A structural basis for BRD2/4-mediated host chromatin interaction and oligomer assembly of Kaposi sarcoma-associated herpesvirus and murine gammaherpesvirus LANA proteins.
A structural basis for BRD2/4-mediated host chromatin interaction and oligomer assembly of Kaposi sarcoma-associated herpesvirus and murine gammaherpesvirus LANA proteins.
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DOI：
10.1371/journal.ppat.1003640
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发表时间：
2013
期刊：
影响因子：
6.7
通讯作者：
Lührs T
中科院分区：
文献类型：
--
作者：
Hellert J;Weidner-Glunde M;Krausze J;Richter U;Adler H;Fedorov R;Pietrek M;Rückert J;Ritter C;Schulz TF;Lührs T
卡波西肉瘤相关疱疹病毒(KSHV)可造成终生潜伏感染，并导致人类多种恶性肿瘤。小鼠疱疹病毒68是一种与γ相关的2型疱疹病毒，常被用作研究γ疱疹病毒体内生物学的模型。KSHV潜伏相关核抗原(KLANA)和MHV68mLANA(Orf73)蛋白是病毒潜伏复制和持续存在所必需的。潜伏的KSHV基因组和KLANA形成核微区，称为LANA斑点，其中还含有细胞染色质蛋白，包括BRD/BET染色质调节剂家族成员BRD2和BRD4。我们解决了KLANA和MHV-68mLANA的C-末端DNA结合域(CTD)的X射线晶体结构。虽然这些结构与Epstein-Barr病毒的EBNA1蛋白具有相同的整体折叠，但它们的表面特征有很大的不同。与脱氧核糖核酸结合位点相反，kLANA和mLANA CTD都含有一个特征的富含赖氨酸的带正电荷的表面斑块，这似乎是γ2疱疹病毒LANA蛋白的一个独特特征。重要的是，kLANA和mLANA CTD二聚体经历了更高阶的齐聚。利用核磁共振波谱，我们确定了BRD2/4的ET结构域在kLANA上的一个特异结合位点。利用多个kLANA突变体进行的功能研究表明，天然kLANA CTD二聚体的寡聚、特有的碱性斑块和kLANA表面的ET结合位点是形成kLANA核斑点和潜在复制所必需的。同样，mLANA上的基础补丁有助于在体内建立MHV-68在脾细胞中的潜伏期。综上所述，我们的数据为高阶LANA低聚体的形成提供了结构基础，这是核斑点形成、潜在复制和病毒持续所必需的。Kaposi肉瘤相关疱疹病毒(KSHV)可引起Kaposi肉瘤、原发性渗出性淋巴瘤和多中心Castleman病的浆细胞变种。它的致癌作用与其在感染者的一生中以潜伏形式存在的能力有关。在潜伏期间，病毒基因组被复制并与感染细胞同步传递给子细胞，而不会形成新的病毒粒子。这一过程中的一个关键病毒蛋白是潜伏期相关核抗原LANA。在潜伏感染的细胞中，病毒基因组和LANA形成了特殊的核微域，称为LANA斑点，其中也包含细胞染色质成分。我们已经解决了KSHVLANA的C末端、DNA结合区(CTD)及其同源物γ2疱疹病毒的同源物mLANA的晶体结构，该病毒经常被用作研究体内潜在持久性的模型。我们还通过核磁共振波谱确定了两个染色质蛋白BRD2/4的结合部位。我们在细胞培养和体内实验中证明了这些结构特征的功能重要性，以及它们对潜伏复制和‘LANA斑点’形成的贡献。我们的结果为组装含有LANA的核结构提供了结构基础，这些结构是潜伏病毒复制和持续所必需的。
Kaposi sarcoma-associated herpesvirus (KSHV) establishes a lifelong latent infection and causes several malignancies in humans. Murine herpesvirus 68 (MHV-68) is a related γ2-herpesvirus frequently used as a model to study the biology of γ-herpesviruses in vivo. The KSHV latency-associated nuclear antigen (kLANA) and the MHV68 mLANA (orf73) protein are required for latent viral replication and persistence. Latent episomal KSHV genomes and kLANA form nuclear microdomains, termed ‘LANA speckles’, which also contain cellular chromatin proteins, including BRD2 and BRD4, members of the BRD/BET family of chromatin modulators. We solved the X-ray crystal structure of the C-terminal DNA binding domains (CTD) of kLANA and MHV-68 mLANA. While these structures share the overall fold with the EBNA1 protein of Epstein-Barr virus, they differ substantially in their surface characteristics. Opposite to the DNA binding site, both kLANA and mLANA CTD contain a characteristic lysine-rich positively charged surface patch, which appears to be a unique feature of γ2-herpesviral LANA proteins. Importantly, kLANA and mLANA CTD dimers undergo higher order oligomerization. Using NMR spectroscopy we identified a specific binding site for the ET domains of BRD2/4 on kLANA. Functional studies employing multiple kLANA mutants indicate that the oligomerization of native kLANA CTD dimers, the characteristic basic patch and the ET binding site on the kLANA surface are required for the formation of kLANA ‘nuclear speckles’ and latent replication. Similarly, the basic patch on mLANA contributes to the establishment of MHV-68 latency in spleen cells in vivo. In summary, our data provide a structural basis for the formation of higher order LANA oligomers, which is required for nuclear speckle formation, latent replication and viral persistence. Kaposi sarcoma-associated herpesvirus (KSHV) causes Kaposi Sarcoma, Primary Effusion lymphoma and the plasma cell variant of Multicentric Castleman's Disease. Its oncogenic effect is linked to its ability to persist in a latent form for the life time of infected individuals. During latency viral genomes are replicated and passed to daughter cells in synchrony with the infected cell without the formation of new virions. A key viral protein in this process is the latency-associated nuclear antigen, LANA. In latently infected cells, viral genomes and LANA form characteristic nuclear microdomains, termed ‘LANA speckles’, which also contain cellular chromatin components. We have solved the crystal structure of the c-terminal, DNA-binding, domain (CTD) of KSHV LANA (kLANA) and its homologue mLANA of a related murine γ2-herpesvirus, which is frequently used as a model to study latent persistence in vivo. We also identified the binding site for two chromatin proteins, BRD2/4, by NMR spectroscopy. We demonstrate the functional importance of these structural features, and their contribution to latent replication and ‘LANA speckle’ formation, in cell culture and in vivo experiments. Our results provide a structural basis for the assembly of LANA-containing nuclear structures that are required for latent viral replication and persistence.
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影响因子：
64.5
作者：
Bochkarev, A;Barwell, JA;Edwards, AM
通讯作者：
Edwards, AM
影响因子：
12.3
作者：
Carpenter AE;Jones TR;Lamprecht MR;Clarke C;Kang IH;Friman O;Guertin DA;Chang JH;Lindquist RA;Moffat J;Golland P;Sabatini DM
通讯作者：
Sabatini DM
DOI：
10.1107/s0907444904019158
发表时间：
2004-12-01
影响因子：
2.2
作者：
Emsley, P;Cowtan, K
通讯作者：
Cowtan, K
影响因子：
4.8
作者：
An, FQ;Compitello, N;Renne, R
通讯作者：
Renne, R
影响因子：
5.4
作者：
CHIANG, CM;DONG, G;CHOW, LT
通讯作者：
CHOW, LT