HSV-1 Remodels Host Telomeres to Facilitate Viral Replication

SummaryTelomeres protect the ends of cellular chromosomes. We show here that infection with herpes simplex virus 1 (HSV-1) results in chromosomal structural aberrations at telomeres and the accumulation of telomere dysfunction-induced DNA damage foci (TIFs). At the molecular level, HSV-1 induces transcription of telomere repeat-containing RNA (TERRA), followed by the proteolytic degradation of the telomere protein TPP1 and loss of the telomere repeat DNA signal. The HSV-1-encoded E3 ubiquitin ligase ICP0 is required for TERRA transcription and facilitates TPP1 degradation. Small hairpin RNA (shRNA) depletion of TPP1 increases viral replication, indicating that TPP1 inhibits viral replication. Viral replication protein ICP8 forms foci that coincide with telomeric proteins, and ICP8-null virus failed to degrade telomere DNA signal. These findings suggest that HSV-1 reorganizes telomeres to form ICP8-associated prereplication foci and to promote viral genomic replication

Timeless Links Replication Termination to Mitotic Kinase Activation

The mechanisms that coordinate the termination of DNA replication with progression through mitosis are not completely understood. The human Timeless protein (Tim) associates with S phase replication checkpoint proteins Claspin and Tipin, and plays an important role in maintaining replication fork stability at physical barriers, like centromeres, telomeres and ribosomal DNA repeats, as well as at termination sites. We show here that human Tim can be isolated in a complex with mitotic entry kinases CDK1, Auroras A and B, and Polo-like kinase (Plk1). Plk1 bound Tim directly and colocalized with Tim at a subset of mitotic structures in M phase. Tim depletion caused multiple mitotic defects, including the loss of sister-chromatid cohesion, loss of mitotic spindle architecture, and a failure to exit mitosis. Tim depletion caused a delay in mitotic kinase activity in vivo and in vitro, as well as a reduction in global histone H3 S10 phosphorylation during G2/M phase. Tim was also required for the recruitment of Plk1 to centromeric DNA and formation of catenated DNA structures at human centromere alpha satellite repeats. Taken together, these findings suggest that Tim coordinates mitotic kinase activation with termination of DNA replication

CTCF Prevents the Epigenetic Drift of EBV Latency Promoter Qp

The establishment and maintenance of Epstein-Barr Virus (EBV) latent infection requires distinct viral gene expression programs. These gene expression programs, termed latency types, are determined largely by promoter selection, and controlled through the interplay between cell-type specific transcription factors, chromatin structure, and epigenetic modifications. We used a genome-wide chromatin-immunoprecipitation (ChIP) assay to identify epigenetic modifications that correlate with different latency types. We found that the chromatin insulator protein CTCF binds at several key regulatory nodes in the EBV genome and may compartmentalize epigenetic modifications across the viral genome. Highly enriched CTCF binding sites were identified at the promoter regions upstream of Cp, Wp, EBERs, and Qp. Since Qp is essential for long-term maintenance of viral genomes in type I latency and epithelial cell infections, we focused on the role of CTCF in regulating Qp. Purified CTCF bound ∼40 bp upstream of the EBNA1 binding sites located at +10 bp relative to the transcriptional initiation site at Qp. Mutagenesis of the CTCF binding site in EBV bacmids resulted in a decrease in the recovery of stable hygromycin-resistant episomes in 293 cells. EBV lacking the Qp CTCF site showed a decrease in Qp transcription initiation and a corresponding increase in Cp and Fp promoter utilization at 8 weeks post-transfection. However, by 16 weeks post-transfection, bacmids lacking CTCF sites had no detectable Qp transcription and showed high levels of histone H3 K9 methylation and CpG DNA methylation at the Qp initiation site. These findings provide direct genetic evidence that CTCF functions as a chromatin insulator that prevents the promiscuous transcription of surrounding genes and blocks the epigenetic silencing of an essential promoter, Qp, during EBV latent infection

A Redox-Sensitive Cysteine in Zta Is Required for Epstein-Barr Virus Lytic Cycle DNA Replication

Epstein-Barr virus (EBV) reactivation from latency is known to be sensitive to redox regulation. The immediate-early protein Zta is a member of the basic-leucine zipper (bZIP) family of DNA binding proteins that stimulates viral and cellular transcription and nucleates a replication complex at the viral lytic origin. Zta shares with several members of the bZIP family a conserved cysteine residue (C189) that confers redox regulation of DNA binding. In this work, we show that replacement of C189 with serine (C189S) eliminated lytic cycle DNA replication function of Zta. The mechanistic basis for this replication defect was investigated. We show that C189S was not significantly altered for DNA binding activity in vitro or in vivo. We also show that C189S was not defective for transcription activation of EBV early gene promoters. C189S was deficient for transcription activation of several viral late genes that depend on lytic replication and therefore was consistent with a primary defect of C189S in activating lytic replication. C189S was not defective in binding methylated DNA binding sites and was capable of activating Rta from endogenous latent viral genomes, in contrast to the previously characterized S186A mutation. C189S was slightly impaired for its ability to form a stable complex with Rta, although this did not prevent Rta recruitment to OriLyt. C189S did provide some resistance to oxidation and nitrosylation, which potently inhibit Zta DNA binding activity in vitro. Interestingly, this redox sensitivity was not strictly dependent on C189S but involved additional cysteine residues in Zta. These results provide evidence that the conserved cysteine in the bZIP domain of Zta plays a primary role in EBV lytic cycle DNA replication

A role for MRE11, NBS1, and recombination junctions in replication and stable maintenance of EBV episomes.

