Expression, purification and characterisation of KSHV's Latency-Associated Nuclear Antigen and its DNA-binding mutant

Latency-Associated Nuclear Antigen (LANA) is a critical protein during the establishment and maintenance of viral persistence and latency in Kaposi’s Sarcoma Associated Herpesvirus (KSHV). Its functions include tethering viral episomes to host chromatin, recruiting cellular DNA replication machinery, regulating viral and cellular transcription, modulating chromatin organization, and suppressing lytic reactivation. The purpose of this study was to obtain a purified wild-type LANA and a mutant protein that carries mutations in its DNA-binding domain. The mutant is designed to impair the interaction between LANA and the LANA binding sites (LBSs) present in the terminal repeat (TR) region of the viral genome. Following protein purification, the DNA-binding properties of LANA wild-type and LANA-mutDBD were analysed and compared in a binding assay. The results suggest a specific binding between the TRs and wild-type LANA, while the DNA-binding domain mutant did not show affinity, thus contributing to the hypothesis of the specificity of the LANA-TR interaction. The purified wild-type and mutant proteins will be the basis for further studies investigating LANA’s role in latent viral replication.