Map of LANA1 subdomain clones.

LANA1 CR2-CR3 fragment retards synthesis in vitro at both the N- and the C-terminal positions. The CR2-CR3 LANA1 fragment fused to GFP either at the N or C terminus prevents translation of heterologously fused protein. In comparison, LANA-N fused at the N-terminal or C-terminal position to GFP is efficiently translated. Representative results of three experiments are shown.

Mechanism for CTL immune evasion by the QED central repeat domain of LANA1. The LANA1 central repeat domain may inhibit peptide processing and MHC-I antigen presentation through two distinct mechanisms: (i) synthesis retardation during initial translation from Orf73 mRNA, in which the QED-containing peptide region slows down protein translation, thereby reducing misfolded protein turnover; and (ii) degradation inhibition, in which mature protein proteosomal processing is inhibited by the QED-containing motifs.

LANA CR2CR3 repeat region inhibits proteosomal processing. (A) The LANA1 CR2CR3 region was cloned as a fusion protein with destabilized enhanced green fluorescent protein (d1EGFP) and expressed in 293 cells. Cycloheximide (CHX) treatment was used to prevent de novo protein synthesis, and protein levels were determined by immunoblotting (IB) with anti-GFP mouse antibody. (B) GFP fluorescence in 293 cells is markedly higher for CR2CR3-d1EGFP expression after 12 h of cycloheximide treatment than for d1EGFP alone. Merged phase-contrast images show similar numbers of cells for both CR2CR3-d1EGFP and d1EGFP expression conditions. Representative results of three experiments are shown.

Disruption of a protein motif located at the junction of CR2 and CR3 reduces translational retardation. (A) Schematic diagram of CR2 and CR3 constructs for in vitro translation experiments. (TAA is a stop codon). (B) Disruption of the junction between CR2 and CR3 increases protein translation efficiency. In this representative experiment (of three), CR2-CR3 translation is compared to CR2-CR3 constructs having a small insert between CR2 and CR3 (CR2-EcoRV-CR3, 6 nt), a large insert (CR2-N-CR3, 1,002 nt), or having the CR3 and CR2 in reverse orientation (CR3-CR2). Each construct has markedly increased translation efficiency compared to that of CR2-CR3. (C) Protein rather than mRNA structure is critical for CR2-CR3 translation retardation. Protein translation was truncated by cloning the TAA stop codon between CR2 and CR3 (*) in the full-length LANA1 gene. Arrowheads show expected size of full-length LANA1 (black) and actual size of LANA1_TAA (white). Translation efficiency for this construct was comparable to that of a construct truncated 3′ to the CR2 sequence (N-CR1-CR2). Representative results of three experiments are shown.

Identification of LANA1 synthesis retardation domain. (A) Map of C-terminal deletion constructs for in vitro translation experiments. Arrows show sites of CR2CR3 junctions. (B) LANA1 mRNAs containing the central repeat region have reduced translation efficiency in uncoupled in vitro translation assays. Results are averages from three experiments. Translation reactions were performed using equimolar amounts of T7 polymerase transcribed-capped RNA. Luciferase RNA was translated (∼61-kDa product) as a positive control. As a negative control, the reaction was performed without added RNA. (C) The amount of [³⁵S]methionine-labeled protein shown in panel B was quantified using ImageQuant software (Molecular Dynamics) and further standardized to the number of methionines in each construct. Lanes 1 to 320, HincII (4 methionines); 1 to 434, AclI (4 methionines); 1 to 842, SapI (4 methionines); 1 to 928, BsmBI (4 methionines); 1 to 980, NruI (5 methionines); 1 to 1162, FL (8 methionines).

LANA1 turnover in KSHV-infected cell line BCBL-1. (A) LANA1 immunoblot after cycloheximide treatment in BCBL-1 cells. BCBL-1 cells were treated with cycloheximide (100 μg/ml) to abolish new protein synthesis at different time points, and immunoblotting (IB) was performed using mouse LANA1 antibody (upper panel). The ∼220- to 230-kDa bands of LANA1 are detectable at 12 h after treatment, but the ∼150- to 180-kDa LANA1 bands (LANA1 isoforms) can be detected for up to 48 h. LANA1 half-life is prolonged (∼24 h) compared to that of cellular IRF3 protein (∼8 h). The membrane was stripped and reprobed with cellular IRF3 antibody for comparison (lower panel). Representative results of two experiments are shown. (B) Immunoprecipitation (IP) of LANA1 from PEL cell lines. LANA1 was immunoprecipitated with mouse monoclonal antibody (CMA-810) and detected with rat monoclonal antibody (ABI). Both the ∼150- to 180-kDa and the ∼222- to 234-kDa bands were detected by both antibodies and show polymorphic variations among cell lines. The BJAB control cell line shows no protein reactivity.

Effect of LANA1 repeat regions on protein synthesis. (A) Maximal translation retardation occurs when CR2 is joined to CR3. Each LANA1 domain was fused at the C terminus to EGFP and translated using equimolar amounts of RNA. All three central repeat domains (EGFP-CR1, EGFP-CR2, and EGFP-CR3) individually show similar rates of translation to the EGFP-NC construct entirely lacking the central repeat domain. Arrows show expected sizes for each construct. Note that the combined CR1CR2 construct may also have reduced translation compared to other comparably sized fragments, but translation retardation is most pronounced for the CR2CR3 construct. (B) CR2CR3 retards in vivo accumulation of a heterologous EGFP fusion protein. 293 cells were transfected with EGFP-fused LANA1 CR1, CR2, CR3, CR1-CR2, and CR2-CR3 to determine the effect of LANA1 fragments on protein synthesis in vivo. Cells were harvested at different time points (0, 4, 8, 12, 16, and 27 h), and lysates were quantitatively assayed for GFP using a fluorescence reader. (C) Translational retardation by LANA1 and EBNA1 occurs in trans at high expression levels. In this experiment, synthesis of constant amounts of LANA1-C (C-terminal region) was measured in the presence of increasing amounts of full-length LANA1 and EBNA1. To ensure equal total amounts of RNA, increasing amounts of full-length LANA1 (upper left panel) or EBNA1 (upper right panel) RNA (0, 0.5, 1.0, 1.5, and 2.0 pM) were added with parallel decreasing amounts (2.0, 1.5, 1.0, 0.5, and 0 pM) of LANA1-NC (lacking the central repeat region) and constant amounts (1 pM) of LANA1-C (C-terminal region). Amounts of [³⁵S]methionine-labeled LANA1-C were quantitated by ImageQuant software and reveal reduced LANA1-C synthesis as full-length LANA1 and EBNA1 amounts are increased (lower panels). Representative results of three experiments are shown.