scholarly journals The 3D structure of Kaposi sarcoma herpesvirus LANA C-terminal domain bound to DNA

2015 ◽  
Vol 112 (21) ◽  
pp. 6694-6699 ◽  
Author(s):  
Jan Hellert ◽  
Magdalena Weidner-Glunde ◽  
Joern Krausze ◽  
Heinrich Lünsdorf ◽  
Christiane Ritter ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Epstein Barr Virus ◽  
Kaposi Sarcoma ◽  
Higher Order ◽  
Host Cells ◽  
Nuclear Antigen ◽  
Arbitrary Sequence ◽  
Homologous Proteins ◽  
Barr Virus ◽  
Epstein Barr

Kaposi sarcoma herpesvirus (KSHV) persists as a latent nuclear episome in dividing host cells. This episome is tethered to host chromatin to ensure proper segregation during mitosis. For duplication of the latent genome, the cellular replication machinery is recruited. Both of these functions rely on the constitutively expressed latency-associated nuclear antigen (LANA) of the virus. Here, we report the crystal structure of the KSHV LANA DNA-binding domain (DBD) in complex with its high-affinity viral target DNA, LANA binding site 1 (LBS1), at 2.9 Å resolution. In contrast to homologous proteins such as Epstein-Barr virus nuclear antigen 1 (EBNA-1) of the related γ-herpesvirus Epstein-Barr virus, specific DNA recognition by LANA is highly asymmetric. In addition to solving the crystal structure, we found that apart from the two known LANA binding sites, LBS1 and LBS2, LANA also binds to a novel site, denoted LBS3. All three sites are located in a region of the KSHV terminal repeat subunit previously recognized as a minimal replicator. Moreover, we show that the LANA DBD can coat DNA of arbitrary sequence by virtue of a characteristic lysine patch, which is absent in EBNA-1 of the Epstein-Barr virus. Likely, these higher-order assemblies involve the self-association of LANA into supermolecular spirals. One such spiral assembly was solved as a crystal structure of 3.7 Å resolution in the absence of DNA. On the basis of our data, we propose a model for the controlled nucleation of higher-order LANA oligomers that might contribute to the characteristic subnuclear KSHV microdomains (“LANA speckles”), a hallmark of KSHV latency.

scholarly journals Structural and Functional Basis for an EBNA1 Hexameric Ring in Epstein-Barr Virus Episome Maintenance

2017 ◽  
Vol 91 (19) ◽  
Author(s):  
Julianna S. Deakyne ◽  
Kimberly A. Malecka ◽  
Troy E. Messick ◽  
Paul M. Lieberman
Keyword(s):  
Crystal Structure ◽  
Dna Binding ◽  
Complex Formation ◽  
Epstein Barr Virus ◽  
Higher Order ◽  
Order Complex ◽  
Barr Virus ◽  
Naturally Occurring ◽  
Epstein Barr

ABSTRACT Epstein-Barr virus (EBV) establishes a stable latent infection that can persist for the life of the host. EBNA1 is required for the replication, maintenance, and segregation of the latent episome, but the structural features of EBNA1 that confer each of these functions are not completely understood. Here, we have solved the X-ray crystal structure of an EBNA1 DNA-binding domain (DBD) and discovered a novel hexameric ring oligomeric form. The oligomeric interface pivoted around residue T585 as a joint that links and stabilizes higher-order EBNA1 complexes. Substitution mutations around the interface destabilized higher-order complex formation and altered the cooperative DNA-binding properties of EBNA1. Mutations had both positive and negative effects on EBNA1-dependent DNA replication and episome maintenance with OriP. We found that one naturally occurring polymorphism in the oligomer interface (T585P) had greater cooperative DNA binding in vitro, minor defects in DNA replication, and pronounced defects in episome maintenance. The T585P mutant was compromised for binding to OriP in vivo as well as for assembling the origin recognition complex subunit 2 (ORC2) and trimethylated histone 3 lysine 4 (H3K4me3) at OriP. The T585P mutant was also compromised for forming stable subnuclear foci in living cells. These findings reveal a novel oligomeric structure of EBNA1 with an interface subject to naturally occurring polymorphisms that modulate EBNA1 functional properties. We propose that EBNA1 dimers can assemble into higher-order oligomeric structures important for diverse functions of EBNA1. IMPORTANCE Epstein-Barr virus is a human gammaherpesvirus that is causally associated with various cancers. Carcinogenic properties are linked to the ability of the virus to persist in the latent form for the lifetime of the host. EBNA1 is a sequence-specific DNA-binding protein that is consistently expressed in EBV tumors and is the only viral protein required to maintain the viral episome during latency. The structural and biochemical mechanisms by which EBNA1 allows the long-term persistence of the EBV genome are currently unclear. Here, we have solved the crystal structure of an EBNA1 hexameric ring and characterized key residues in the interface required for higher-order complex formation and long-term plasmid maintenance.

