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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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Related in: MedlinePlus

Analysis of DNA replication mediated by alanine substitution mutants using a short-term replication assay. LANADBD-expressing constructs were co-transfected with pPuro/4TR into 293 cells; 10 % of the extracted DNA (Hirt, 1967) was digested with HindIII as input (a, lanes 1–8, and b, lanes 1–6) and the remaining DNA was double digested with HindIII and DpnI (a, lanes 9–16, and b, lanes 7–12). The DNA was detected by Southern blotting with a radiolabelled 4TR probe. Full-length LANA was transfected as a positive control. The arrow indicates the position of full-length pPuro-4TR.
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f5: Analysis of DNA replication mediated by alanine substitution mutants using a short-term replication assay. LANADBD-expressing constructs were co-transfected with pPuro/4TR into 293 cells; 10 % of the extracted DNA (Hirt, 1967) was digested with HindIII as input (a, lanes 1–8, and b, lanes 1–6) and the remaining DNA was double digested with HindIII and DpnI (a, lanes 9–16, and b, lanes 7–12). The DNA was detected by Southern blotting with a radiolabelled 4TR probe. Full-length LANA was transfected as a positive control. The arrow indicates the position of full-length pPuro-4TR.

Mentions: Mutagenesis of the LBS1/2 showed that the replication efficiency of TR-containing plasmids is dependent on the LBS1 (Garber et al., 2002). To test the inverse, we chose a subset of mutants with reduced DNA binding or dimerization and performed transient replication assays as described previously (Garber et al., 2002; Hu et al., 2002). Briefly, a plasmid containing four copies of the TR was co-transfected with plasmid expressing wt or mutant LANADBD into 293 cells. Replicating DNA was extracted and analysed by Southern blotting after DpnI digestion. As described previously, LANADBD replicated with about 20 % efficiency compared with full-length LANA (compare Fig. 5a, lanes 9–11, and Fig. 5b, lanes 7–9) (Hu et al., 2002).


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)

Analysis of DNA replication mediated by alanine substitution mutants using a short-term replication assay. LANADBD-expressing constructs were co-transfected with pPuro/4TR into 293 cells; 10 % of the extracted DNA (Hirt, 1967) was digested with HindIII as input (a, lanes 1–8, and b, lanes 1–6) and the remaining DNA was double digested with HindIII and DpnI (a, lanes 9–16, and b, lanes 7–12). The DNA was detected by Southern blotting with a radiolabelled 4TR probe. Full-length LANA was transfected as a positive control. The arrow indicates the position of full-length pPuro-4TR.
© Copyright Policy
Related In: Results  -  Collection

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

f5: Analysis of DNA replication mediated by alanine substitution mutants using a short-term replication assay. LANADBD-expressing constructs were co-transfected with pPuro/4TR into 293 cells; 10 % of the extracted DNA (Hirt, 1967) was digested with HindIII as input (a, lanes 1–8, and b, lanes 1–6) and the remaining DNA was double digested with HindIII and DpnI (a, lanes 9–16, and b, lanes 7–12). The DNA was detected by Southern blotting with a radiolabelled 4TR probe. Full-length LANA was transfected as a positive control. The arrow indicates the position of full-length pPuro-4TR.
Mentions: Mutagenesis of the LBS1/2 showed that the replication efficiency of TR-containing plasmids is dependent on the LBS1 (Garber et al., 2002). To test the inverse, we chose a subset of mutants with reduced DNA binding or dimerization and performed transient replication assays as described previously (Garber et al., 2002; Hu et al., 2002). Briefly, a plasmid containing four copies of the TR was co-transfected with plasmid expressing wt or mutant LANADBD into 293 cells. Replicating DNA was extracted and analysed by Southern blotting after DpnI digestion. As described previously, LANADBD replicated with about 20 % efficiency compared with full-length LANA (compare Fig. 5a, lanes 9–11, and Fig. 5b, lanes 7–9) (Hu et al., 2002).

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