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Crystal Structure of the Gamma-2 Herpesvirus LANA DNA Binding Domain Identifies Charged Surface Residues Which Impact Viral Latency

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Crystal Structure of the Gamma-2 Herpesvirus LANA DNA Binding Domain Identifies Charged Surface Residues Which Impact Viral Latency Crystal Structure of the Gamma-2 Herpesvirus LANA DNA Binding Domain Identifies Charged Surface Residues Which Impact Viral Latency The Harvard community has made this article openly available. Please share how this access benefits you. Your story matters Citation Correia, B., S. A. Cerqueira, C. Beauchemin, M. Pires de Miranda, S. Li, R. Ponnusamy, L. Rodrigues, et al. 2013. “Crystal Structure of the Gamma-2 Herpesvirus LANA DNA Binding Domain Identifies Charged Surface Residues Which Impact Viral Latency.” PLoS Pathogens 9 (10): e1003673. doi:10.1371/journal.ppat.1003673. http://dx.doi.org/10.1371/journal.ppat.1003673. Published Version doi:10.1371/journal.ppat.1003673 Citable link http://nrs.harvard.edu/urn-3:HUL.InstRepos:11878899 Terms of Use This article was downloaded from Harvard University’s DASH repository, and is made available under the terms and conditions applicable to Other Posted Material, as set forth at http:// nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of- use#LAA Crystal Structure of the Gamma-2 Herpesvirus LANA DNA Binding Domain Identifies Charged Surface Residues Which Impact Viral Latency Bruno Correia1., Sofia A. Cerqueira2., Chantal Beauchemin3, Marta Pires de Miranda2, Shijun Li3, Rajesh Ponnusamy1,Le´nia Rodrigues2, Thomas R. Schneider4, Maria A. Carrondo1*, Kenneth M. Kaye3*, J. Pedro Simas2*, Colin E. McVey1* 1 Instituto de Tecnologia Quı´mica e Biolo´gica, Universidade Nova de Lisboa, Oeiras, Portugal, 2 Instituto de Microbiologia e Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Lisboa, Portugal, 3 Departments of Medicine, Brigham and Women’s Hospital and Harvard Medical School, Boston, Massachusetts, United States of America, 4 EMBL c/o DESY, Hamburg, Germany Abstract Latency-associated nuclear antigen (LANA) mediates c2-herpesvirus genome persistence and regulates transcription. We describe the crystal structure of the murine gammaherpesvirus-68 LANA C-terminal domain at 2.2 A˚ resolution. The structure reveals an alpha-beta fold that assembles as a dimer, reminiscent of Epstein-Barr virus EBNA1. A predicted DNA binding surface is present and opposite this interface is a positive electrostatic patch. Targeted DNA recognition substitutions eliminated DNA binding, while certain charged patch mutations reduced bromodomain protein, BRD4, binding. Virus containing LANA abolished for DNA binding was incapable of viable latent infection in mice. Virus with mutations at the charged patch periphery exhibited substantial deficiency in expansion of latent infection, while central region substitutions had little effect. This deficiency was independent of BRD4. These results elucidate the LANA DNA binding domain structure and reveal a unique charged region that exerts a critical role in viral latent infection, likely acting through a host cell protein(s). Citation: Correia B, Cerqueira SA, Beauchemin C, Pires de Miranda M, Li S, et al. (2013) Crystal Structure of the Gamma-2 Herpesvirus LANA DNA Binding Domain Identifies Charged Surface Residues Which Impact Viral Latency. PLoS Pathog 9(10): e1003673. doi:10.1371/journal.ppat.1003673 Editor: Shou-Jiang Gao, University of Southern California Keck School of Medicine, United States of America Received April 26, 2013; Accepted August 16, 2013; Published October 17, 2013 Copyright: ß 2013 Correia et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The work was supported by grants from the Fundac¸a˜o para a Cieˆncia e a Tecnologia (FCT) Harvard Medical School Portugal Program in Translational Research and Information (JPS, CEM, MAC and KMK) HMSP-ICT/0021/2010. This work was also supported by grants from the National Cancer Institute (CA082036) (KMK) and the U.S. Department of Defense (PR093491) (KMK). CEM is a holder of a Cieˆncia 2008 position from the Portuguese Ministry of Science. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: [email protected] (MAC); [email protected] (KMK); [email protected] (JPS); [email protected] (CEM) . These authors contributed equally to this work. Introduction episomes (plasmids). Only a small subset of viral genes is expressed during latency. In the case of gamma-herpesvirus sub-family Herpesviruses are ubiquitous viruses, which infect many members, latent infection is predominantly established in B mammalian species and are a leading cause of human viral lymphocytes. Intransal infection of mice with MHV-68 causes disease. There are two known human viruses from the gamma an acute self-limiting respiratory infection, followed by the herpesvirus sub-family. The Epstein-Barr virus (EBV), a gamma-1 establishment of splenic latency, which peaks 14 days after herpesvirus (Lymphocryptovirus), and the Kaposi’s sarcoma- infection [4,5]. Splenic germinal centre (GC) B