Alison J. Sinclair

Induction of p16INK4a Is the Major Barrier to Proliferation when Epstein-Barr Virus (EBV) Transforms Primary B Cells into Lymphoblastoid Cell Lines

To explore the role of p16INK4a as an intrinsic barrier to B cell transformation by EBV, we transformed primary B cells from an individual homozygous for a deletion in the CDKN2A locus encoding p16INK4a and p14ARF. Using recombinant EBV-BAC viruses expressing conditional EBNA3C (3CHT), we developed a system that allows inactivation of EBNA3C in lymphoblastoid cell lines (LCLs) lacking active p16INK4a protein but expressing a functional 14ARF-fusion protein (p14/p16). The INK4a locus is epigenetically repressed by EBNA3C – in cooperation with EBNA3A – despite the absence of functional p16INK4a. Although inactivation of EBNA3C in LCLs from normal B cells leads to an increase in p16INK4a and growth arrest, EBNA3C inactivation in the p16INK4a-null LCLs has no impact on the rate of proliferation, establishing that the repression of INK4a is a major function of EBNA3C in EBV-driven LCL proliferation. This conditional LCL system allowed us to use microarray analysis to identify and confirm genes regulated specifically by EBNA3C, independently of proliferation changes modulated by the p16INK4a-Rb-E2F axis. Infections of normal primary B cells with recombinant EBV-BAC virus from which EBNA3C is deleted or with 3CHT EBV in the absence of activating ligand 4-hydroxytamoxifen, revealed that EBNA3C is necessary to overcome an EBV-driven increase in p16INK4a expression and concomitant block to proliferation 2–4 weeks post-infection. If cells are p16INK4a-null, functional EBNA3C is dispensable for the outgrowth of LCLs.

Bioimpedance Analysis for the Characterization of Breast Cancer Cells in Suspension

The bioimpedance spectroscopy (BIS) technique is potentially a useful tool to differentiate malignancy based on the variation of electrical properties presented by different tissues and cells. The different tissues and cells present variant electrical resistance and reactance when excited at different frequencies. The main purpose of this area of research is to use impedance measurements over a low-frequency bandwidth ranging from 1 kHz to 3 MHz to 1) differentiate the pathological stages of cancer cells under laboratory conditions and 2) permit the extraction of electrical parameters related to cellular information for further analysis. This provides evidence to form the basis of bioimpedance measurement at the cellular level and aids the potential future development of rapid diagnostics from biopsy materials. Three cell lines, representing normal breast epithelia and different pathological stages of breast cancer, have been measured using a standard impedance analyzer driving a four-electrode chamber filled with different cell suspensions. We identify the specific BIS profile for each cell type and determine whether these can be differentiated. In addition, the electrical parameters, e.g., the intracellular conductivity, membrane capacitance/capacity, characteristic frequency, are extracted by the use of equivalent circuit models and physical models to provide details of the cell electric signatures for further analysis of cancer cell pathology.

The efficient and rapid import of a peptide into primary B and T lymphocytes and a lymphoblastoid cell line

Lymphocytes are notoriously difficult to transfect. The favoured technique, electroporation, has three major disadvantages: it is highly disruptive, causing large scale cell death; it is inefficient; quiescent primary lymphocytes are refractory to electroporation unless they have been partially activated. We have investigated the cellular import of the third helix of the Antennapedia homeodomain protein (pAntp) as an alternative method for introducing peptides into primary lymphocytes and lymphoid cell lines. The pAntp peptide is taken up rapidly into the cytoplasm and nucleus of the cells where it is retained for at least 48 h. The system displays none of the disadvantages of electroporation; no toxicity of the pAntp peptide was detected at the concentrations tested and the process was efficient with up to 95% of lymphocytes importing the pAntp peptide. Finally, quiescent primary lymphocytes were as efficient as activated primary lymphocytes and a lymphoid cell line at importing the pAntp peptide. This demonstrates that the pAntp peptide delivery system has major advantages over electroporation as a method of delivering molecules to lymphocytes and a lymphoid cell line.

Methylated DNA recognition during the reversal of epigenetic silencing is regulated by cysteine and serine residues in the Epstein-Barr virus lytic switch protein.

