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J Virol. 2016 February 1; 90(3): 1231–1243.
Published online 2016 January 15. Prepublished online 2015 November 11. doi:  10.1128/JVI.02617-15
PMCID: PMC4719599

Occupancy of RNA Polymerase II Phosphorylated on Serine 5 (RNAP S5P) and RNAP S2P on Varicella-Zoster Virus Genes 9, 51, and 66 Is Independent of Transcript Abundance and Polymerase Location within the Gene

R. M. Sandri-Goldin, Editor

ABSTRACT

Regulation of gene transcription in varicella-zoster virus (VZV), a ubiquitous human neurotropic alphaherpesvirus, requires coordinated binding of multiple host and virus proteins onto specific regions of the virus genome. Chromatin immunoprecipitation (ChIP) is widely used to determine the location of specific proteins along a genomic region. Since the size range of sheared virus DNA fragments governs the limit of accurate protein localization, particularly for compact herpesvirus genomes, we used a quantitative PCR (qPCR)-based assay to determine the efficiency of VZV DNA shearing before ChIP, after which the assay was used to determine the relationship between transcript abundance and the occupancy of phosphorylated RNA polymerase II (RNAP) on the gene promoter, body, and terminus of VZV genes 9, 51, and 66. The abundance of VZV gene 9, 51, and 66 transcripts in VZV-infected human fetal lung fibroblasts was determined by reverse transcription-linked quantitative PCR. Our results showed that the C-terminal domain of RNAP is hyperphosphorylated at serine 5 (S5P) on VZV genes 9, 51, and 66 independently of transcript abundance and the location within the virus gene at both 1 and 3 days postinfection (dpi). In contrast, phosphorylated serine 2 (S2P)-modified RNAP was not detected at any virus gene location at 3 dpi and was detected at levels only slightly above background levels at 1 dpi.

IMPORTANCE Regulation of herpesvirus gene transcription is an elaborate choreography between proteins and DNA that is revealed by chromatin immunoprecipitation (ChIP). We used a quantitative PCR-based assay to determine fragment size after DNA shearing, a critical parameter in ChIP assays, and exposed a basic difference in the mechanism of transcription between mammalian cells and VZV. We found that hyperphosphorylation at serine 5 of the C-terminal domain of RNAP along the lengths of VZV genes (the promoter, body, and transcription termination site) was independent of mRNA abundance. In contrast, little to no enrichment of serine 3 phosphorylation of RNAP was detected at these virus gene regions. This is distinct from the findings for RNAP at highly regulated host genes, where RNAP S5P occupancy decreased and S2P levels increased as the polymerase transited through the gene. Overall, these results suggest that RNAP associates with human and virus transcriptional units through different mechanisms.

INTRODUCTION

Chromatin immunoprecipitation (ChIP) has been used to determine the association of proteins with DNA in cells of humans (1), mice (2), Drosophila melanogaster (3), Caenorhabditis elegans (4), and Saccharomyces cerevisiae yeast (5) and has shown that gene regulation requires the interaction of multiple nuclear proteins, including RNA polymerase II (RNAP), with various transcription factors across the genome. ChIP has also been used to explore the phosphorylation status of the C-terminal domain (CTD) of RNAP at different positions along a transcription unit and showed that in eukaryotes, phosphorylation of serine 5 (S5P) residues is enriched at the beginning of genes, whereas the ends of genes are enriched with RNAP phosphorylated on serine 2 (S2P) residues (6). ChIP assays can also map the genomic location of posttranslationally modified histone proteins and chromosomal insulator elements that influence host and virus gene transcription (7,9). ChIP is a multistep procedure that requires experimental optimization. Whether sensitivity is measured by sequencing, microarray analysis, or quantitative PCR (qPCR), the sensitivity of a ChIP experiment depends on enrichment of protein-bound DNA fragments among a background of unbound fragments. Thus, antibody quality and the immunoprecipitation procedure are critical parameters. While the number and quality of ChIP-grade antibodies are increasing and immunoprecipitation is becoming increasingly standardized (10), chromatin fragmentation is a critical step that is receiving increased attention (11). Chromatin is fragmented by enzyme digestion or sonication, with the approximate size range of DNA fragments being determined by agarose gel electrophoresis. Typically, shearing of formaldehyde-cross-linked chromatin produces random DNA fragments ranging from 100 to 1,000 bp in length. Such fragmentation is necessary to show increased enrichment of protein binding to specific sites over unbound genomic regions and is a critical parameter when mapping adjacent protein binding sites. In particular, determination of the size of DNA fragments is important when investigating small genomes with compact transcriptional units.