Recombination-like structures formed at origins of DNA replication may contribute to replication fidelity, sister chromatid cohesion, chromosome segregation, and overall genome stability. The Epstein-Barr Virus (EBV) origin of plasmid replication (OriP) provides episomal genome stability through a poorly understood mechanism. We show here that recombinational repair proteins MRE11 and NBS1 are recruited to the Dyad Symmetry (DS) region of OriP in a TRF2- and cell cycle-dependent manner. Depletion of MRE11 or NBS1 by siRNA inhibits OriP replication and destabilized viral episomes. OriP plasmid maintenance was defective in MRE11 and NBS1 hypomorphic fibroblast cell lines and only integrated, non-episomal forms of EBV were detected in a lympoblastoid cell line derived from an NBS1-mutated individual. Two-dimensional agarose gel analysis of OriP DNA revealed that recombination-like structures resembling Holliday-junctions form at OriP in mid S phase. MRE11 and NBS1 association with DS coincided with replication fork pausing and origin activation, which preceded the formation of recombination structures. We propose that NBS1 and MRE11 promote replication-associated recombination junctions essential for EBV episomal maintenance and genome stability

Repair of DNA strand breaks in a minichromosome in vivo: kinetics, modeling, and effects of inhibitors.

To obtain an overall picture of the repair of DNA single and double strand breaks in a defined region of chromatin in vivo, we studied their repair in a ~170 kb circular minichromosome whose length and topology are analogous to those of the closed loops in genomic chromatin. The rate of repair of single strand breaks in cells irradiated with γ photons was quantitated by determining the sensitivity of the minichromosome DNA to nuclease S1, and that of double strand breaks by assaying the reformation of supercoiled DNA using pulsed field electrophoresis. Reformation of supercoiled DNA, which requires that all single strand breaks have been repaired, was not slowed detectably by the inhibitors of poly(ADP-ribose) polymerase-1 NU1025 or 1,5-IQD. Repair of double strand breaks was slowed by 20-30% when homologous recombination was supressed by KU55933, caffeine, or siRNA-mediated depletion of Rad51 but was completely arrested by the inhibitors of nonhomologous end-joining wortmannin or NU7441, responses interpreted as reflecting competition between these repair pathways similar to that seen in genomic DNA. The reformation of supercoiled DNA was unaffected when topoisomerases I or II, whose participation in repair of strand breaks has been controversial, were inhibited by the catalytic inhibitors ICRF-193 or F11782. Modeling of the kinetics of repair provided rate constants and showed that repair of single strand breaks in minichromosome DNA proceeded independently of repair of double strand breaks. The simplicity of quantitating strand breaks in this minichromosome provides a usefull system for testing the efficiency of new inhibitors of their repair, and since the sequence and structural features of its DNA and its transcription pattern have been studied extensively it offers a good model for examining other aspects of DNA breakage and repair

Repair of single strand breaks in linear minichromosome DNA.

<p>(A) DNA synthesis (incorporation of [¹⁴C]thymidine) in irradiated and control cells in the conditions used for repair; error bars show SEM from three independent experiments. (B) Fragmentation by nuclease S1 of linear minichromosome DNA isolated immediately after irradiation (50 Gy) or after repair for 2 h. Linear DNA was isolated from a gel of total cell DNA and incubated without or with nuclease S1 for 15 h and the fragments produced were separated by PFGE. For these experiments sufficient linear DNA could be conserved for 2 h only if repair of double strand breaks was arrested; this was achieved by including the DNA-PK inhibitor NU7441 during repair as described in the Section "Pathways for repair of double strand breaks". (C) Scans of the hybridisation signal from lanes in (B) (nuclease S1 100 u/ml); the position of full-length linear molecules is indicated by the vertical dashed line.</p

Effect of inhibiting HR-mediated repair of double strand breaks.

<p>(A) Phosphorylation of ATM on Ser1981 (green) in cells irradiated and incubated without or with caffeine (10 mM) or KU55933 (20 µM), assayed by immunofluorescence; DNA was stained by DRAQ (red). Below, quantitation of the signal from ATM1981S-P (green pixel intensity/nuclear area). (B) Repair of minichromosome DNA in cells incubated without or with caffeine (10 mM) or KU55933 (20 µM), inhibitors of ATM kinase, or (C) with mirin (100 µM) which prevents activation of ATM without affecting its kinase activity. (D) Repair in cells transfected with siRNA to silence expression of Rad51 or with a control siRNA; cells were irradiated 48 h later and incubated for repair. Rad51 protein was detected in cell lysates by Western blot, with actin as a sample loading control. All error bars show SEM from three independent experiments.</p