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Department of Microbiology and Immunology, Brody School of Medicine, East Carolina University, Greenville, NC 27834, USA
Correspondence
Shaw M. Akula
akulas{at}mail.ecu.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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An Excel spreadsheet showing raw microarray data for one representative trial is available as supplementary material in JGV Online.
These authors contributed equally to this work. 
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Since the discovery of KSHV in 1994, the entire virus genome has been sequenced successfully (Neipel et al., 1997; Russo et al., 1996). However, the biology of KSHV infection, with a special emphasis on the reactivation of latency, is far from being understood completely. This is in part due to the lack of a good model system to analyse changes occurring in cells that are crucial for the reactivation process.
A lytic cycle of KSHV infection of BCBL-1 cells (a PEL-derived cell line) can be conditionally initiated by treating with 12-O-tetradecanoyl-phorbol-13-acetate (TPA) (Renne et al., 1996). In a recently published work, the effectiveness of TPA in inducing a lytic cycle of infection was analysed by using a conventional method of synchronizing cells in the G0 phase of the cell cycle by serum-starving for 24 h prior to treating with fetal bovine serum (FBS) to induce re-entry into the cell cycle. TPA was added to these cells at 0, 6, 16 and 24 h post-release from G0 block and monitored for the expression of early-lytic and late-lytic protein markers at the end of 48 h. It was determined that the S phase of the cell cycle was reached by 12–16 h after addition of FBS and, interestingly, the highest expression of KSHV lytic and late proteins was observed at 16 h post-TPA treatment. Accordingly, it was concluded that TPA was effective in stimulating a lytic cycle of KSHV infection in BCBL-1 cells that were predominantly in the S phase of the cell cycle (McAllister et al., 2005).
Cellular signalling plays a major role in different aspects of virus infection and pathogenesis (Akula et al., 2005; Shackelford & Pagano, 2004; Whitman et al., 2004). Deciphering such signalling, which is crucial for reactivation, will help us understand KSHV pathogenesis better. Here, the use and effectiveness of flow cytometry in sorting cells for analysing the cellular signalling crucial for TPA-induced KSHV reactivation are described. We provide evidence for the existence of an apt environment in cells derived from the S phase of the cell cycle to support an active lytic infection.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Antibodies.
Anti-PKMYT1 (Myt1; H-300), anti-phospho-Cdc2 (p-Cdc2 p34; Tyr 15) and anti-Cdc2 p34 (Cdc2; C-19) purchased from Santa Cruz Biotechnology Inc. were used in this study. We also used mouse anti-actin antibodies (clone AC-74; Sigma) in the Western blotting experiments.
Sorting of cells in different phases of the cell cycle by using flow cytometry.
BCBL-1 cells cultured in RPMI medium containing 10 % FBS were stimulated with 20 ng TPA ml–1. After 36 h, the cells were washed once in RPMI+10 % FBS and resuspended at a rate of 2x106 cells (ml RPMI+10 % FBS)–1 in a 15 ml blue-capped tube. A 10 µg ml–1 concentration of Hoechst dye 33342 (Sigma) was added to the cell suspension and incubated further at 37 °C for 45 min. These cells were analysed by using a FACScan flow cytometer (Becton Dickinson) to discriminate cells in different stages of the cell cycle.
Immunofluorescence assay (IFA).
Cells were washed once in PBS, spotted onto clean glass slides, air-dried, fixed in ice-cold acetone, stained for the KSHV early lytic protein ORF59 with a mAb developed by Dr Bala Chandran (The University of Kansas Medical Center, Kansas City, MO, USA) followed by goat anti-mouse fluorescein isothiocyanate-labelled secondary antibody (KPL) and examined under a fluorescent Nikon microscope that was attached to a SPOT Advanced digital-imaging system (Diagnostic Instruments Inc.). The number of cells positive for ORF59 expression was counted.
RT-PCR.