Varicella-zoster virus (VZV), a ubiquitous neurotropic herpesvirus, has the smallest genome among the human alphaherpesviruses (12). To date, 74 open reading frames (ORFs) have been identified within the 124,884-bp VZV genome. While transcripts (13,15) and proteins (16) mapping to these ORFs have been detected, few of their promoters have been identified (17) and even fewer associated transcription factors have been characterized (18). Distal VZV gene regulatory elements, including transcriptional enhancers, remain to be identified, and most identified transcription regulatory regions are located proximal to the virus ORFs. Each VZV ORF is separated by an average of 272 bp (19). Since 146 bp of DNA is packaged into a nucleosome core particle (20), regulation of VZV transcription may involve posttranslational modifications of histones enriched on the gene promoters. Histone proteins containing posttranslational modifications indicative of permissive chromatin are bound to promoters of VZV genes transcribed during both latent and lytic infection (21).

Due to the high density of the VZV genome coding sequence with a close proximity of regulatory regions to associated genes, it is important to accurately quantify DNA fragmentation efficiency during ChIP. Thus, we developed a qPCR assay to rapidly determine DNA shearing efficiency. Using this assay and ChIP-qPCR, we investigated changes in RNAP density and CTD phosphorylation during transcription of VZV genes 9, 51, and 66 in human fetal lung (HFL) fibroblasts harvested at 1 and 3 days postinfection (dpi).

Unlike RNAP CTD phosphorylation in mammalian gene transcription, we found that the RNAP CTD heptad repeat contained no S2P in VZV promoter regions, suggesting that serine 2 phosphorylation is not involved in the exit of polymerase from the promoter region. In contrast, RNAP near the promoter displayed high levels of S5P. However, unlike their levels during host gene transcription, RNAP S5P levels did not decrease as polymerase transcribed beyond the promoter region into the gene body but were constant throughout the length of the VZV gene region and independent of transcript levels. These results suggest that the dynamic changes in CTD phosphorylation patterns seen during the transcription of VZV genes are distinct from those seen during the transcription of human genes.

MATERIALS AND METHODS

Virus and cells.

The VZV Ellen strain was used to infect HFL cells (ATCC CCL-153) propagated in Dulbecco's modified Eagle's medium (Invitrogen, Grand Island, NY) supplemented with 10% fetal bovine serum (HyClone, Logan, UT) (22).

Preparation of sheared chromatin from VZV-infected HFL cells.

VZV-infected HFL cells in 10-cm plates were fixed in 1% formaldehyde at room temperature for 10 min, quenched with 0.25 M glycine at room temperature for 5 min, washed with ice-cold phosphate-buffered saline (PBS) containing proteinase and phosphatase inhibitors (Pierce Biotechnology, Rockford, IL), scraped, pelleted, and stored at −80°C. For sonication, cell pellets were resuspended in 1 ml sonication buffer {5 mM PIPES [piperazine-N,N′-bis(2-ethanesulfonic acid)], 85 mM KCl, 10 μl/ml NP-40} supplemented with proteinase and phosphatase inhibitors. Total DNA from 2 × 106 cells (approximately 20 μg) was sonicated at −7°C (maintained by immersion in NaCl-saturated ice water) in 15 ml polymethylpentene conical tubes (Diagenode, Denville, NJ) using the microprobe, at a setting of 4 (40% maximal power output), of a Sonifer W140D cell disrupter (Heat Systems-Ultrasonics, Farmingdale, NY). After sonication, samples were centrifuged for 30 min at 40,000 × g and 4°C to eliminate nucleocapsids containing virus DNA (23).