cells have a key associated herpesvirus (KSHV), a gamma-2 herpesvirus (Rhadi- role both in the establishment and maintenance of viral latency novirus). KSHV has an etiologic role in Kaposi’s Sarcoma, the [6]. Like EBV [7], MHV-68 establishes latency predominantly in leading AIDS malignancy, as well as primary effusion lymphoma activated germinal centre B cells. This strategy favours physiolog- and multicentric Castleman’s disease. EBV is also associated with ical access of the virus to the memory B-cell pool, the major human cancers. These human viruses do not readily infect small reservoir of long-term latency. laboratory animals. The murine gammaherpesvirus 68 (MHV-68 KSHV latency-associated nuclear antigen (kLANA), a 1,162 or murid herpesvirus 4), a rhadinovirus that was isolated from amino acid protein encoded by ORF73 of the viral genome, is the naturally infected rodents, is structurally and functionally related predominant gene expressed in latent infection. LANA mediates to human gamma-herpesviruses and readily infects mice, thus persistence of episomal DNA and thus is required for long-term providing a mouse model for the investigation of gammaherpes- maintenance of viral genomes in dividing cells, a role central to virus pathogenesis [1–3]. viral latency. LANA promotes episomal replication and segrega- A key feature of herpesvirus infection is their lifelong persistence tion to progeny nuclei after mitosis. The C-terminal region of in the host in the form of latency. During latent infection, viral kLANA harbours a DNA-binding domain (DBD) that self- genomes persist as multi-copy, circularized, extrachromosomal associates and binds to terminal repeat (TR) DNA sequences of PLOS Pathogens | www.plospathogens.org 1 October 2013 | Volume 9 | Issue 10 | e1003673 Crystal Structure of mLANA DBD Author Summary B (NF-kB) [31]. The mechanism involves the assembly of an Elongin C/Cullin5/SOCS (suppressors of cytokine signalling)-like Herpesviruses establish life-long latent infections. During complex, mediated by an unconventional viral SOCS-box motif latency, gammaherpesviruses, such as Kaposi’s sarcoma- (residues 199–206), homologous to the kLANA Cul5 box, present associated herpesvirus (KSHV), persist as multicopy, circu- in the C-terminal domain of mLANA [30,31]. Another motif also larized genomes in the cell nucleus and express a small within the C-terminal region of mLANA (QAKKLK motif, subset of viral genes. KSHV latency-associated nuclear residues 226–231) has been shown to bind to several members of antigen (LANA) is the predominant gene expressed during the BET (Bromodomain and Extra Terminal domain) family of latent infection. C-terminal LANA binds KSHV terminal proteins, including BRD2, BRD3 and BRD4 [33,34], that interact repeat (TR) DNA to mediate DNA replication. TR DNA with acetylated histones. mLANA interaction with BET proteins binding also allows tethering of the viral genome to leads to activation of cell cycle promoters. mitotic chromosomes to mediate DNA segregation to Secondary structure predictions have suggested that the C- daughter nuclei. We describe here the crystal structure of the murine gammaherpesvirus 68 LANA DNA binding terminal regions of both mLANA and kLANA have similarity to domain, which is homologous to that of KSHV LANA. The the X-ray structure of the DNA-binding domain of the EBV structure revealed a dimer and we identified residues nuclear antigen 1 protein (EBNA1) [35]. As a first step towards involved in the interaction with viral DNA. Mutation of understanding the mechanism by which the C-terminal region of these residues abolished DNA binding and viable latency mLANA exerts its functions, we have determined the crystal establishment in a mouse model of infection. We also structure of this mLANA domain (amino acids 140–272). identified a positively charged patch on the dimer surface mLANA140–272 forms a b-barrel induced dimer and has an overall opposite to the DNA binding region and found this patch fold with an a-helix arrangement similar to that of the EBNA1 and exerts an important role in the virus’s ability to expand E2 latency protein structures [36,37]. However, the structure latent infection in vivo. This work elucidates the structure reveals unique features that exert important effects on viral of the LANA DNA binding domain and identifies a novel persistence in an animal model. surface feature that is critical for viral latent infection, likely by acting through a host cell protein. Results the viral episome [8–14]. LANA cooperatively binds to two sites X-ray crystal structure of C-terminal mLANA reveals an within the TR DNA, LBS1 and LBS2. LBS1 is a high affinity site alpha+beta fold that assembles to form a dimer capable of facilitating the cooperative binding of LANA to LBS2. The structure of the C-terminal domain of mLANA was Both sites contribute to the ability of LANA to suppress determined at 2.2 A˚ resolution. Data collection and refinement transcription and to facilitate DNA replication [10]. An N- statistics are reported in Table 1.
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