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

Latency-associated nuclear antigen (LANA) mediates γ2-herpesvirus genome persistence and regulates transcription. We describe the crystal structure of the murine gammaherpesvirus-68 LANA C-terminal domain at 2.2 Å 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).潜伏相关核抗原（Latency-associated nuclear antigen，LANA）可介导γ2疱疹病毒的基因组维持并调控转录。本研究解析了鼠γ疱疹病毒68型LANA C端结构域的2.2埃（Å）分辨率晶体结构。该结构呈现αβ折叠模体，组装为二聚体，其结构特征与EB病毒（Epstein-Barr virus）EBNA1相似。结构中存在一处预测的DNA结合界面，与该界面相对的区域带有正静电补丁。定向的DNA识别位点氨基酸替换突变可消除LANA的DNA结合能力，而部分带电补丁区域的突变则会降低其与溴结构域蛋白BRD4（bromodomain protein, BRD4）的结合活性。携带DNA结合功能丧失型LANA的病毒无法在小鼠体内建立可存活的潜伏感染。带电补丁区域外围发生突变的病毒在潜伏感染扩增过程中表现出显著缺陷，而中心区域的氨基酸替换突变则几乎无影响，且该缺陷与BRD4无关。上述结果阐明了LANA DNA结合结构域的三维结构，并揭示了一个独特的带电区域，该区域在病毒潜伏感染中发挥关键作用，其功能可能通过宿主细胞蛋白介导。