Total RNA was isolated from the target cells by using a Nucleospin RNA II kit (Clontech) as per the manufacturer's recommendations. Extracted RNA was examined for the presence of viral RNA transcripts by RT-PCR as described previously (Hamden et al., 2004). Briefly, a 2·5 µg sample of each RNA was incubated with 2 U DNase I (Invitrogen) and reverse-transcribed in a solution containing 250 ng random hexadeoxynucleotides and 50 U Superscript reverse transcriptase (First-Strand cDNA Synthesis System for RT-PCR; Invitrogen) in a final volume of 20 µl. A 2 µl sample of the cDNA was subjected to PCR analysis with specific primers to determine the expression of KSHV ORF50, ORF8 and the mannose-6-phosphate receptor (M6PR) gene. We used M6PR as an internal control, because no significant difference in the expression levels was observed between cells obtained from the G0/1 and S phases of the cell cycle (refer to raw data from microarrays, available in JGV Online). The primer set used to amplify ORF50 was 5'-TGGTGGAAGATGTGTGCATT-3' (sense) and 5'-CTCCGTGAGGATCCGAATAA-3' (antisense); that for ORF8 was 5'-AGTGAGGATCCACAATGACTCCCAGG-3' (sense) and 5'-TCCGAATTCTCAGAGCTCGTACCACATTAGGTTGTC-3' (antisense) and that for M6PR was 5'-ACTCCAGTTTCCCACGACAC-3' (sense) and 5'-ACTGAGGAAGAGGCTGGACA-3' (antisense). A 3 µl sample from the PCR was resolved in a 1·5 % agarose gel at 20, 25 and 30 cycles, respectively. The agarose gels were stained with ethidium bromide, bands were scanned and the band intensities were assessed by using the ImageQuaNT software program (Molecular Dynamics).
Microarray analysis.
BCBL-1 cells cultured in RPMI+10 % FBS were treated with 20 ng TPA ml–1 at 37 °C. After 36 h incubation, the cells were stained with Hoechst dye 33342 and sorted by a FACScan flow cytometer to obtain cells in the G0/1 and S phases of the cell cycle. The sorted cells (a total of 106 cells) were collected by centrifuging at 1000 r.p.m. for 10 min at 4 °C and total RNA was isolated by using a Nucleospin RNA II kit (Clontech) as per the manufacturer's recommendations. The quality of total RNA was evaluated by measuring the A260/A280 ratio, which was at least 1·9, and by gel-electrophoresis pattern, which revealed two major bands of 28S and 18S RNA. The RNA quality was further assessed by Agilent Bioanalyser (Agilent Technologies) and submitted to the SuperArray Bioscience Corporation (Frederick, MD, USA) for further analysis. Briefly, Oligo GEArray Human Cancer Microarray (OHS-802; SuperArray Bioscience Corporation) was used for the analysis and comparison of the expression of cancer genes in the G0/1 and S phases of the cell cycle (Zeng et al., 2003). The Oligo GEArray contains gene-specific oligonucleotides (60 bp) and uses a nylon matrix for its high nucleic acid-binding capacity. Briefly, biotin–cRNA was prepared from total RNA by using TrueLabelling-AMP 2.0 for hybridization to the array. The resulting cRNA was purified on a spin column and quantified by UV absorbance. For each reaction, 8 µg biotinylated cRNA was used for hybridization. Data acquisition and quantification of spot intensities were performed by using the GEArrayT Expression Analysis suite. Data for cells in the G0/1 phase of the cell cycle were set as the baseline and data for cells in the S phase of the cell cycle as the experiment. Experiment data were compared with baseline data. The raw data were filtered so that individual spots had to pass a number of quality criteria, including minimum-intensity levels and minimum signal-to-background ratios. Genes that passed these criteria were used for further data analysis. Each gene signal was then normalized by 10 % of the M6PR signal in the same membrane, and only those gene signals (
10 % of the M6PR signal) that were well above background were considered specific gene signals. Genes were considered to be expressed differentially if they were found to be altered by 
2·0-fold or 
0·5-fold in cells derived from the S phase of the cell cycle compared with those obtained from the G0/1 phase. Other comparative data analysis was done in an Excel spreadsheet (available in JGV Online). Duplicate RNA samples for each treatment obtained on two different days were used and compared for the microanalysis. Accordingly, a table was obtained with mean signal (n=2) showing the fold change in the expression pattern of genes in the experiment set (S phase of the cell cycle) compared with the baseline (G0/1 phase of the cell cycle).
