Herpesviruses have long been a menace to society. Derived from the Greek word herpein, meaning “to creep,” the first mentions of sores on the lips, a common symptom resulting from herpesviruses, occurred during the rule of Emperor Tiberius (42 BC- 37 AD) as first recorded by Aulus Cornelius Celsus. In an attempt to stop the spread of these sores, Emperor Tiberius banned the act of promiscuous kissing while others like Celsus suggested applying a red hot iron to the sore. While these measures were not the best means of dealing with viral spread, they are indicative of man’s first toiling with the virus. Since then we have greatly expand our understanding of herpesviruses and now recognize a classification of 25 viruses falling within the family of Herpesviridae.
Qualities of this virus family distinguish them as double-stranded linear DNA viruses. Within this class viruses posses a lytic (rapid viral replication, efficient cell destruction) phase along with a dormant, latent phase (low amounts of viral replication) that can transform host cells (immortalize as in cancer). These latently infected cells can also be reactivated into the lytic phase upon stimulation by different cues (e.g. stress) as demonstrated in the image below. The Herpesviridae family is further divided into the following three subfamilies, each described by which type of cell the latent viral phase exists in: alphaherpesviridae (neurons), betaherpesviridae (monocytes, (T) lymphocytes), and gammaherpesviridae (B lymphocytes). From this last subfamily, the viral menace faced by the Romans continues to persist as Epstein-Barr Virus (EBV). Associated with several carcinomas (Burkitt’s and Hodgkin’s lymphoma) and autoimmune disorders (PTLD, post-transplant lymphoproliferative disorder), it is more widely associated with infectious mononucleosis or, the “kissing disease.”
(Rochford et al. 2005.)
A primary goal of EBV, like many of its family members, is to establish a steady, lasting infection in the host system, one that can switch between a lytic and latent viral phase as needed. In order to begin this process, a major hurdle is faced by the virus: entry into the host cell nucleus. Here it will encounter an antiviral nuclear protein complex, the promyelocytic leukemia nuclear body (PML-NBs, figure below). These protein complexes are composed mostly of the proteins PML (a tumor suppressor), Sp100 (a nuclear antigen), Daxx (protein binding partner associated with transcription, apoptosis and chromatin remodeling), and ATRX (involved in chromatin remodeling). PML-NBs as a complex regulate cellular transcription and may act to repress viral transcription. As a consequence, PML-NBs are also a major localization site of many DNA viruses. Knock down of particular components of PML-NBs has also been shown to increase viral infectivity (as in HCMV and HSV).
For herpesviridae, entry into the nucleus means access to replication and transcriptional machinery. In order to face this barrier, the virus utilizes tegument proteins studding the surface of the viral nucleocapsid that encompasses the viral genome. These tegument proteins, pre-existing and packaged in infectious virions released from an infected cell, have been shown to disrupt the PML-NB complex. Human Cytomegalovirus (HCMV), a betaherpesvirus, utilizes its tegument protein, pp71, to displace ATRX and promote degradation of Daxx. Other viruses, like the alphaherpesvirus Herpes Simplex Virus-1 (HSV-1), utilize proteins made upon entry into the nucleus (immediate early gene product, ICP0) to degrade PML instead of those delivered by the tegument. This provides interest as it suggests that viral association with the PML-NB complex takes place during the lytic stages (or reactivation of lytic) of herpesvirus infections, rather than latent. As shown by a study in which EBV genomes associated with PML-NBs during lytic, but not latent, infection, the aforementioned statement is supported. It is interesting then to think Herpesviridae proteins targeting the PML-NBs may be responsible for regulation of early viral replication and transcription. Studies in which HSV-1 and HCMV were deficient for ICP0 or pp71, respectively, showed that viral infection was decreased and viral gene expression was severely inhibited, creating a latent viral state. While this suggests a role for these viral proteins in PML-NB disruption and early viral replication, much less is known about the tools gammaherpesviridae utilize to overcome this cellular block.
In EBV, the major tegument protein, BNRF1, has been shown as necessary for the establishment of latent infection (transformation of B-cells/lymphocytes). While these BNRF1 deficient viruses were able to reactivate from latency and produce a steady normal infection, they had decreased infectivity of B cells and showed little B cell transformation during the establishment of latency.
