KSHV LANA acetylation-selective acidic domain reader sequence mediates virus persistence

Abstract

Viruses modulate biochemical cellular pathways to permit infection. A recently described mechanism mediates selective protein interactions between acidic domain readers and unacetylated, lysine-rich regions, opposite of bromodomain function. Kaposi´s sarcoma (KS)-associated herpesvirus (KSHV) is tightly linked with KS, primary effusion lymphoma, and multicentric Castleman's disease. KSHV latently infects cells, and its genome persists as a multicopy, extrachromosomal episome. During latency, KSHV expresses a small subset of genes, including the latency-associated nuclear antigen (LANA), which mediates viral episome persistence. Here we show that LANA contains two tandem, partially overlapping, acidic domain sequences homologous to the SET oncoprotein acidic domain reader. This domain selectively interacts with unacetylated p53, as evidenced by reduced LANA interaction after overexpression of CBP, which acetylates p53, or with an acetylation mimicking carboxyl-terminal domain p53 mutant. Conversely, the interaction of LANA with an acetylation-deficient p53 mutant is enhanced. Significantly, KSHV LANA mutants lacking the acidic domain reader sequence are deficient for establishment of latency and persistent infection. This deficiency was confirmed under physiological conditions, on infection of mice with a murine gammaherpesvirus 68 chimera expressing LANA, where the virus was highly deficient in establishing latent infection in germinal center B cells. Therefore, LANA's acidic domain reader is critical for viral latency. These results implicate an acetylation-dependent mechanism mediating KSHV persistence and expand the role of acidic domain readers.

Keywords: Kaposi´s sarcoma herpesvirus; acetylation-regulated interaction; acidic domain reader; latency-associated nuclear antigen; virus persistence.

Fig. 1.

Schematic representation of KSHV LANA and LANA mutants. The amino terminal bipartite nuclear localization signal (NLS) at residues 24 to 30 and 41 to 47 (9, 10) is indicated by the two vertical black bands. LANA regions identified include proline-rich (P), aspartate- and glutamate-rich (DE), glutamine-rich (Q), glutamate- and glutamine-rich (EQE), and putative leucine zipper (LZ). The DE, Q, LZ, and EQE regions each contain imperfect repeat elements. Residues 5 to 13 mediate chromosome association through interaction with histones H2A/H2B (11, 12). Residues 996 to 1,139 bind TR DNA, mediate self-association, and associate with mitotic chromosomes. The capabilities for DNA replication, episome segregation, LANA mediated episome persistence, and KSHV persistence in cells are summarized at the right. *Data from refs. and . NT, not tested.

Fig. 2.

BAC LANA-R Δ332–929 is abolished, and BAC LANA-R Δ262–320 is impaired for latently infected cell outgrowth. (A) BACΔLANA, BAC LANA-R, and BAC containing LANA mutants were transfected into 293T cells. The cells were harvested after 48 h, and LANA expression was assessed by Western blot analysis using a polyclonal Ab that recognizes the LANA internal repeat elements. (B) BACΔLANA, BAC LANA-R, and LANA mutants were each transfected into 293T cells and harvested after 24 h. DNA was extracted by Hirt extraction (33). RNA was extracted with TRIzol and reverse-transcribed. The level of LANA cDNA compared with KSHV DNA, as determined by real-time qPCR, was used to determine expression levels. (C and D) BAC LANA-R and mutants were transfected in 293T cells. At 48 h posttransfection, the number of GFP-expressing cells (i.e., GFP expressed from the recombinant BAC) was assessed by flow cytometry, and a cell suspension containing 1,000 green cells was plated and placed under selection, for which resistance is encoded by the BAC, in 150-mm dishes. After 2.5 wk of selection, colonies were stained with crystal violet and counted. A representative experiment is shown in C, and average colony outgrowths are shown in D. All data are from at least three independent experiments. ns, P > 0.05. **P < 0.01, independent-samples t test.

Fig. 3.