DNA fragment size determination.

Formaldehyde cross-links were reversed by exposure to 65°C for 1.5 h in 0.2 M NaCl, and DNA was extracted by affinity chromatography (E.Z.N.A. tissue DNA kit; Omega Bio-Tek, Norcross, GA) and quantified by determination of the optical absorbance at 260 nm (NanoDrop spectrophotometer; Thermo Scientific). The size of the DNA was estimated using capillary gel electrophoresis (Bioanalyzer; Agilent Technologies, Santa Clara, CA). Equal amounts of sonicated DNA were analyzed by qPCR (7500 Fast PCR machine; Applied Biosystems, Foster City, CA) using virus- and cell-specific primers (Integrated DNA Technologies, Coralville, IA) (Table 1) with the following cycling conditions: 15 min at 95°C (activation), followed by 40 cycles at 95°C for 30 s (denaturation), 55°C for 30 s (annealing), and 72°C for 90 s (extension). Amplified DNA was resolved on 2.0% agarose gels, visualized with ethidium bromide, and imaged on a FluorChem Q imager (R&D Systems, Minneapolis, MN). The amount of PCR-amplifiable DNA at each region and each sonication time point was quantified by comparison to the amounts for dilutions of intact (unsonicated) DNA analyzed in the same PCR. The efficiency of VZV DNA shearing was calculated as follows: [(N0Nt)/N0] × 100, where N0 is the initial VZV DNA copy number and Nt is the VZV DNA copy number at time t.

TABLE 1
Oligonucleotide primers

Chromatin immunoprecipitation.

Immunoprecipitations were carried out using a Pierce magnetic ChIP kit (Thermo Scientific, Rockford, IL) according to the manufacturer's instructions and overnight antibody incubations with mouse anti-RNA polymerase II S2P IgG (Diagenode, Denville, NJ) or mouse anti-RNA polymerase II S5P IgG (Thermo Scientific, Rockford, IL). qPCR was used to determine the amount of VZV DNA immunoprecipitated, after normalization of the results to the amount of input DNA. The GAPDH (glyceraldehyde-3-phosphate dehydrogenase) gene promoter served as a positive ChIP control for RNAP S5P enrichment, while two regions on HFL cell chromosome 19 known to lack RNAP S5P were used as negative controls (see Table 2). A region 4 kbp downstream from the transcriptional start site of the GAPDH gene was selected as a positive ChIP control for S2P enrichment on the basis of published Encode data from A549 (human lung carcinoma) cells and human umbilical vein endothelial cells (HUVEC) (http://genome.ucsc.edu). Further ChIP controls included normal mouse IgG (AbCam, Eugene, OR) and no IgG (beads alone). ChIP data were analyzed by analysis of variance (ANOVA) and multiple t tests using the Bonferroni correction to adjust for type 1 error.

TABLE 2
RNAP S5P and S2P occupancy on VZV genes 9, 51, and 66 and GAPDH gene

Quantitative RNA analysis.

Total RNA was extracted (TriPure isolation reagent; Roche, San Francisco, CA) from VZV-infected HFL cells at 1 dpi before a virus-induced cytopathic effect (CPE) and 3 dpi, when the CPE was at its height, and first-strand cDNA was synthesized (Transcriptor first-strand cDNA synthesis kit, Roche). Transcripts were quantified by qPCR as described above using VZV- and cell-specific primers (Table 1). Each VZV qPCR mixture contained dilutions of VZV DNA at known concentrations to determine the abundance of the virus transcripts (24).

RESULTS

Efficiency of VZV DNA shearing.

