Alanine scanning mutagenesis of the LANA1 N terminus. LANA1 residues 1 to 32 are shown with the indicated alanine substitutions below. The NLS and previously defined chromosome association region are indicated (31). The chromosome binding abilities of GFP LANA1 1-32 and GFP LANA1 1-1162 containing each indicated mutation are summarized at the right.

LANA1 amino acids 5 to 13 are essential and sufficient for chromosome targeting. GFP fusion proteins were expressed in BJAB cells and confocal microscopy was performed after metaphase arrest and counterstaining with propidium iodide. Overlay of GFP (green) and chromosomes (red) generates yellow. (A) GFP NLS (GFP), GFP LANA1 1-32 (1-32), GFP LANA1 1-32 ₅GMR₇→ AAA (GMR), GFP LANA1 1-32 ₈LRS₁₀→ AAA (LRS), GFP LANA1 1-32 ₁₁GRS₁₃→ AAA (GRS), GFP LANA1 1-32 ₁₄TG₁₅→ AA (TG), GFP LANA1 1-32 ₁₇PLT₁₉→ AAA (PLT), and GFP LANA1 1-32 ₂₀RGS₂₂→ AAA (RGS). (B) GFP NLS (GFP), GFP LANA1 (LANA), GFP LANA1 ₅GMR₇→ AAA (GMR), GFP LANA1 ₈LRS₁₀→ AAA (LRS), GFP LANA1 ₁₁GRS₁₃→ AAA (GRS), GFP LANA1 ₁₄TG₁₅→ AA (TG), GFP LANA1 ₁₇PLT₁₉→ AAA (PLT), and GFP LANA1 ₂₀RGS₂₂ → AAA (RGS). (C) GFP NLS (GFP), GFP LANA1 1-32 (1-32), GFP LANA1 5-13 (5-13), and GFP LANA1 5-14 (5-14). Arrows indicate small amounts of GFP LANA1 fusions dispersed between chromosomes for GFP LANA1 1-32 ₁₄TG₁₅→ AA, GFP LANA1 ₁₄TG₁₅→ AA, GFP LANA1 5-13, and GFP LANA1 5-14. Nontransfected cells are red only. Magnification, ×630.

Confocal microscopy of G418-resistant BJAB cells expressing LANA1 mutants. LANA1 (green) was detected with anti-LANA1 monoclonal antibody, and interphase nuclei (red) (left) and metaphase chromosomes (red) (right) were detected with propidium iodide. Overlay of green and red generates yellow. BJAB cells with only pSG5 do not express LANA1. BJAB cells expressing F-LANA1 (LANA), LANA1 ₅GMR₇→ AAA (GMR), LANA1 ₈LRS₁₀→ AAA (LRS), LANA1 ₁₁GRS₁₃→ AAA (GRS), and LANA1 ₁₄TG₁₅→ AA-3 (TG-3) and -4 (TG-4) are shown. Magnification, ×630.

LANA1 residues 5 to 13 are essential for episome persistence. Gardella gel analysis was performed to assay for episomes; 2.5 × 10⁶ cells were lysed in situ in gel wells, DNA was resolved by electrophoresis and detected by Southern blotting with the TR probe. (A) After 26 days of G418 selection, independent BJAB cell lines expressing F-LANA1 and LANA1 mutants were assayed for TR episomes. Lane 1, BCBL-1 primary effusion lymphoma cells; lanes 2 to 4, BJAB cells containing pSG5; lanes 5 to 7, BJAB cells expressing F-LANA1; lanes 8 to 10, BJAB cells expressing LANA1 ₅GMR₇→ AAA; lanes 11 to 13, BJAB cells expressing LANA1 ₈LRS₁₀→ AAA; lanes 14 to 16, BJAB cells expressing LANA1 ₁₁GRS₁₃→ AAA; lanes 17 to 20, BJAB cells expressing LANA1 ₁₄TG₁₅→ AA; lane 21, p8TR plasmid DNA. E, BCBL-1 KSHV episomes (lane 1), L, linear, replicating BCBL-1 KSHV (lane 1). The asterisk indicates episomes in F-LANA1-expressing cells (lanes 5 to 7). More slowly migrating episomal DNA is also present in lanes 5 to 7. Brackets indicate episomal DNA in LANA1 ₁₄TG₁₅→ AA-expressing cells (lanes 17 and 19). (B) After 53 days of G418 selection, BJAB cell lines expressing F-LANA1 and LANA1 ₁₄TG₁₅→ AA were again assayed for TR episomes. Lanes 1 to 3, BJAB cells expressing F-LANA1; lanes 4 to 7, BJAB expressing LANA1 ₁₄TG₁₅→ AA; lane 8, p8TR plasmid. Nicked and covalently closed circular (ccc) p8TR plasmid is indicated. O, gel origin. This figure is representative of four experiments.

LANA1 residues 5 to 13 are essential for DNA replication and residues 14 to 15 have a role in DNA replication. Assay of DNA replication of LANA1 mutants. BJAB cells containing the pSG5 vector or stably expressing F-LANA1 or LANA1 mutants were transfected with p8TR. After 48 h, low-molecular-weight DNA was digested with BglII or with BglII and DpnI. Digested DNA was resolved in agarose gels, transferred to nylon membranes, and detected with the TR probe. (A) Lane 1, p8TR plasmid; lane 2, F-LANA1; lane 3, LANA1 ₁₁GRS₁₃→ AAA; lane 4, LANA1 ₁₄TG₁₅→ AA-a; lane 5, LANA1 ₁₄TG₁₅→ AA-b; lane 6, F-LANA1; lane 7, LANA1 ₁₁GRS₁₃→ AAA; lane 8, LANA1 ₁₄TG₁₅→ AA-a; lane 9, LANA1 ₁₄TG₁₅→ AA-b. Signal below linearized p8TR but above the DpnI-digested fragments in lanes 6, 8, and 9 is due to partially replicated p8TR DNA. (B) Lane 1, pSG5; lane 2, F-LANA1; lane 3, LANA1 ₅GMR₇→ AAA; lane 4, LANA1 ₈LRS₁₀→ AAA; lane 5, pSG5; lane 6, F-LANA1; lane 7, LANA1 ₅GMR₇→ AAA; lane 8, LANA1 ₈LRS₁₀→ AAA. The arrow indicates linearized p8TR. This figure is representative of at least three experiments.

Alanine substitutions at the LANA1 N terminus do not inhibit binding to TR DNA. Radiolabeled probe TR-13 was incubated with in vitro-translated F-LANA1 (lanes 1 and 2), LANA1 ₅GMR₇→ AAA (lane 3), LANA1 ₈LRS₁₀→ AAA (lane 4), LANA1 ₁₁GRS₁₃→ AAA (lane 5), or LANA1 ₁₄TG₁₅→ AA (lane 6), and electrophoretic mobility shift assays were performed. Addition of a 100-fold excess of unlabeled competitor TR-13 is indicated (lane 2). Arrows indicate specific complexes. NSB, nonspecific bands. Free probe was run off the gel.