BAC LANA-R Δ331–495 is severely impaired for latently infected cell outgrowth. (A) BAC LANA-R and BAC LANA-R Δ331–495 were transfected into 293T cells. The cells were harvested after 48 h, and LANA expression was assessed by Western blot analysis. (B and C) BAC LANA-R and BAC LANA-R Δ331–495 were each transfected into 293T cells. At 48 h after transfection, the cells were plated and placed under selection as described in Fig. 2. Data in C are from at least three independent experiments. *P < 0.05; **P < 0.01.

Fig. 4.

The LANA SET homology sequence interacts with p53 and acetylation reduces LANA–p53 interaction. (A) Schematic of LANA with acidic SET homology regions indicated below. Sequence alignments were determined with LALIGN. Identity and similarity are shown. (B) GFP LANA 275–435 interacts with p53. GFP, GFP-LANA, or GFP-LANA 275–435 and Flag-p53 were cotransfected into 293T cells. At 48 h after transfection, Flag p53 was immunoprecipitated with M2-Flag Ab, and Western blot analysis was performed. (C) Expression of CBP, which acetylates p53, reduces the LANA–p53 interaction. pT7LANA, Flag-p53, and HA-CBP were transfected into 293T cells. At 48 h after transfection, Flag p53 was immunoprecipitated using M2-Flag Ab, and Western blot analysis was performed. Data in B and C are representative of at least three independent experiments.

Fig. 5.

Decreased LANA interaction with acetylation mimicking p53^KQ and increased LANA interaction with acetylation-deficient p53^KR. GFP LANA (Left) and GFP LANA 275–435 (Right) were coexpressed with Flag p53, p53^KQ, or p53^KR (p53 K acetylation sites 370, 372 to 373, 381 to 382, and 386 mutated to Q or R, respectively) in 293T cells. At 48 h posttransfection, Flag p53 was immunoprecipitated, and Western blot analysis was performed. Results are representative of at least three experiments. Quantitation of GFP LANA or GFP LANA 275–435 bands are shown. *Heavy chain, which comigrates with p53. V, Flag vector.

Fig. 6.

Deletion of kLANA residues 332 to 929 abolishes kLANA-MHV-68 latency in vivo. (A) Relative levels of kLANA transcripts from BHK-21 cells infected with 5 PFU/cell for 8 h. Expression of MHV68 M3, a lytic viral product, was also assessed as a measure of the level of infection. Bars indicate mean fold changes ± SD. (B) Growth curves. BHK-21 cells were infected with 0.01 PFU/cell for 1 h and, after removal of inoculum and washing, incubated at 37 °C. Total virus titers at the indicated times were determined by plaque assay. Time 0 represents the viral titer after washing inoculum. (C and D) Latent infection in the spleen at day 14 after intranasal infection with 10⁴ PFU. (C) Infectious center titers following reactivation from latency (closed circles) and preformed virus (open circles). Circles represent individual mice. The horizontal bar indicates the mean, and the dashed line indicates the limit of detection. (D) Frequency of viral DNA-positive cells (bars) ± 95% CIs in total splenocytes (Left) and FACS-sorted germinal center B cells (CD19⁺CD95⁺GL7⁺) (Right). Data are from pools of three to five spleens per infection group. ND, not detectable. i denotes independent clone. NS, not significant; **P < 0.01; ***P < 0.001, Mann–Whitney U test.

Fig. 7.

LANA residues 331 to 495 are required for efficient establishment of viral latency in infected mice. (A) Detection of kLANA Δ331–495 by Western blot in infected BHK-21 cells (3 PFU/cell; 6 h postinfection), using an Ab specific for the repeat region. mLANA was detected specifically in v-WT–infected cells. Anti-actin immunoblots confirmed equivalent amounts of cellular extracts among the samples. (B) Growth curves in BHK-21 cells following infection with 0.01 PFU/cell performed as in Fig. 6B. (C and D) Latent infection in the spleen at 14 d after intranasal infection with 10⁴ PFU. (C and D, Left), Infectious center titers (closed circles) and preformed virus (open circles). Circles represent individual mice. The horizontal bar indicates the mean, and the dashed line represents the limit of detection. (D, Right) Frequency of viral DNA-positive cells (bars) ± 95% CIs in total splenocytes. Data are from pools of five spleens per infection group. i and ii denote independent clones.*P < 0.05; **P < 0.01, Mann–Whitney U test.