Initial experiments showed that most sheared cell DNA averaged ~300 bp in size, but ChIP routinely demonstrated a high nonspecific background (data not shown). While a high ChIP background can be attributed to many factors, we reasoned that virus DNA was insufficiently sheared, since shorter DNA fragments would be expected to have fewer sites for nonspecific binding. Thus, PCR primers that spanned 100% of VZV ORF 9 and 45% of ORF 51 were used to determine the extent of VZV DNA shearing by sonication (Fig. 1). For VZV ORF 9 (Fig. 1B) or ORF 51 (Fig. 1C), one forward primer and three reverse primers were selected to yield nested PCR products of ~250 bp (short fragments), 550 bp (fragments of medium length), and 1,100 bp (long fragments) (Table 1). VZV ORFs 9 and 51 represent two distinct regions of the virus genome (Fig. 1A) separated by ~77 kbp. To ensure that the SYBR-based quantitative PCR conformed to the minimum information for publication of quantitative real-time PCR experiments (MIQE guidelines for PCR specificity, sensitivity, and efficiency) (25), PCR amplification of known amounts of VZV DNA revealed single bands of the expected size for each primer pair (Fig. 2, top), and each PCR assay detected as few as 10 copies of VZV DNA with little variation in 6 independent trials (Fig. 2, bottom).

FIG 1
Locations of oligonucleotide primers on the VZV transcription map. (A) The VZV genome consists of unique long (UL) and unique short (US) DNA segments, each of which is bounded by inverted repeat regions (open boxes). A total of 74 ORFs, 39 on the top ...
FIG 2
Long-range qPCR. (Top) Log10 dilutions of VZV DNA (106 to 101 virus DNA copies) were amplified with PCR primers that generated long (1,108 bp for ORF 9 and 1,138 bp for ORF 51), medium-length (569 bp for ORF 9 and 504 bp for ORF 51), and short (218 bp ...

The efficiency of VZV DNA shearing was determined by analysis of formaldehyde-cross-linked chromatin from virus-infected HFL cells after sonication for 0, 4, 8, 12, and 20 min. The amount of host DNA with lengths of between 100 and 1,000 bp increased with the time of sonication and peaked at ~200 bp after 20 min of sonication (Fig. 3A). Bioanalyzer analysis provided a convenient and sensitive estimate of the DNA fragmentation pattern within the size range of the capillary gel. DNA fragments larger than the capillary exclusion limit (1,500 bp) were concentrated at the top of the gel and obscured by the 1,500-bp DNA marker added to each sample. While Southern blotting of sheared DNA probed with labeled VZV DNA could be used to determine the size range of the virus DNA fragments, we used long-range qPCR to determine the efficiency of shearing of the virus DNA among the background of host DNA fragments. The amount of VZV DNA remaining following sonication was determined by long-range PCR with both ORF 9- and ORF 51-specific primer sets, and the results from two independent biological replicates were averaged and showed that nucleocapsid-free VZV DNA was sensitive to sonication (Fig. 3A, bottom). After 4 min, 30% of virus DNA fragments of <~550 bp remained intact. However, a 20-min sonication was required to reduce the amount of VZV DNA fragments of <250 bp that remained intact to <20% of the starting quantity. A 15-min sonication was selected for subsequent ChIP experiments, since 6 independent trials showed that this amount of time was sufficient to reduce the amount of VZV DNA fragments of <1,100 bp to <5% of the initial quantity and VZV DNA fragments of <550 bp to <10% of the initial quantity by PCR amplification. An example of the DNA fragmentation achieved following 15 min of sonication of formaldehyde-cross-linked VZV-infected HFL cell DNA is shown in Fig. 3B. The Bioanalyzer analysis (Fig. 3B, top, left) showed an increase in the amount of DNA fragments of <1,000 bp obtained following 15 min of sonication. Similar results were observed when the same DNA was analyzed by standard electrophoresis on 2% agarose gels (Fig. 3B, top, right). The percentage of VZV DNA sheared was calculated from the amount of PCR product remaining, as described in Materials and Methods, and indicated that 91% of the VZV DNA was sheared to fragments of <1,100 bp, 86% of the VZV DNA was sheared to fragments of <550 bp, and 69% of the VZV DNA was sheared to fragments of <250 bp (Fig. 3B, bottom).

FIG 3
Sheared chromatin size. (A) Formaldehyde-fixed DNA from VZV-infected HFL cells was sonicated for 0, 4, 8, 12, and 20 min. (Top) The abundance of DNA fragments with lengths of between 50 bp and 1,500 bp was determined by capillary gel electrophoresis after ...

