Identifying the Cellular Interactome of Epstein-Barr Virus Lytic Regulator Zta Reveals Cellular Targets Contributing to Viral Replication

Abstract

The human gammaherpesvirus Epstein-Barr virus (EBV) (human herpesvirus 4 [HHV4]) infects most adults and is an important contributor to the development of many types of lymphoid and epithelial cancers. Essential contributions of viral genes to viral replication are known, but the potential contributions of cell genes are less well delineated. A key player is the viral protein Zta (BZLF1, ZEBRA, or Z). This sequence-specific DNA-binding protein can disrupt EBV latency by driving the transcription of target genes and by interacting with the EBV lytic origin of replication. Here, we used an unbiased proteomics approach to identify the Zta-interactome in cells derived from Burkitt’s lymphoma. Isolating Zta and associated proteins from Burkitt’s lymphoma cells undergoing EBV replication, followed by tandem mass tag (TMT) mass spectrometry, resulted in the identification of 39 viral and cellular proteins within the Zta interactome. An association of Zta with the cellular protein NFATc2 was validated in independent experiments. Furthermore, the ability of Zta to attenuate the activity of an NFAT-dependent promoter was shown, which suggests a functional consequence for the association. The expression of Zta is itself regulated through NFAT activity, suggesting that Zta may contribute to a feedback loop that would limit its own expression, thus aiding viral replication by preventing the known toxic effects of Zta overexpression. IMPORTANCE Epstein-Barr virus infects most people across the world and causes several kinds of cancer. Zta is an important viral protein that makes the virus replicate by binding to its DNA and turning on the expression of some genes. We used a sensitive, unbiased approach to isolate and identify viral and cellular proteins that physically interact with Zta. This revealed 39 viral and cellular proteins. We found that one protein, termed NFATc2, was already known to be important for a very early step in viral replication. We identify that once this step has occurred, Zta reduces the effectiveness of NFATc2, and we suggest that this is important to prevent cells from dying before viral replication is complete and the mature virus is released from the cells.

Oncogenic Properties of the EBV ZEBRA Protein

“…ZEBRA directly binds B cell specific transcription factors Pax5 and Oct2 via bZIP domain; this inhibits ZEBRA activity, however, the reciprocal inhibition was proven only for Pax5 [72,73]. Unidirectional inactivation of the family of nuclear factor of activated T cells (NFAT) transcription factors, involved in calcium signal transduction, by direct interaction with ZEBRA was also reported [74]. Presumably, the same mechanism related to ZEBRA inhibitory binding to host transcriptional factor is involved in the class II transactivator (CIITA) repression, however, it involves the TAD and a transcriptional factor inhibited by ZEBRA was not identified [67].…”

“…In summary, ZEBRA binds specific DNA motifs and/or interacts with other proteins, either recruiting them to DNA binding sites or altering their activity. However, ZEBRA direct interactions with many other cellular proteins [74] are much less studied as compared to interaction with chromatin-binding proteins.…”

“…ZEBRA interaction with other proteins: the cellular interactome of ZEBRA needs further investigation to explain the functional significance of ZEBRA interaction network [74]. ZEBRA is extensively modified in vivo [52], however, the enzymes (viral and cellular) and signaling pathways involved in its post-translational modifications are largely unknown, as well as the effect of these modifications on ZEBRA activity.…”

TMT-based quantitative proteomics analysis reveals the attenuated replication mechanism of Newcastle disease virus caused by nuclear localization signal mutation in viral matrix protein

“…In recent years, tandem mass tag (TMT) combined with liquid chromatography tandem-mass spectrometry (LC-MS/MS) analysis has become a powerful tool in the identification, characterization, and quantitation analysis of the proteomic profiles [24,25]. This approach has been successfully applied to many virus-host interaction studies, such as in human immunodeficiency virus (HIV) [26,27], Bombyx mori nucleopolyhedrovirus [28], canine parvovirus [29], HIV/TM co-infected patients [30], and Epstein-Barr virus [31]. These studies have revealed the dynamic interactions between virus and host, and provided a better understanding of the pathogenesis involved in viral infection and replication.…”

Chlamydia trachomatis suppresses host cell store-operated Ca2+ entry and inhibits NFAT/calcineurin signaling

“…African swine fever virus inhibits NFAT-regulated transcription of immunomodulatory proteins by synthesis of a protein, A238L, that directly binds to and inhibits the calcineurin phosphatase activity required for NFAT activation [44]. Reactivation of latent Epstein-Barr virus to a lytic state via a Ca 2+ /calcineurin dependent activation of NFAT by a complex mechanism has been proposed to represent a negative feedback loop involving a viral protein, 14 Zta, that directly binds to and attenuates NFAT [45]. Helicobacter pylori is associated with peptic ulcers and gastric adenocarcinoma.…”