scholarly journals Transcriptional Regulatory Properties of Epstein-Barr Virus Nuclear Antigen 3C Are Conserved in Simian Lymphocryptoviruses

2003 ◽  
Vol 77 (10) ◽  
pp. 5639-5648 ◽  
Author(s):  
Bo Zhao ◽  
Rozenn Dalbiès-Tran ◽  
Hua Jiang ◽  
Ingrid K. Ruf ◽  
Jeffery T. Sample ◽  
...  
Keyword(s):  
Nonhuman Primate ◽  
Transcriptional Activation ◽  
Epstein Barr Virus ◽  
Leucine Zipper ◽  
Nuclear Antigen ◽  
Homologous Proteins ◽  
Basic Leucine Zipper ◽  
Barr Virus ◽  
C Terminus ◽  
Epstein Barr

ABSTRACT Epstein-Barr virus (EBV) nuclear antigen 3C (EBNA-3C) is a large transcriptional regulator essential for EBV-mediated immortalization of B lymphocytes. We previously identified interactions between EBNA-3C and two cellular transcription factors, Jκ and Spi proteins, through which EBNA-3C regulates transcription. To better understand the contribution of these interactions to EBNA-3C function and EBV latency, we examined whether they are conserved in the homologous proteins of nonhuman primate lymphocryptoviruses (LCVs), which bear a strong genetic and biological similarity to EBV. The homologue of EBNA-3C encoded by the LCV that infects baboons (BaLCV) was found to be only 35% identical in sequence to its EBV counterpart. Of particular significance, this homology localized predominantly to the N-terminal half of the molecule, which encompasses the domains in EBNA-3C that interact with Jκ and Spi proteins. Like EBNA-3C, both BaLCV and rhesus macaque LCV (RhLCV) 3C proteins bound to Jκ and repressed transcription mediated by EBNA-2 through its interaction with Jκ. Both nonhuman primate 3C proteins were also able to activate transcription mediated by the Spi proteins in the presence of EBNA-2. Like EBNA-3C, a domain encompassing the putative basic leucine zipper motif of the BaLCV-3C protein directly interacted with both Spi-1 and Spi-B. Surprisingly, a recently identified motif in EBNA-3C that mediates repression was not identifiable in the BaLCV-3C protein. Finally, although the C terminus of BaLCV-3C bears minimal homology to EBNA-3C, it nonetheless contains a C-terminal domain rich in glutamine and proline that was able to function as a potent transcriptional activation domain, as does the C terminus of EBNA-3C. The conservation of these functional motifs despite poor overall homology among the LCV 3C proteins strongly suggests that the interactions of EBNA-3C with Jκ and Spi do indeed play significant roles in the life cycle of EBV.

scholarly journals Structural Basis for Cooperative Binding of EBNA1 to the Epstein-Barr Virus Dyad Symmetry Minimal Origin of Replication

2019 ◽  
Vol 93 (20) ◽  
Author(s):  
Kimberly A. Malecka ◽  
Jayaraju Dheekollu ◽  
Julianna S. Deakyne ◽  
Andreas Wiedmer ◽  
Ursula D. Ramirez ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Epstein Barr Virus ◽  
Latent Infection ◽  
Nuclear Antigen ◽  
Plasmid Replication ◽  
Origin Of Replication ◽  
Replication Machinery ◽  
Barr Virus ◽  
Epstein Barr ◽  
Dyad Symmetry