After optimization of VZV DNA fragmentation, ChIP was conducted to ensure that excess antibody was present. Serial 8-fold dilutions of fragmented chromatin from duplicate biological replicates were immunoprecipitated with 5 μg purified anti-RNAP S5P antibody. qPCR amplification of input VZV DNA with VZV ORF 9- and ORF 51-specific primers reflected the 8-fold dilutions (Fig. 4). The amount of VZV DNA immunoprecipitated with anti-RNAP S5P antibody also resulted in similar 8-fold reductions. For all dilutions and at all 3 locations within both VZV ORF 9 and VZV ORF 51, the same ratio of input VZV DNA to immunoprecipitated virus DNA was obtained (ORF 9, 6.5% ± 1.7%; ORF 51, 5.0% ± 1.1%). Overall, 5.77% ± 1.57% of input VZV DNA was immunoprecipitated with anti-RNAP S5P IgG. On the basis of the results presented above, antibody excess was clearly evident.

FIG 4
ChIP under conditions of excess antibody. Serial log8 dilutions of sonicated chromatin from formaldehyde-fixed, VZV-infected HFL cells were immunoprecipitated with equal amounts of mouse anti-RNAP S5P-specific IgG. The amount of VZV DNA added to each ...

RNAP S5P and RNAP S2P enrichment on VZV genes at height of CPE.

qPCR primers were designed to amplify regions within the promoter, the approximate ORF center, and the terminus of VZV ORFs 9, 51, and 66 (Table 1) and used to determine RNAP S5P and RNAP S2P occupancy (Fig. 5, top). All primer pairs, regardless of product length (38 to 143 bp), detected as few as 10 copies of VZV DNA and produced a single discrete band on agarose gels, as well as sharp melting temperature profiles (data not shown).

FIG 5
Comparison of RNAP S5P and RNAP S2P occupancy to VZV mRNA abundance at 1 and 3 days postinfection. VZV ORFs 9, 51, and 66 were investigated for RNAP S5P and RNAP S2P occupancy by ChIP (top), and mRNA abundance was determined by RT-qPCR at 1 and 3 dpi ...

Table 2 lists all individual ChIP data along with the overall results obtained for each location in VZV genes 9, 51, and 66 as well as three cellular regions (the GAPDH gene promoter, ZFP154, and ZFP180). No statistically significant difference (ANOVA, P = 0.92) in RNAP S5P occupancy in the promoter, ORF center, or transcription terminus was detected for VZV genes 9, 51, and 66. However, the amount of VZV DNA immunoprecipitated with anti-RNAP S5P antibody was significantly greater (t test, P < 0.001) than that recovered using an isotype control antibody or when antibody was omitted from the reaction mixture (background). With respect to the host control regions, significant enrichment of RNAP S5P on the GAPDH gene promoter compared to that for the isotype control was detected (ANOVA, P = 0.05; t test, P = 0.05), while no significant RNAP S5P enrichment (ANOVA, P = 0.56) at the two host negative-control regions relative to the amount seen in the ChIP control immunoprecipitations was detected (Table 2). Since both ChIP assays for determination of the amount of background (assays with nonspecific, isotype control IgG and assays in which IgG was omitted altogether) gave the same results (ANOVA, P = 0.55), subsequent assays used only the nonspecific, isotype antibody as the ChIP background control.

No significant difference in the occupancy of RNAP S2P at the promoter, ORF center, or transcription terminus was detected for VZV genes 9, 51, and 66 in virus-infected HFL cells harvested at 1 and 3 dpi (ANOVA, P = 0.93) and with respect to that obtained with nonspecific isotype control IgG (ANOVA, P = 0.06) (Table 2).

