Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Lisbon, Portugal.
Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Oeiras, Portugal.
J Virol. 2018 Oct 12;92(21). doi: 10.1128/JVI.01251-18. Print 2018 Nov 1.
The latency-associated nuclear antigen from Kaposi's sarcoma-associated herpesvirus (KSHV), kLANA, and its homolog from the murid herpesvirus 4 (MuHV-4), mLANA, are essential for viral latency. kLANA is nearly four times the size of mLANA, mainly due to an extensive central repeat region that is absent in mLANA. Both proteins harbor a C-terminal DNA binding domain (DBD). The DBD binds the terminal repeat (TR) DNA sequences of the viral genome to mediate persistence. Despite structural conservation, the kLANA and mLANA DBDs differ in sequence and mode of oligomerization. kLANA DBD oligomers are flexible and bent, while mLANA DBD oligomers bind DNA in a rigid, linear conformation. We previously reported that kLANA and mLANA acted reciprocally on TR sequences. Furthermore, a MuHV-4 expressing kLANA instead of mLANA (v-kLANA) established latency in mice, albeit at a lower magnitude than the wild-type (WT) virus. Here, we asked if kLANA can accommodate the mLANA DBD and generated a fusion protein which contains kLANA but with the mLANA C-terminal region in place of that of kLANA. We report a recombinant MuHV-4 (v-KM) encoding this LANA fusion protein instead of mLANA. The fusion protein was expressed in lytic infection and assembled nuclear LANA dots in infected splenocytes. Results demonstrated that kLANA functionally accommodated mLANA's mode of DNA binding, allowing MuHV-4 chimeric virus to establish latency Notably, v-KM established latency in germinal center B cells more efficiently than did v-kLANA, although levels were reduced compared to WT MuHV-4. KSHV is a human oncogenic virus for which there is no tractable, immunocompetent animal model of infection. MuHV-4, a related rodent gammaherpesvirus, enables pathogenesis studies in mice. In latency, both viruses persist as extrachromosomal, circular genomes (episomes). LANA proteins encoded by KSHV (kLANA) and MuHV-4 (mLANA) contain a C-terminal DNA binding domain (DBD) that acts on the virus terminal repeats to enable episome persistence. mLANA is a smaller protein than kLANA. Their DBDs are structurally conserved but differ strikingly in the conformation of DNA binding. We report a recombinant, chimeric MuHV-4 which contains kLANA in place of mLANA, but in which the DBD is replaced with that of mLANA. Results showed that kLANA functionally accommodated mLANA's mode of DNA binding. In fact, the new chimeric virus established latency more efficiently than MuHV-4 expressing full-length kLANA.
卡波西肉瘤相关疱疹病毒 (KSHV) 的潜伏相关核抗原 (kLANA) 及其来自鼠疱疹病毒 4 (MuHV-4) 的同源物 mLANA 对病毒潜伏至关重要。kLANA 的大小几乎是 mLANA 的四倍，主要是由于中央重复区域广泛，而 mLANA 中不存在该区域。这两种蛋白质都含有 C 末端 DNA 结合域 (DBD)。DBD 结合病毒基因组的末端重复 (TR) 序列，以介导持续性。尽管结构保守，但 kLANA 和 mLANA 的 DBD 在序列和寡聚化模式上存在差异。kLANA DBD 寡聚体灵活且弯曲，而 mLANA DBD 寡聚体以刚性、线性构象结合 DNA。我们之前报道过 kLANA 和 mLANA 可以相互作用于 TR 序列。此外，一种表达 kLANA 而不是 mLANA 的 MuHV-4（v-kLANA）在小鼠中建立了潜伏，尽管其程度低于野生型 (WT) 病毒。在这里，我们询问 kLANA 是否可以容纳 mLANA DBD，并生成一种融合蛋白，其中包含 kLANA，但带有 mLANA 的 C 末端区域而不是 kLANA 的 C 末端区域。我们报告了一种编码这种 LANA 融合蛋白的重组 MuHV-4 (v-KM)，而不是 mLANA。融合蛋白在裂解感染中表达，并在感染的脾细胞中组装核 LANA 点。结果表明，kLANA 可以适应 mLANA 的 DNA 结合模式，使 MuHV-4 嵌合病毒能够建立潜伏。值得注意的是，v-KM 在生发中心 B 细胞中建立潜伏的效率比 v-kLANA 更高，尽管与 WT MuHV-4 相比，水平降低。KSHV 是一种人类致癌病毒，目前尚无可行的、免疫功能正常的动物感染模型。MuHV-4 是一种相关的啮齿动物γ疱疹病毒，可在小鼠中进行发病机制研究。在潜伏中，两种病毒都以染色体外的环状基因组 (episomes) 形式存在。由 KSHV (kLANA) 和 MuHV-4 (mLANA) 编码的 LANA 蛋白都含有一个 C 末端 DNA 结合域 (DBD)，该域作用于病毒末端重复序列，使外染色体体持久存在。mLANA 是比 kLANA 小的蛋白。它们的 DBD 在结构上保守，但在 DNA 结合构象上存在显著差异。我们报告了一种嵌合的重组 MuHV-4，其中 kLANA 取代了 mLANA，但 DBD 被 mLANA 的取代。结果表明，kLANA 可以适应 mLANA 的 DNA 结合模式。事实上，新的嵌合病毒建立潜伏的效率比表达全长 kLANA 的 MuHV-4 更高。