Epstein-Barr virus (EBV) causes infectious mononucleosis and is associated with various malignancies, including Burkitts lymphoma and nasopharyngeal carcinoma. Like all herpesviruses, the EBV life cycle alternates between latency and lytic replication. During latency, the viral genome is largely silenced by host-driven methylation of CpG motifs and, in the switch to the lytic cycle, this epigenetic silencing is overturned. A key event is the activation of the viral BRLF1 gene by the immediate-early protein Zta. Zta is a bZIP transcription factor that preferentially binds to specific response elements (ZREs) in the BRLF1 promoter (Rp) when these elements are methylated. Ztas ability to trigger lytic cycle activation is severely compromised when a cysteine residue in its bZIP domain is mutated to serine (C189S), but the molecular basis for this effect is unknown. Here we show that the C189S mutant is defective for activating Rp in a Burkitts lymphoma cell line. The mutant is compromised both in vitro and in vivo for binding two methylated ZREs in Rp (ZRE2 and ZRE3), although the effect is striking only for ZRE3. Molecular modeling of Zta bound to methylated ZRE3, together with biochemical data, indicate that C189 directly contacts one of the two methyl cytosines within a specific CpG motif. The motifs second methyl cytosine (on the complementary DNA strand) is predicted to contact S186, a residue known to regulate methyl-ZRE recognition. Our results suggest that C189 regulates the enhanced interaction of Zta with methylated DNA in overturning the epigenetic control of viral latency. As C189 is conserved in many bZIP proteins, the selectivity of Zta for methylated DNA may be a paradigm for a more general phenomenon.

Dynamic Chromatin Environment of Key Lytic Cycle Regulatory Regions of the Epstein-Barr Virus Genome

ABSTRACT The ability of Epstein-Barr virus (EBV) to establish latency allows it to evade the immune system and to persist for the lifetime of its host; one distinguishing characteristic is the lack of transcription of the majority of viral genes. Entry into the lytic cycle is coordinated by the viral transcription factor, Zta (BZLF1, ZEBRA, and EB1), and downstream effectors, while viral genome replication requires the concerted action of Zta and six other viral proteins at the origins of lytic replication. We explored the chromatin context at key EBV lytic cycle promoters (BZLF1, BRLF1, BMRF1, and BALF5) and the origins of lytic replication during latency and lytic replication. We show that a repressive heterochromatin-like environment (trimethylation of histone H3 at lysine 9 [H3K9me3] and lysine 27 [H3K27me3]), which blocks the interaction of some transcription factors with DNA, encompasses the key early lytic regulatory regions. Epigenetic silencing of the EBV genome is also imposed by DNA methylation during latency. The chromatin environment changes during the lytic cycle with activation of histones H3, H4, and H2AX occurring at both the origins of replication and at the key lytic regulatory elements. We propose that Zta is able to reverse the effects of latency-associated repressive chromatin at EBV early lytic promoters by interacting with Zta response elements within the H3K9me3-associated chromatin and demonstrate that these interactions occur in vivo. Since the interaction of Zta with DNA is not inhibited by DNA methylation, it is clear that Zta uses two routes to overcome epigenetic silencing of its genome.

Epigenetic Control of Viral Life-Cycle by a DNA-Methylation Dependent Transcription Factor

Epstein-Barr virus (EBV) encoded transcription factor Zta (BZLF1, ZEBRA, EB1) is the prototype of a class of transcription factor (including C/EBPalpha) that interact with CpG-containing DNA response elements in a methylation-dependent manner. The EBV genome undergoes a biphasic methylation cycle; it is extensively methylated during viral latency but is reset to an unmethylated state following viral lytic replication. Zta is expressed transiently following infection and again during the switch between latency and lytic replication. The requirement for CpG-methylation at critical Zta response elements (ZREs) has been proposed to regulate EBV replication, specifically it could aid the activation of viral lytic gene expression from silenced promoters on the methylated genome during latency in addition to preventing full lytic reactivation from the non-methylated EBV genome immediately following infection. We developed a computational approach to predict the location of ZREs which we experimentally assessed using in vitro and in vivo DNA association assays. A remarkably different binding motif is apparent for the CpG and non-CpG ZREs. Computational prediction of the location of these binding motifs in EBV revealed that the majority of lytic cycle genes have at least one and many have multiple copies of methylation-dependent CpG ZREs within their promoters. This suggests that the abundance of Zta protein coupled with the methylation status of the EBV genome act together to co-ordinate the expression of lytic cycle genes at the majority of EBV promoters.

The Epstein-Barr virus lytic cycle activator Zta interacts with methylated ZRE in the promoter of host target gene egr1.