With respect to the results obtained with the host control regions (the GAPDH gene promoter and the two zinc finger protein genes), no significant amounts of RNAP S2P above the amount of isotypic control IgG were immunoprecipitated (ANOVA, P = 0.98). Since these regions were specifically selected for their RNAP S5P binding characteristics, Encode ChIP-DNA sequencing data were searched for phosphorylated RNAP binding, and combination of the information for human umbilical endothelial cells (HUVEC) with that for human lung carcinoma (A549) cells provided three regions within the GAPDH gene that showed differences in RNAP S5P and RNAP S2P occupancy (Fig. 6 bottom).

FIG 6
Comparison of RNAP S5P and RNAP S2P occupancy on the GAPDH gene. DNA from VZV-infected HFL cells was harvested at 1 and 3 dpi. Formaldehyde-cross-linked, sheared chromatin was immunoprecipitated with mouse anti-RNAP S5P IgG (top) and mouse anti-RNAP S2 ...

Before quantitative ChIP analysis, each new GAPDH gene-specific primer pair was shown to amplify HFL cell DNA with a similar efficiency and specificity and produced a single discrete amplicon (data not shown). For all GAPDH gene-specific primers and at both 1 and 3 dpi, RNAP S2P and RNAP S5P occupancy on HFL DNA was significantly more than that of the isotype control antibody (t test, P < 0.01). Within the GAPDH gene, RNAP S5P occupancy decreased from the promoter to the transcription termination site in HFL cell chromatin harvested at both 1 and 3 dpi. More RNAP S5P was present at each location at 1 dpi than at 3 dpi. However, RNAP S2P occupancy on the GAPDH gene was maximal at the transcription termination site at 1 dpi. At 3 dpi, the RNAP S2P occupancy at the GAPDH gene promoter was reduced by 48%. The RNAP S2P occupancy was similar (0.08-fold ± 0.002-fold the input amount) at the GAPDH gene promoter and gene body but 8-fold above that for the respective background (isotype) controls (Fig. 6, top).

RNAP S5P and RNAP S2P enrichment on VZV genes early during VZV infection.

Since RNAP S2P occupancy at the GAPDH transcription termination site decreased from 1 to 3 dpi (Fig. 6) and at 3 dpi no significant RNAP S2P enrichment was detected on VZV genes 9, 51, and 66 (Fig. 5, top), posttranslationally modified RNAP occupancy on VZV DNA was investigated in virus-infected HFL cells harvested early (1 dpi). No significant difference (ANOVA, P = 0.06) in the occupancy of RNAP S2P at the promoter, ORF center, and transcription terminus of VZV genes 9, 51, and 66 was detected; however, the amount of VZV DNA immunoprecipitated at these regions was 2.8-fold above the background, which was a significant increase (t test, P < 0.01).

While the amount of RNAP S5P specifically immunoprecipitated was significantly above the background level (ANOVA, P < 0.01; t test, P < 0.01), the occupancy of RNAP S5P at the promoter, ORF center, and transcription terminus was the same within each gene (ANOVA, P < 0.01; t test, P < 0.01). Since RNAP S5P co-occupancy within each gene was the same at each location tested, each gene pair was compared. Gene 66 contained the most RNAP S5P (t test, P < 0.01), and genes 9 and 51 contained the same amount of RNAP S5P (t test, P = 0.12).

Steady-state VZV mRNA abundance.

The steady-state mRNA levels present at the promoter, ORF center, and transcription terminus of VZV ORFs 9, 51, and 66 RNA, as determined by reverse transcription-qPCR (RT-qPCR), was 46-fold ± 16-fold more at 3 dpi than at 1 dpi for all sites except the ORF 51 transcription termination site, whereas at 3 dpi it was only 6-fold more than that at 1 dpi (Fig. 5, middle section, top). Using gene body PCR results as an indication of gene transcript abundance, the levels of the ORF 9 transcripts were increased 30- and 40-fold over those of the ORF 51 transcripts at 1 and 3 dpi, respectively, which are similar to the 29- and 21-fold increases in the levels of the ORF 9 transcripts over those of the ORF 51 and 66 transcripts at 3 dpi, respectively. The abundance of ORF 51 and ORF 66 transcripts was the same at 1 dpi (t test, P = 0.27), while at 3 dpi, the amount of ORF 66 transcripts was twice that of the ORF 51 transcripts, which is a significant difference (t test, P < 0.001). The compactness of VZV transcriptional units was shown by the qPCR data and suggested continuation of ORF 9A into ORF 9 and overlap of the 5′ region of VZV ORF 51 with the 5′ untranslated region of ORF 50. In addition, the qPCR data indicated that ORF 66 mRNA terminates at least 200 nucleotides (nt) beyond the termination codon for ORF 66.