In the article entitled “EBV Tegument Protein BNRF1 Disrupts Daxx-ATRX to Activate Viral Early Gene Transcription,” Tsai et al (2011) attempted to uncover the function BNRF1 plays in tackling the PML-NB problem. As Daxx and ATRX interaction (components of PML-NBs) was shown to inhibit latent viral reactivation, it is relevant to understand the role BNRF1 plays in disrupting PML-NBs. While the BNRF1 was shown to interact with the PML-NB complex, specifically with Daxx (western blot (B) below on left; V= empty vector, B=FLAG-tagged BNRF1), it was surprising to discover that BNRF1 utilized a unique interaction domain, a sequence unmatched by any other known protein. This Daxx interaction domain (a 240bp region; Figures A&B below on right) was even different from regions homologous to cellular proteins shared by all BNRF1 orthologues, the FGARAT (GAT_1) and AIR synthetase (AIR_S) domains (enzymes involved in steps of purine biosynthesis).
While its interaction with Daxx is interesting, the downstream effects associated with disrupting the chromatin remodeling complex composed of Daxx and ATRX provides greater information. Co-immunoprecipitations showed that when the minimal Daxx interaction domain (DID) was expressed, ATRX did not precipitate with Daxx as it did when other unassociated regions of BNRF1 were expressed (Figure C below). Despite the visible decrease in ATRX association in the co-IP of the DID, the comparison with WT BNRF1 expression shows that ATRX is still somewhat associated with Daxx in the presence of just DID. As suggested by the authors, interaction with Daxx is necessary, but not sufficient for complete disruption of the Daxx-ATRX complex. This is corroborated by data in which FGARAT and AIR synthase homology regions were shown to be necessary for complete disruption of Daxx and ATRX in a Co-IP, perhaps playing a regulatory role for Daxx-BNRF1 interaction
Just as we are able to glean the interaction demonstrated by BNRF1 with the PML-NB, we now have insight into its effect on PML-NB localization as well. Due to its interaction with Daxx, it was determined that BNRF1 co-localized with Daxx to the PML nuclear bodies (nuclear foci)(Figure B below). As a result, ATRX was seen to be dispersed from nuclear bodies when comparing WT/DID BRNF1 and mutants lacking the DID (Figure C below). While the disruption of PML-NB components is shared by other herpesviridae, it was interesting to note that EBV disruption of PML-NBs via BNRF1 did not involve the degradation or modification of PML-NB components (e.g. Daxx and ATRX) as in the case of other herpesviridae (HSV-1, ICP0; HCMV, pp71). As indicated by its nuclear localization to PML-NBs, the authors suggested the DID of BNRF1 may play a role in nuclear localization. While this was somewhat supported by western blots of cellular extracts infected with varying EBV BNRF1 mutants, more work needs to be done to support a more conclusive statement.
Having established a role for BNRF1 on a molecular level, it is relevant to look at the effects of BNRF1 on viral cycle efficacy. As mentioned previously, BNRF1 is relevant for B-cell proliferation, a major step toward establishing latency. In complementation studies, it was shown that WT BNRF1 restored efficiency of primary infection while a mutant variation lacking the DID region did not. Although this established the necessity of BNRF1 for primary infection, it is curious as to why the authors did not utilize a complementation with a BNRF1 mutant containing only the DID region as they used in so many other experiments.
With so little known about BNRF1 (ten published papers on PubMed), the data presented in the paper certainly paved a road for work done with the protein. It will be interesting to see if any further interactions are relevant with other PML-NB proteins. Furthermore, it would be interesting to explore some of the downstream effects of ATRX expulsion from PML nuclear bodies as a result of Daxx interaction. While some details are understood about its relevancy for the EBV viral life cycle, it seems that less is known about the cellular effects. In addition, as mentioned by the authors, information about this DID is an interesting route for antiviral drug exploration, especially if similar routes may be explored with other gammaherpesviridae (e.g. Kaposi-Sarcoma, etc), albeit by differently associated PML-NB domains.
References:
Hunt, Richard (2010) Virology- Chapter Eleven Herpes Viruses. http://pathmicro.med.sc.edu/virol/herpes.htm (27 Dec 2011)
Liebniz Institute for Age Research, Fritz Lipmann Institute (FLI). (2011) http://www.fli-leibniz.de/www_imaging/PML_en.php (27 Dec 2011)
Miller, Sheldon (2007) What is the History of Herpes and How is it Spread? http://ezinearticles.com/?What-is-The-History-of-Herpes-and-How-is-it-Spread?&id=591001 (27 Dec 2011)
Rochford R, Cannon MJ, & Moormann AM (2005). Endemic Burkitt's lymphoma: a polymicrobial disease? Nature reviews. Microbiology, 3 (2), 182-7 PMID: 15685227
Tsai K, Thikmyanova N, Wojcechowskyj JA, Delecluse HJ, & Lieberman PM (2011). EBV Tegument Protein BNRF1 Disrupts DAXX-ATRX to Activate Viral Early Gene Transcription. PLoS pathogens, 7 (11) PMID: 22102817