Activation of the host gene egr1 is essential for the lytic replication of Epstein–Barr virus (EBV). egr1 is activated by Zta (BZLF1, ZEBRA). Zta interacts directly with DNA through a series of closely related Zta-response elements (ZREs). Here we dissect the mechanism used by Zta to interact with the egr1 promoter and identify a weak interaction with egr1ZRE that is dependent on the distal part of egr1ZRE. Furthermore, we demonstrate that the ability of Zta to interact with egr1ZRE is enhanced at least tenfold by methylation. The ability of Zta to transactivate a reporter construct driven by the egr1 promoter can be enhanced by methylation. As the ability of Zta to interact with a methylated ZRE in the EBV genome correlates with its ability to activate the expression of the endogenous viral gene BRLF1, this suggests that Zta may also have the capability to overturn epigenetic control of egr1.

Functional Interaction between Epstein-Barr Virus Replication Protein Zta and Host DNA Damage Response Protein 53BP1

ABSTRACT Epstein-Barr virus (EBV; human herpesvirus 4) poses major clinical problems worldwide. Following primary infection, EBV enters a form of long-lived latency in B lymphocytes, expressing few viral genes, and it persists for the lifetime of the host with sporadic bursts of viral replication. The switch between latency and replication is governed by the action of a multifunctional viral protein Zta (also called BZLF1, ZEBRA, and Z). Using a global proteomic approach, we identified a host DNA damage repair protein that specifically interacts with Zta: 53BP1. 53BP1 is intimately connected with the ATM signal transduction pathway, which is activated during EBV replication. The interaction of 53BP1 with Zta requires the C-terminal ends of both proteins. A series of Zta mutants that show a wild-type ability to perform basic functions of Zta, such as dimer formation, interaction with DNA, and the transactivation of viral genes, were shown to have lost the ability to induce the viral lytic cycle. Each of these mutants also is compromised in the C-terminal region for interaction with 53BP1. In addition, the knockdown of 53BP1 expression reduced viral replication, suggesting that the association between Zta and 53BP1 is involved in the viral replication cycle.

Mutation of a Single Amino Acid Residue in the Basic Region of the Epstein-Barr Virus (EBV) Lytic Cycle Switch Protein Zta (BZLF1) Prevents Reactivation of EBV from Latency

ABSTRACT Zta, the product of the BZLF1 gene carried by Epstein-Barr virus (EBV), is crucial for reactivation of EBV from latency. Zta is a member of the bZIP family of transcription factors, and in common with many of these, Zta possesses a conserved cysteine residue in its basic region (C189) and a further cysteine residue in its ZIP region (C222). We demonstrate that C189 is required to reactivate EBV from latency but C222 is not and that this single amino acid affects two independent functions of Zta, (i) binding to a Zta-responsive site and (ii) manipulating the cell cycle.

Exploitation of a non-apoptotic caspase to regulate the abundance of the cdkI p27(KIP1) in transformed lymphoid cells.

Expression of the cyclin dependent kinase inhibitor p27KIP1 is intimately linked to the control of proliferation, and is itself regulated by transcription, translation, phosphorylation, protein stability or sequestration. p27KIP1 is also regulated during apoptosis; cleavage occurs at DPSD139S and ESQD108V, by a sub-set of Z-VAD-fmk-sensitive caspases. We have identified a related but distinct mechanism that regulates p27KIP1 in proliferating lymphoid cell lines. In a B-lymphoid cell line (BJAB), the abundance of p27KIP1 oscillates inversely to proliferation; loss of full-length p27KIP1 correlates with the appearance of a truncated version corresponding to cleavage at DPSD139S. A direct correlation exists between the appearance of truncated p27KIP1 and the presence of an activity able to cleave peptides representing DPSD139S and a caspase-8 substrate (Ac-IETD-AMC) in vitro. This activity is inhibited by Ac-IETD-CHO but not Z-VAD-fmk in vitro. Furthermore a requirement for caspase-8 has been excluded. The activity differs from the apoptosis related p27KIP1-cleaving activity; indeed few cells undergoing apoptosis are present in the population of proliferating cells. The activity is further distinguished by its inability to cleave a peptide based on ESQD108V in vitro, together with the lack of a corresponding cleavage product in vivo. Inhibition of the caspase activity in vivo promotes an accumulation of full length p27KIP1, as well as a decrease in cell proliferation. Together these studies highlight the importance of non-apoptotic caspases in regulating p27KIP1 in transformed lymphoid cells.