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

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.

Fig. 1.

Sequence alignments of LANA_DBDs of gammaherpesviruses and EBNA1_DBD reveals structural conservation. (a) Multiple alignments of amino acid sequences among LANA_DBDs of KSHV, RFHVMn and RRV using the praline and T-Coffee programs. Conserved amino acids among the OBPs are labelled in bold above the sequences. (b) Binary amino acid alignments between EBV EBNA1_DBD and the LANA_DBDs of KSHV, RFHVMn and RRV using the 3d-pssm program. Conserved helices among the proteins are shown as shaded dark grey boxes. Proline loops are indicated in italic within light grey boxes. Numbers in parentheses refer to corresponding amino acid numbers from BC-1 KSHV LANA (Kelley-Clarke et al., 2007). Short dashes indicate missing amino acids; + and − indicate similarity or no similarity between amino acids.

Fig. 2.

Computational model of the LANA_DBD and multimer structure bound to DNA. The figure shows the LANA_DBD model with the specific DNA-binding site predicted by the m-zdock program based on alignment with the structure of EBNA1_DBD. (a) The tetramer formed by combining two dimers bound to their respective LBS1/2 (DNA helix in light blue and green). The β-barrel bundle is made of four β-strands from each monomer at the dimer interface. (b) Each monomer is composed of four β-strands and three helices (helix 1 in red, helix 2 in blue and helix 3 in green). (c, d) Crucial amino acids for DNA contact or dimerization are shown in yellow: ⁸⁷¹K and ⁸⁷⁵Q for helix 1, ⁹⁶³W and ⁹⁶⁴E for helix 3 (c) and ⁹⁰⁷Y, ⁹¹⁰K and ⁹¹¹K for helix 2 (d). The monomer pictures were generated using ViewerLite 4.2 (Accelrys).

Fig. 3.

DNA-binding activity of LANA_DBD mutants. Purified LANA_DBD wt and mutant proteins were incubated with radiolabelled LBS1 or LBS1/2 as described previously (Garber et al., 2001). The DNA-binding affinity is represented as the percentage for mutants compared with wt LANA_DBD, which was set to 100 %. In each assay, all mutants were tested for DNA-binding activity with LBS1 (a–c) or LBS1/2 (d). EMSA results are shown for helix 1 mutants (a), helix 2 mutants (b), helix 3 mutants (c) and adapted mutants from each helix (d). Arrows indicate specific protein–DNA complexes. NC, Probe alone as a negative control; wt, wt LANA_DBD. Results on graphs are shown as means±sd from three independent experiments.

Fig. 4.

Co-immunoprecipitation assays with alanine substitution mutants. The dimerization ability of Flag-tagged wt or mutant LANA_DBDs with HA-tagged wt LANA_DBD was tested. Dimerization activity for each mutant was normalized based on the expression level of Flag-tagged wt or mutant LANA_DBD 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.

Fig. 5.

Analysis of DNA replication mediated by alanine substitution mutants using a short-term replication assay. LANA_DBD-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.

Fig. 6.

Analysis of the activity of LANA-dependent transcriptional repression by alanine substitution. Graphical representation of data from luciferase reporter assays. pGL3/7TR luciferase reporter and wt or mutant LANA_DBD plasmid were co-transfected. RLU values were normalized to total protein concentration as described previously (Renne et al., 2001). The percentage of suppression activity was compared with that of LANA_DBD wt, which was set to 100 %. Results are shown as means±sd from three independent experiments. (−)LANA, Negative control with no LANA_DBD.