Quantitative real-time PCR (qRT-PCR).
Total RNA was isolated from cells sorted in the G0/1 and S phases of the cell cycle by using a Nucleospin RNA II kit (Clontech) as per the manufacturer's recommendations. cDNA was synthesized from the total RNA (500 ng) by using the First-Strand cDNA synthesis system (Invitrogen). qRT-PCR was performed by using the synthesized cDNA in single wells of a 96-well plate (Bio-Rad) in a 25 µl reaction volume to analyse the expression of various transcripts and 
-actin mRNA as described previously (Ford et al., 2005). The 25 µl reaction mix contained 12·5 µl iQ SYBR Green supermix (Bio-Rad), 1 µl forward primer, 1 µl reverse primer, 2 µl cDNA and 8·5 µl sterile water. The primer sets used for PCR were as follows: for MYBL2, 5'-AAAACAGTGAGGAGGAAC-3' (sense) and 5'-CAGGGAGGTCAAATGTAC-3' (antisense) (Raschellà et al., 1999); for PKMYT1, 5'-GACTCCAAACTGCCTTGCTC-3' (sense) and 5'-CTCCAAAGAGGCCACAGAAG-3' (antisense); for TK1, 5'-CCTGAGGATGGCCTGGAGTCA-3' (sense) and 5'-ATTTCATAAGCTACAGCAGAG-3' (antisense) (Karbownik et al., 2005); for PPARD, 5'-CTCTATCGTCAACAAGGACG-3' (sense) and 5'-GTCTTCTTGATCCGCTGCAT-3' (antisense) (Dunlop et al., 2005); and for M6PR, 5'-GACACACCCTAGCGGACAAT-3' (sense) and 5'-CATTCCTTTGGCTCCCACTA-3' (antisense). The thermocycling program consisted of 95 °C for 3 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 45 s. The specificity and purity of the amplification reaction were determined by performing a melting-curve analysis.
Silencing PKMYT1 RNA (short interfering RNA).
Expression of PKMYT1 was inhibited by the transfection of double-stranded RNA oligos as described previously (Akula et al., 2005; Hamden et al., 2004). The oligos used in this experiment were 5'-CCUUUGACUACGCAAGUUUtt-3' (sense) and 5'-AAACUUGCGUAGUCAAAGGtg-3' (antisense) (Ambion). The lower-case letters at the end of the oligos indicate the 3'-terminal dinucleotide overhangs. The non-specific (NS) short interfering RNAs (siRNAs) used were those described previously (Akula et al., 2005). Briefly, 1x106 cells were washed twice in RPMI medium and incubated in phenol red-free RPMI medium supplemented with 5 % FBS at 37 °C. After 24 h incubation (considered as 0 h for the experiments in Fig. 4), the target cells were transfected with either double-stranded siRNAs or the NS controls by using Lipofectamine 2000 as per the manufacturer's recommendations (Invitrogen) as described previously (Ford et al., 2004; Hamden et al., 2004). These cells were treated with 20 ng TPA ml–1 4 h post-transfection. At 0, 12, 24 and 48 h post-transfection, total RNA was isolated from the cells and subjected to Northern blotting to monitor the expression of PKMYT1 and -actin mRNA.
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-actin expression was performed by using a DIG Luminescent Detection kit (Roche) as per the manufacturer's recommendations (Akula et al., 2004, 2005).
Western blotting.