DISCUSSION

ChIP is a powerful tool used to decipher transcription regulation in alphaherpesviruses. Indeed, ChIP has shown that during productive herpes simplex virus 1 (HSV-1) infection, virus gene transcription is regulated, in part, by binding of virus (26) or host (27) proteins to specific DNA sequences within gene promoters or to protein components of the transcriptional apparatus (28). Importantly, ChIP analyses of HSV-1-infected neurons have shown that posttranslational modification of histone proteins is a critical factor that maintains the unique transcriptional pattern of virus genes during latency. Most of the ~80 HSV-1 genes expressed during productive infection are transcriptionally silent during latent infection, and their promoters are associated with nucleosomes containing posttranslationally modified histones indicative of transcriptionally silent heterochromatin. Conversely, the promoter of the single HSV-1 gene transcribed during latency is associated with histones containing posttranslational modifications indicative of actively transcribed euchromatin (29, 30). ChIP has shown that transcriptionally distinct regions of the virus genome are maintained by chromosomal insulators, most notably, the CCCTC-binding factor, CTCF (31). During HSV-1 reactivation, these chromatin boundary elements are released from the virus DNA (32, 33). Since the release of CTCF from the virus genome is an attractive mechanism connecting external stress to HSV-1 reactivation (34, 35) and the critical CCCTC-binding sites are separated by at least 3,300 nt (31), the HSV-1 DNA fragment size is not a critical aspect of these ChIP analyses. However, determination of the efficiency of virus DNA fragmentation is critical when locating transcription factors on the virus genome where promoters are closely linked to the genes that they control. Although computer algorithms that analyze DNA sequence data obtained by ChIP are continually updated to better locate protein binding domains on the genome (36), our long-distance PCR assay is a quick, convenient, and sensitive means to determine the efficiency of size fractionations of virus DNA within a large background of cell chromatin. The long-distance PCR assay can also be applied to determine the DNA fragmentation generated by single-strand-specific endo-exonuclease (micrococcal nuclease [MNase]) or DNase I, the well-established “gold standard” method for probing chromatin accessibility (37). Additionally, Southern blot analysis requires >2 days and significant amounts of sample, and it is difficult to accurately quantify the results. The <4-h PCR-based assay requires minimal sample (<10 ng input DNA), does not require an isotope, and is completed in a time frame that permits further sonication, if required.

In tissue culture and at the single-cell level, VZV productive infection takes 9 to 12 h, during which time immediate early, early, and late proteins are sequentially synthesized (38). However, VZV is a highly cell-associated virus, and titers of cell-free virus sufficient to infect cultures at multiplicities of >1 are unattainable. Consequently, in vitro infections are initiated by cocultivation of virus-infected cells with uninfected cells. Under these conditions, the CPE progresses slowly and transcription analysis does not reveal the sequential synthesis of individual classes of virus transcripts but, rather, reveals an overall increase in the number of transcripts of all classes (13). This was seen in the uniform increase in ORF 9 (late), ORF 51 (early), and ORF 66 (late) transcripts from 1 to 3 dpi. Also, since high-multiplicity VZV infections are not feasible, assignment of virus genes into kinetic classes (immediate early, early, and late) is done by analogy to the genes of herpes simplex virus 1, the prototype alphaherpesvirus. Transcripts for all annotated VZV genes have been detected by RT-qPCR (15) and quantified on PCR-based arrays (13) and long-oligonucleotide-based arrays (14), as well as strand-specific cDNA deep sequencing (22). These studies do not reveal the sequential transcription of different classes of virus genes, as is seen in other herpesviruses (39), but they do show a variation in the steady-state abundance of VZV transcripts. For example, the ratio of VZV ORF 9 to ORF 66 transcripts in virus-infected HFL cells at 3 dpi determined in this study was 21:1 and compared favorably to previous findings for VZV-infected HFL cells (30:1) (22) and virus-infected malignant melanoma cells (22:1) (13). Both VZV ORF 9 and ORF 66 transcripts have similar decay constants, indicating that mRNA stability does not account for the difference in ORF 9 and ORF 66 abundance (40). Instead, we reasoned that the number of VZV DNA templates involved in transcribing these genes differed and used RNAP ChIP analysis to determine polymerase occupancy.

