The latency-associated nuclear antigen (LANA) of Kaposi's sarcoma-associated herpesvirus functions as an origin-binding protein (OBP) and transcriptional regulator. LANA binds the terminal repeats via the C-terminal DNA-binding domain (DBD) to support latent DNA replication. To date, the structure of LANA has not been solved. Sequence alignments among OBPs of gammaherpesviruses have revealed that the C terminus of LANA is structurally related to EBNA1, the OBP of Epstein–Barr virus. Based on secondary structure predictions for LANA and published structures of EBNA1, this study used bioinformatics tools to model a putative structure for LANA bound to DNA. To validate the predicted model, 38 mutants targeting the most conserved motifs, namely three -helices and a conserved proline loop, were constructed and functionally tested. In agreement with data for EBNA1, residues in helices 1 and 2 mainly contributed to sequence-specific DNA binding and replication activity, whilst mutations in helix 3 affected replication activity and multimer formation. Additionally, several mutants were isolated with discordant phenotypes, which may aid further studies into LANA function. In summary, these data suggest that the secondary and tertiary structures of LANA and EBNA1 DBDs are conserved and are critical for (i) sequence-specific DNA binding, (ii) multimer formation, (iii) LANA-dependent transcriptional repression, and (iv) DNA replication.


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vol. , part 9, pp. 2203 - 2215

Western blot analysis of purified wt and mutant LANADBD proteins

Oligonucleotide primers used to generate amino acid substitution mutants [Single PDF file](117 KB)

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