Cell lysates were prepared by using cells grown in T25 flasks as described previously (Akula et al., 2004). Equal protein loading (25 µg) was maintained for all Western blotting experiments. Western blots were probed first with rabbit anti-PKMYT1 antibodies, then stripped and reprobed sequentially with the primary antibodies used in this study, which included rabbit anti-phospho-Cdc2 antibodies, rabbit anti-Cdc2 antibodies and mouse anti-actin antibodies. Bands were scanned and the band intensities were assessed by using the ImageQuaNT software program (Molecular Dynamics).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). We determined, by trypan blue exclusion test, that the proportions of dead cells in the R3-gated and non-R3-gated cells were 6·0±1 and 28·0±4·5 %, respectively. The majority of cells were in the G0/1 phase (73 %), followed by the G2/M (19 %) and S (8 %) phases of the cell cycle (Fig. 1b). Cells from the G0/1, S and G2/M phases of the cell cycle were sorted into separate tubes. These cells were analysed for the expression of ORF59 (early gene) by performing IFA. There was a significantly higher number of cells that were positive for ORF59 expression in the S phase of the cell cycle compared with those obtained from the G0/1 and G2/M phases (Fig. 1c, d). IFA staining for the early-lytic viral protein ORF59 confirms that there is enhanced KSHV lytic replication in cells derived from the S phase of the cell cycle compared with those obtained from the G0/1 and G2/M phases.
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). There was no significant difference in the levels of M6PR detected between cells derived from different phases of the cell cycle (Fig. 2a). No detectable signal was observed with reactions performed in the absence of reverse transcriptase or with no template (Fig. 2a), demonstrating the specificity of the RT-PCR performed. The results indicate that BCBL-1 cells in the S phase of the cell cycle provide an apt environment for initiation of a KSHV lytic cycle of infection by TPA. Taken together, our results based on experiments performed on flow cytometry-sorted cells corroborate those obtained in earlier studies (McAllister et al., 2005).
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2·0-fold or 0·5-fold) in cells from the S phase of the cell cycle compared with those in the G0/1 phase (Table 1). The raw data for one representative trial are available in JGV Online.
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) were also found to be increased in qRT-PCR studies (Fig. 3). The changes in expression detected by microarray analysis were generally lower than those derived by qRT-PCR. This could be explained by the different detection ranges of these two techniques. In fact, it has been demonstrated that microarrays tend to have a low dynamic range, which could lead to under-representations of changes in gene expression, whereas qRT-PCR has a high dynamic range (Mutch et al., 2002). As the majority of genes listed in Table 1 were not confirmed by qRT-PCR, the significance of their differential expression should be interpreted with caution.
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). To investigate a possible role for PKMYT1 in the initiation of a KSHV lytic cycle of infection by TPA, we monitored ORF59 expression in cells treated with siRNA specific for PKMYT1. Briefly, BCBL-1 cells were untransfected or transfected with siRNA specific for PKMYT1 or with (NS)siRNA. After 4 h, these cells were treated with 20 ng TPA ml–1 and further incubated at 37 °C. Northern blotting was performed with RNA isolated at 0, 12, 24 and 48 h after transfection to monitor PKMYT1 expression (Fig. 4a). The level of PKMYT1 was suppressed significantly by siRNA in TPA-induced BCBL-1 cells compared with the (NS)siRNA control (Fig. 4b). PKMYT1 mRNA inhibition of 25±6, 86±4 and 56±4 % was observed in BCBL-1 cells at 12, 24 and 48 h post-siRNA transfection, respectively (Fig. 4b). PKMYT1 expression levels were not altered significantly by the (NS)siRNA controls, demonstrating the specificity of the siRNA used (Fig. 4b). In order to further confirm the effect of silencing the PKMYT1 gene, we monitored the expression of PKMYT1 and the extent of Cdc2 phosphorylation (Cdc2-P) by Western blotting at 24 h post-transfection. PKMYT1 inhibition by specific siRNA resulted in a significant inhibition of PKMYT1 expression and Cdc2 phosphorylation in target cells (Fig. 4c). Transfection of cells with (NS)siRNA did not significantly alter the PKMYT1 expression levels or the extent of Cdc2 phosphorylation. In contrast, levels of endogenous Cdc2 (Cdc2-T) or -actin remained mainly unchanged, even in siRNA-transfected cells (Fig. 4c). Taken together, transfection of cells with siRNA specific for PKMYT1 was able to silence PKMYT1 expression and lower Cdc2 phosphorylation (Fig. 4d).
