature for 3 hr). The coupling reaction was purified using the QIAquick PCR purification kit (Qiagen). The probe was eluted in 100 μl DNAse/RNase-free water and analyzed using a Beckman spectrophotometer to measure dye incorporation and nucleotides per dye.
Slides were rinsed in 0.1% SDS and denatured in boiling water. Labeled probes (Cy-3/Cy-5) were resuspended in water and salmon sperm DNA and denatured at 95°C. Hybridization solution (50% formamide, 5X SSC, 0.1% SDS, and 0.2 mg/mL bovine serum albumin) was added and the probes were incubated for 20 min at the appropriate hybridization temperature. Probes were allowed to hybridize overnight at 42°C and 48°C for RNA and DNA experiments, respectively. Slides for DNA experiments were washed sequentially in (i) 1X SSC, 0.2% SDS at 48°C; (ii) 0.1X SSC, 0.1% SDS at room temperature; and (iii) 0.1X SSC at room temperature. Each wash was for 4 min. Slides for RNA experiments were washed sequentially in (i) 2X SSC, 0.1% SDS at 42°C; (ii) 0.1X SSC, 0.1% SDS at room temperature; (iii) 0.1X SSC at room temperature; and (iv) 0.01X SSC at room temperature. Each wash was for 4 min. The slides were scanned with an Axon-4000B scanner and images saved as paired single-color TIFF images.
Determination of HHV-6 copy number
Genomic DNA from infected cells was isolated according to the manufacturer's directions (Gentra System) and the concentration was adjusted to 10 ng/μl. Immediate early gene sequences were amplified using a TaqMan assay . A standard curve was generated using a known concentration of variant-specific HHV-6 plasmids. Results were plotted and sorted using the Sequence Detector System (Perkin Elmer). Results were normalized using a human genomic β-actin calibration curve. Absolute viral and β-actin DNA copy number was assessed and final viral DNA load per 106 cells was calculated by the following formula: [HHV-6 DNA copy number/(β-actin DNA copy number/2)] × 106.
Chromatin immunoprecipitation (ChIP) was performed as described previously  with slight modifications. Briefly, cells were cross-linked with 1% formaldehyde. Nuclei, prepared by hypotonic lysis, were resuspended in lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl, pH 8.1), sonicated to reduce DNA length to 200–1000 bp, and debris removed by centrifugation. The chromatin solution was precleared on protein A/G beads pre-adsorbed with sonicated salmon sperm DNA. The chromatin solution was then incubated with an antibody to histone H3 phosphorylated on serine 10 (Upstate Cell Signaling Solutions) or no antibody overnight at 4°C. Immune complexes were collected with protein beads pre-adsorbed with sonicated salmon sperm DNA. Following washes and elution, cross-linking was reversed by heating at 65°C for 4 to 5 hours, and DNA was recovered by digestion of proteins with proteinase K followed by phenol-chloroform extraction and ethanol precipitation. DNA was labeled directly as described above for genomic DNA, hybridized (chip), and washed accordingly.
As a first step, in-slide replicate analyses were performed by calculating the geometric mean of the spots corresponding to each gene. For the DNA experiments where infected (test) versus uninfected (control) host cell DNA was used in the hybridizations, a linear normalization was performed based on the assumption that the ratio of the host cell genes should be equal to 1. In genomic DNA hybridizations, for each host cell gene represented on the array, the geometric mean of the measured fluorescence intensities was calculated for both the experimental and control and the ratio of these was used as a scaling factor to adjust the values of all fluorescence measures for the viral genes represented on the array. For the expression studies (cDNA hybridizations), data was generated by dye-swap replication experiments. Total normalization was performed followed by flip-dye consistency checking using the TIGR Microarray Data Analysis System  or by one-class T-test analysis. For the plasmid experiments, where the same DNA was labeled with both dyes, an iterative log mean centering normalization was performed using MIDAS v2.17 with the following parameters: global mode, +/- 3 S.D. outlier range, Cy3 . We treated the Cy3 and adjusted Cy5 intensities as technical replicates and calculated the mean of these values. The ratio of this mean on the average of the intensity across the array set was then obtained. A ratio greater than 2 indicates that there was hybridization to a specific gene 2-fold above the background intensity across the whole array.
Elodie Ghedin and Anne Pumfery contributed equally to the work and should be regarded as joint first authors. We would like to thank Dr. C. Wood (University of Nebraska) for various reagents used in this study and Dr. Claire Fraser for her support of the work at TIGR. The work at The Institute for Genomic Research (TIGR) was supported by an internal grant to Elodie Ghedin; the work at The George Washington University was supported by a grant from the National Institute for Allergy and Infectious Diseases, National Institutes of Health (NIAID/NIH) to Fatah Kashanchi. Fatah Kashanchi and Steve Jacobson share senior authorship.
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