ABSTRACTEpstein-Barr virus is associated with several human malignancies, including nasopharyngeal carcinoma, gastric cancer, and lymphoma. Latently infected cells carry a circularized EBV episome where the origin of replication (oriP) is comprised of two elements: the family of repeats (FR) and dyad symmetry (DS). The viral protein Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA1) binds to FR and DS to promote EBV episome maintenance and DNA replication during latent infection in proliferating cells. EBNA1 binding to the DS constitutes a minimal origin of DNA replication. Here we report the crystal structure of two EBNA1 DNA-binding domain dimers bound to a DS half-site. This structure shows that the DNA is smoothly bent, allowing for stabilizing interactions between the dimers. The dimer-dimer interface requires an intricate hydrogen bonding network involving residues R491 and D581. When this interface is disrupted, we note loss of stable dimer-dimer complex formation on the DNA, compromisedoriP-containing plasmid replication in cells, and impaired recruitment of the MCM3 complex to theoriP. Surface conservation analysis reveals that these residues are part of a larger conserved surface that may be critical for recruitment of replication machinery to theoriP. Our results reveal a new region of EBNA1 critical for its activity and one that may be exploited by targeted small molecules to treat EBV-associated disease.IMPORTANCEEpstein-Barr virus (EBV) is a causative agent of various malignancies and may also contribute to autoimmune disease. The latent and episomal form of the virus is known to drive EBV-associated oncogenesis. Persistence of the viral episome in proliferating tumor cells requires the interaction of Epstein-Barr virus nuclear antigen 1 (EBNA1) with the viral origin of plasmid replication (oriP). The dyad symmetry (DS) element inoriPis the essential minimal replicator oforiP. Here we report the X-ray crystal structure of EBNA1 bound to DS. The structure reveals a previous unrecognized interface formed between dimers of EBNA1 necessary for cooperative DNA binding, recruitment of cellular replication machinery, and replication function. These findings provide new insights into the mechanism of EBNA1 function at the replication origin and new opportunities to inhibit EBV latent infection and pathogenesis.

scholarly journals Epstein–Barr virus enhances genome maintenance of Kaposi sarcoma-associated herpesvirus

2018 ◽  
Vol 115 (48) ◽  
pp. E11379-E11387 ◽  
Author(s):  
Rachele Bigi ◽  
Justin T. Landis ◽  
Hyowon An ◽  
Carolina Caro-Vegas ◽  
Nancy Raab-Traub ◽  
...  
Keyword(s):  
Epstein Barr Virus ◽  
Primary Effusion Lymphoma ◽  
Kaposi Sarcoma ◽  
B Cell Lymphoma ◽  
Nuclear Antigen ◽  
Genome Maintenance ◽  
Barr Virus ◽  
Single Positive ◽  
Infected Cells ◽  
Epstein Barr

Primary effusion lymphoma (PEL) is a B cell lymphoma that is always associated with Kaposi’s sarcoma-associated herpesvirus (KSHV) and in many cases also with Epstein–Barr virus (EBV); however, the requirement for EBV coinfection is not clear. Here, we demonstrate that adding exogenous EBV to KSHV+single-positive PEL leads to increased KSHV genome maintenance and KSHV latency-associated nuclear antigen (LANA) expression. To show that EBV was necessary for naturally coinfected PEL, we nucleofected KSHV+/EBV+PEL cell lines with an EBV-specific CRISPR/Cas9 plasmid to delete EBV and observed a dramatic decrease in cell viability, KSHV genome copy number, and LANA expression. This phenotype was reversed by expressing Epstein–Barr nuclear antigen 1 (EBNA-1)in trans, even though EBNA-1 and LANA do not colocalize in infected cells. This work reveals that EBV EBNA-1 plays an essential role in the pathogenesis of PEL by increasing KSHV viral load and LANA expression.

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