The KSHV lytic cycle of infection in the above siRNA-transfected cells was monitored by performing IFA to monitor ORF59 expression, a good indicator of the KSHV lytic cycle of infection (Glaunsinger & Ganem, 2004). BCBL-1 cells were untransfected or transfected with siRNA specific for PKMYT1 or (NS)siRNA and TPA-induced as described before. These cells were stained with Hoechst dye 36 h post-TPA induction and sorted to obtain cells from the G0/1, S and G2/M phases of the cell cycle by using a FACScan flow cytometer as described in Fig. 1. Cells from the G0/1, S and G2/M phases of the cell cycle were sorted into separate tubes. These cells were analysed for the expression of ORF59 by performing IFA. In untransfected cells, there was a significantly higher number of cells that were positive for ORF59 expression in the S phase of the cell cycle compared with those obtained from the G0/1 and G2/M phases (Fig. 5). In cells that were transfected with siRNA specific for PKMYT1, we observed a sharp decline in the number of cells positive for ORF59 expression in the S phase of the cell cycle (Fig. 5). There was a 60 % drop in the total number of cells (inclusive of those in the G0/1, S and G2/M phases of the cell cycle) expressing ORF59 due to siRNA transfection compared with untransfected cells. IFA staining for the ORF59 protein confirms a crucial role for PKMYT1 expression in the TPA-induced induction of the KSHV lytic cycle of infection.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). The ORF50 gene product possesses the ability to initiate the switch between latent- and lytic-gene expression of KSHV (Lukac et al., 1998). Subsequent studies have confirmed the ability of ORF50 to activate the entire KSHV lytic cycle of infection (Chang et al., 2002; Deng et al., 2002). KSHV ORF50 is expressed very early (<4 h) in reactivation, prior to the other lytic genes (Sun et al., 1998). In addition, expression of ORF59 and ORF8 is the best indicator of a lytic cycle of KSHV infection (Glaunsinger & Ganem, 2004; Jenner et al., 2001). Our data indicate that cells in the S phase of the cell cycle have their machinery tuned to provide signals that promote cell survivability, active DNA replication and increased lipid metabolism, while blocking cell-cycle progression to M phase. Based on the array data, we propose a simplified model explaining the cellular environment existing in the S phase of the cell cycle with the ability to support a lytic cycle of KSHV infection (Fig. 6).
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; Detta et al., 2003; Wawra et al., 1981; Zhang et al., 2001) and all of these genes play a role in enhancing DNA replication and, accordingly, making KSHV replication easier. Cellular TK1 is one of the key enzymes in the thymidine nucleotide-salvage pathway. TK catalyses a reaction forming thymidine 5'-phosphate (TMP), the precursor of thymidine 5'-triphosphate (TTP), which is required for the synthesis of DNA (Dobrovolsky et al., 2003). Herpesviruses encode a TK homologue that plays a crucial role in reactivation of its latent state (Griffiths et al., 2003). However, the protein encoded by ORF21 in KSHV has relatively low TK activity compared with other human herpesvirus TKs (Gustafson et al., 2000). RRM1 encodes the regulatory subunit of ribonucleotide reductase that catalyses the rate-limiting step of deoxyribonucleotide formation. It is the only enzyme in human cells capable of such synthesis, critical for the synthesis and repair of DNA (Bepler et al., 2005). PCNA is also identified to play a major role in the DNA-replication process, especially in herpes simplex virus (HSV) infections (Brown et al., 1997; Detta et al., 2003). It was demonstrated by the above studies that, after HSV infection, the neurovirulence factor ICP34.5 complexes with PCNA either directly or indirectly, allowing cellular DNA replication to continue. In the absence of ICP34.5, the PCNA complex is not formed and hence the cell goes into a growth-arrest state, failing to provide sufficient machinery for the virus to go through the replication cycle.
