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. 2015 May 26;112(21):6694-9.
doi: 10.1073/pnas.1421804112. Epub 2015 May 6.

The 3D structure of Kaposi sarcoma herpesvirus LANA C-terminal domain bound to DNA

Jan Hellert  1 , Magdalena Weidner-Glunde  2 , Joern Krausze  1 , Heinrich Lünsdorf  3 , Christiane Ritter  1 , Thomas F Schulz  4 , Thorsten Lührs  5
Affiliations

The 3D structure of Kaposi sarcoma herpesvirus LANA C-terminal domain bound to DNA

Jan Hellert et al. Proc Natl Acad Sci U S A. .

Abstract

Kaposi sarcoma herpesvirus (KSHV) persists as a latent nuclear episome in dividing host cells. This episome is tethered to host chromatin to ensure proper segregation during mitosis. For duplication of the latent genome, the cellular replication machinery is recruited. Both of these functions rely on the constitutively expressed latency-associated nuclear antigen (LANA) of the virus. Here, we report the crystal structure of the KSHV LANA DNA-binding domain (DBD) in complex with its high-affinity viral target DNA, LANA binding site 1 (LBS1), at 2.9 Å resolution. In contrast to homologous proteins such as Epstein-Barr virus nuclear antigen 1 (EBNA-1) of the related γ-herpesvirus Epstein-Barr virus, specific DNA recognition by LANA is highly asymmetric. In addition to solving the crystal structure, we found that apart from the two known LANA binding sites, LBS1 and LBS2, LANA also binds to a novel site, denoted LBS3. All three sites are located in a region of the KSHV terminal repeat subunit previously recognized as a minimal replicator. Moreover, we show that the LANA DBD can coat DNA of arbitrary sequence by virtue of a characteristic lysine patch, which is absent in EBNA-1 of the Epstein-Barr virus. Likely, these higher-order assemblies involve the self-association of LANA into supermolecular spirals. One such spiral assembly was solved as a crystal structure of 3.7 Å resolution in the absence of DNA. On the basis of our data, we propose a model for the controlled nucleation of higher-order LANA oligomers that might contribute to the characteristic subnuclear KSHV microdomains ("LANA speckles"), a hallmark of KSHV latency.

Keywords: DNA-binding protein; KSHV LANA; X-ray crystallography; gammaherpesvirinae; viral latency.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Structure of KSHV LANA bound to LBS1 DNA. (A) Crystal structure of an oligomerization-deficient LANA(1008–1146) multiple-point mutant bound to 20-bp LBS1 DNA. (B) Close-up of the primary site of DNA recognition. (C) Close-up of the symmetry-related secondary site. (D) Structure-derived interaction chart. LBS1 sequence numbering corresponds to a previous study (17).
Fig. 2.
Fig. 2.
The three LANA binding sites of the KSHV minimal replicator. (A) Sequence of the minimal replicator with the helical phase for standard B-DNA shown above. The primary sites of DNA recognition are shaded in gray. Nucleotide positions identical to those in LBS1 are shown in red letters. Sequence numbering corresponds to a previous study (44) (GenBank: U86666.1). (B) Sequence alignment of the three LANA binding sites. Sequence numbering is as in Fig. 1. (C) EMSA using wild-type LANA(934–1162) (+) or GST as a control (−). An interpretation of the band patterns is provided on the right. Bands of unknown composition are marked with asterisks. These bands may reflect complexes with DNA bound to the lysine patch of LANA (compare Fig. 3). The left gel was exposed with higher sensitivity to visualize the weak bands of the low-affinity core sites. (D) EMSA using the oligomerization-deficient LANA(1008–1146) mutant (+) or GST as a control (−). An interpretation of the bands is provided on the right. The left gel was exposed with higher sensitivity. (E) Model of three LANA dimers bound to the minimal replicator, assuming that binding to LBS2 and LBS3 is structurally similar to LBS1.
Fig. 3.
Fig. 3.
Structure of the LANA spiral. (A) Top view of the spiral crystal structure of wild-type LANA(1013–1149) shown in cartoon representation (Left) and in an electrostatic surface potential representation (Right). (B) Side view of a full helical repeat of 24 dimers showing the electrostatic surface potential on the outside (Left) and inside (Right) of the spiral. (C and D) Negative-stain EM of a specific DNA-binding-deficient LANA(1013–1162) multiple-point mutant bound to 207 bp/70 nm nonspecific DNA. Kinks in the regular complexes are marked in D. (E) A LANA DBD mutant as in C and D additionally carrying the A1121E mutation loosely bound to 207 bp/70 nm DNA. (F) A LANA DBD mutant as in C and D additionally carrying the mutations K1055E, K1109E, and K1138E in the presence of 207 bp/70 nm DNA. (G) The same mutant as in C and D in the absence of DNA.
Fig. 4.
Fig. 4.
Hypothetical model for LANA spiral assembly. (A) A nucleator is formed once three LANA dimers bound to the minimal replicator catch a remote segment of free DNA on their lysine patches. (B) Recruitment of more LANA molecules leads to spiral formation in trans. Histones and chromatin structure are omitted for clarity.
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