RNAP CTD serves as a large flexible docking site for the coordinated assembly of factors involved in pre-mRNA capping, splicing, and 3′ polyadenylation at different stages in transcription of nascent RNA molecules (41). In eukaryotes ranging from yeast to humans, the phosphorylation of serines 2 and 5 of the RNAP CTD heptad repeat coordinates distinct phases of the polymerase complex assembly. Accordingly, CTD undergoes a cycle of phosphorylation and dephosphorylation during transcription initiation, elongation, and termination (42). During transcription initiation, cyclin-dependent kinase 7 (CDK7) phosphorylates serine 5 residues at gene promoters (43,45) which enhance the association and activity of mRNA capping complexes (46, 47). RNAP is released from its promoter-paused position by pTEFb-dependent phosphorylation of RNAP at serine 2 (48). As RNAP transits the gene body, during transcriptional elongation, CDK9 further phosphorylates serine 2 (49, 50), while phosphatases progressively dephosphorylate S5P (41).

Consequently, RNAP phosphorylation on serine 5 generally decreases and is replaced by serine 2 phosphorylation as the polymerase transits the gene body (51). While the RNAP S5P-to-S2P switch typically present during eukaryotic transcription (52) was seen on the HFL cell GAPDH gene, RNAP S5P levels on VZV genes 9, 51, and 66 remained the same at the promoter, ORF center, and transcription terminus at both 1 and 3 dpi. Additionally, RNAP S2P levels did not differ within VZV genes 9, 51, and 66 at 1 dpi and showed no enrichment above the background at 3 dpi. RNAP S2P is inhibited during HSV-1 infection (26, 53), suggesting that herpesvirus transcription differs from the typical progression of RNAP C-terminal domain phosphorylation patterns seen in eukaryotic cells, a notion consistent with our finding regarding RNAP S2P. A possible explanation is that since RNA splicing is rare in VZV genes, the lack of RNAP S2P could reduce splicing and favor virus gene transcription; RNAP S2P is a signal for splicing factor accretion (54). In addition, the steady-state amounts of the VZV gene 9 and 66 transcripts are independent of the amount of RNAP S5P and RNAP S2P present at any location within these virus genes, suggesting that the rate of transcription may be a key component in virus transcript accumulation.

Overall, we have developed an accurate and sensitive PCR-based assay to quantify the efficiency of VZV DNA shearing. The assay showed significant and similar occupancy of RNAP S5P at three distinct regions within three virus genes whose steady-state mRNA abundance differs by ~20-fold. Determining the factors involved in maintaining mRNA steady-state levels is critical to a full understanding of virus gene transcription. While RNAP occupancy is a predictor of mRNA abundance (55), our findings indicate that mRNA degradation, RNAP S5P, or the transition to RNAP S2P occupancy does not determine VZV transcript abundance during productive infection. Future studies will investigate RNAP processivity as a factor influencing VZV mRNA accumulation.

ACKNOWLEDGMENTS

We thank Wenhua Ren and Bifeng Gau in the Genomics and Microarray Core at the University of Colorado School of Medicine for technical assistance, Marina Hoffman for editorial assistance, and Cathy Allen for manuscript preparation.

Funding Statement

This work was supported in part by Public Health Service grants AG032958 (D.G. and R.J.C.), NS093716 (D.G.), and NS082228 (R.J.C.) from the National Institutes of Health. H.B. and N.L.B. were supported by training grant NS007321 to D.G. from the National Institutes of Health.

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