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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.

Han SJ, Hu J, Pierce B, Weng Z, Renne R - J. Gen. Virol. (2010)

Bottom Line: The latency-associated nuclear antigen (LANA) of Kaposi's sarcoma-associated herpesvirus functions as an origin-binding protein (OBP) and transcriptional regulator.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL 32610-3633, USA.

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.

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Co-immunoprecipitation assays with alanine substitution mutants. The dimerization ability of Flag-tagged wt or mutant LANADBDs with HA-tagged wt LANADBD was tested. Dimerization activity for each mutant was normalized based on the expression level of Flag-tagged wt or mutant LANADBD proteins. L, Cell lysate, IP; immunoprecipitated samples; Wt (N), HA-tagged wt only as a negative control; Wt (P), Flag-tagged and HA-tagged wt as a positive control.
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f4: Co-immunoprecipitation assays with alanine substitution mutants. The dimerization ability of Flag-tagged wt or mutant LANADBDs with HA-tagged wt LANADBD was tested. Dimerization activity for each mutant was normalized based on the expression level of Flag-tagged wt or mutant LANADBD proteins. L, Cell lysate, IP; immunoprecipitated samples; Wt (N), HA-tagged wt only as a negative control; Wt (P), Flag-tagged and HA-tagged wt as a positive control.

Mentions: Mutants HIF876AAA and YR879AA in helix 1, which showed drastically reduced DNA-binding affinities, did dimerize at a level comparable to wt (Fig. 4a, lanes 5–8). RF881AA and Q875A reduced dimerization only (Fig. 4b, lanes 5 and 6, and Table 2), further suggesting that most helix 1 residues contribute directly to DNA binding but not to dimer formation.


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.

Han SJ, Hu J, Pierce B, Weng Z, Renne R - J. Gen. Virol. (2010)

Co-immunoprecipitation assays with alanine substitution mutants. The dimerization ability of Flag-tagged wt or mutant LANADBDs with HA-tagged wt LANADBD was tested. Dimerization activity for each mutant was normalized based on the expression level of Flag-tagged wt or mutant LANADBD proteins. L, Cell lysate, IP; immunoprecipitated samples; Wt (N), HA-tagged wt only as a negative control; Wt (P), Flag-tagged and HA-tagged wt as a positive control.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3066550&req=5

f4: Co-immunoprecipitation assays with alanine substitution mutants. The dimerization ability of Flag-tagged wt or mutant LANADBDs with HA-tagged wt LANADBD was tested. Dimerization activity for each mutant was normalized based on the expression level of Flag-tagged wt or mutant LANADBD proteins. L, Cell lysate, IP; immunoprecipitated samples; Wt (N), HA-tagged wt only as a negative control; Wt (P), Flag-tagged and HA-tagged wt as a positive control.
Mentions: Mutants HIF876AAA and YR879AA in helix 1, which showed drastically reduced DNA-binding affinities, did dimerize at a level comparable to wt (Fig. 4a, lanes 5–8). RF881AA and Q875A reduced dimerization only (Fig. 4b, lanes 5 and 6, and Table 2), further suggesting that most helix 1 residues contribute directly to DNA binding but not to dimer formation.

Bottom Line: The latency-associated nuclear antigen (LANA) of Kaposi's sarcoma-associated herpesvirus functions as an origin-binding protein (OBP) and transcriptional regulator.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL 32610-3633, USA.

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.

Show MeSH
Related in: MedlinePlus