There are three isoforms of peroxisome proliferator-activated receptors (PPARs). They are PPAR
, PPAR or PPARD (also referred to as 
) and PPAR
. PPARs are members of the nuclear-receptor superfamily, which regulates gene expression in response to the binding of fatty acids and their metabolites. Expression of PPAR is primarily observed in tissues that have a high level of fatty acid catabolism, such as liver, brown fat, kidney, heart and skeletal muscle (Braissant et al., 1996). PPARD is expressed ubiquitously and PPAR has a restricted pattern of expression, mainly in white and brown adipose tissues, whereas other tissues, such as skeletal muscle and heart, contain limited amounts (Jové et al., 2005). In this study, we observed significant upregulation of only the PPARD isoform in TPA-induced cells derived from the S phase of the cell cycle. There was no significant change in the expression levels of PPAR in cells from the G0/1 or S phases of cell cycle; PPAR was not tested (refer to raw microarray data, available in JGV Online). The role of PPARD in KSHV infection has not yet been analysed. We hypothesize a key role for PPARD in the KSHV entry and egress processes, especially in cells undergoing reactivation of latency, as both of these events are at their peak. The biological membranes involved in signal transduction and viral DNA nuclear import and the cellular machinery aiding in virus egress are all made up of a phospholipid bilayer and cholesterol. In fact, fatty acylation of viral and host-cell proteins is required to direct enveloped viruses to the membranes (Raulin, 2002). Our future studies are directed to understanding the role of PPARD in KSHV infection of cells.
MYBL2 or B-myb has an anti-apoptotic function, facilitating cell survivability for enhanced KSHV replication. MYBL2 is expressed during late G1 and early S phases of the cell cycle. It has the ability to overcome a block induced by both p53 and p107 when overexpressed, suggesting a role in tumorigenesis (Sala et al., 1996; Ziebold et al., 1997).
Finally, and most importantly, PKMYT1 or Myt1 causes G2/M arrest in the case of DNA damage by phosphorylating Thr14 and Tyr15 of Cdc2 of the Cdc2–cyclin B1 complex (Chen & Gardner, 2004; Liu et al., 1997; Wells et al., 1999). Such an arrest at the G2 phase of the cell cycle has been a common strategy utilized by several viruses, such as Simian virus 40, polyoma virus, human T-lymphotrophic retrovirus, adenovirus and human immunodeficiency virus type 1 (Janssens & Goris, 2001; Zhao & Elder, 2005). Interestingly, KSHV seems to use a similar strategy during TPA-induced reactivation from latency. PKMYT1 seems to play a crucial role in the TPA-induced reactivation of KSHV. Inhibition of PKMYT1 lowers phosphorylation of Cdc2, resulting in an enhanced Cdc2 activity and leading cells to progress from the G2 to the M phase of the cell cycle (Fig. 4c). This phenomenon results in a significant decrease in the efficiency of TPA-induced BCBL-1 cells in the S phase of the cell cycle to support a lytic cycle of KSHV infection (Fig. 5). We hypothesize that the increase in PKMYT1 expression allows cells more time in the S and G2 phases of the cell cycle, which provides ample opportunity for a successful and productive TPA-induced KSHV lytic replication.
Under normal conditions, cells pass through phases of the cell cycle, producing cellular proteins aiding in a variety of functions. Upon entry, KSHV proceeds to utilize the host-cell machinery to maximize its chances of establishing a latent infection. In vivo, the majority of cells are latently infected, whilst 2–5 % of cells spontaneously undergo reactivation (Mesri et al., 1996; Renne et al., 1998). In the cell cycle, DNA synthesis occurs in the S phase. Due to the host-cell machinery being primed for DNA replication, this seems to be the ideal phase for KSHV reactivation (Fig. 6).
Our analysis has identified key changes in the expression of cancer-related genes occurring in TPA-induced BCBL-1 cells in the S phase of the cell cycle. Overall, the results reported here describe the first use of flow cytometry-sorted cells in studying the cellular events required to support a lytic cycle of KSHV infection. This method will prove useful in analysing cellular proteins that may play a role in reactivation of KSHV latency. To date, all of the antivirals used to treat herpesvirus infections target only the lytic cycle of infection. Hence, these antivirals can never get rid of the virus completely. In the long run, understanding the molecular switch critical for the reactivation of latent infections will definitely open the door for developing new strategies capable of controlling, and perhaps eradicating, latent viral infections.
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ACKNOWLEDGEMENTS |
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Received 12 October 2005;
accepted 7 November 2005.
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