Mutational analysis of the latency-associated nuclear antigen DNA-binding domain of Kaposi's sarcoma-associated herpesvirus reveals structural conservation among gammaherpesvirus origin-binding proteins.

TitleMutational analysis of the latency-associated nuclear antigen DNA-binding domain of Kaposi's sarcoma-associated herpesvirus reveals structural conservation among gammaherpesvirus origin-binding proteins.
Publication TypeJournal Article
Year of Publication2010
AuthorsHan, S-J, Hu, J, Pierce, B, Weng, Z, Renne, R
JournalJ Gen Virol
Volume91
IssuePt 9
Pagination2203-15
Date Published2010 Sep
ISSN1465-2099
KeywordsAmino Acid Sequence, Amino Acid Substitution, Animals, Antigens, Viral, Binding Sites, Cell Line, Cercopithecus aethiops, Conserved Sequence, DNA Replication, DNA, Viral, Electrophoretic Mobility Shift Assay, Epstein-Barr Virus Nuclear Antigens, Evolution, Molecular, Gammaherpesvirinae, Herpesvirus 8, Human, Humans, Models, Molecular, Molecular Sequence Data, Mutant Proteins, Mutation, Nuclear Proteins, Protein Multimerization, Protein Structure, Quaternary, Protein Structure, Tertiary, Sequence Homology, Amino Acid, Structural Homology, Protein
Abstract

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(DBD) and published structures of EBNA1(DBD), this study used bioinformatics tools to model a putative structure for LANA(DBD) bound to DNA. To validate the predicted model, 38 mutants targeting the most conserved motifs, namely three alpha-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.

DOI10.1099/vir.0.020958-0
Alternate JournalJ. Gen. Virol.
PubMed ID20484563
PubMed Central IDPMC3066550
Grant ListR01 CA088763 / CA / NCI NIH HHS / United States
R01 CA119917 / CA / NCI NIH HHS / United States
R01 GM084884 / GM / NIGMS NIH HHS / United States
R01 CA88763 / CA / NCI NIH HHS / United States