Full-Length Isoforms of Kaposi's Sarcoma-Associated Herpesvirus Latency-Associated Nuclear Antigen Accumulate in the Cytoplasm of Cells Undergoing the Lytic Cycle of Replication

Abstract

The latency-associated nuclear antigen (LANA) of the Kaposi's sarcoma-associated herpesvirus (KSHV) performs a variety of functions to establish and maintain KSHV latency. During latency, LANA localizes to discrete punctate spots in the nucleus, where it tethers viral episomes to cellular chromatin and interacts with nuclear components to regulate cellular and viral gene expression. Using highly sensitive tyramide signal amplification, we determined that LANA localizes to the cytoplasm in different cell types undergoing the lytic cycle of replication after de novo primary infection and after spontaneous, tetradecanoyl phorbol acetate-, or open reading frame 50 (ORF50)/replication transactivator (RTA)-induced activation. We confirmed the presence of cytoplasmic LANA in a subset of cells in lytically active multicentric Castleman disease lesions. The induction of cellular migration by scratch-wounding confluent cell cultures, culturing under subconfluent conditions, or induction of cell differentiation in primary cultures upregulated the number of cells permissive for primary lytic KSHV infection. The induction of lytic replication was characterized by high-level expression of cytoplasmic LANA and nuclear ORF59, a marker of lytic replication. Subcellular fractionation studies revealed the presence of multiple isoforms of LANA in the cytoplasm of ORF50/RTA-activated Vero cells undergoing primary infection. Mass spectrometry analysis demonstrated that cytoplasmic LANA isoforms were full length, containing the N-terminal nuclear localization signal. These results suggest that trafficking of LANA to different subcellular locations is a regulated phenomenon, which allows LANA to interact with cellular components in different compartments during both the latent and the replicative stages of the KSHV life cycle.IMPORTANCE Kaposi's sarcoma-associated herpesvirus (KSHV) causes AIDS-related malignancies, including lymphomas and Kaposi's sarcoma. KSHV establishes lifelong infections using its latency-associated nuclear antigen (LANA). During latency, LANA localizes to the nucleus, where it connects viral and cellular DNA complexes and regulates gene expression, allowing the virus to maintain long-term infections. Our research shows that intact LANA traffics to the cytoplasm of cells undergoing permissive lytic infections and latently infected cells in which the virus is induced to replicate. This suggests that LANA plays important roles in the cytoplasm and nuclear compartments of the cell during different stages of the KSHV life cycle. Determining cytoplasmic function and mechanism for regulation of the nuclear localization of LANA will enhance our understanding of the biology of this virus, leading to therapeutic approaches to eliminate infection and block its pathological effects.

Keywords: KSHV; LANA; activation; cytoplasm; mass spectrometry; migration; viral replication.

FIG 1

Differential localization of nuclear and cytoplasmic LANA in KS and MCD lesions. Paraffin-embedded tissue sections were prepared for immunohistochemical detection of KSHV LANA protein using anti-LANA monoclonal antibody LN53. (A) KS lesion. The arrowheads indicate spindle-like tumor cells with punctate LANA staining in the nucleus. (B) Montage of different cells from two MCD lesions. The arrowheads indicate small cells with punctate LANA staining in the nucleus, similar to that seen in the KS lesion. The arrows indicate larger cells with LANA localized to the nucleus and cytoplasm. Scale bar, 20 μm.

FIG 2

Differential localization of nuclear and cytoplasmic LANA in KSHV-infected primary HOK and Vero cell cultures. (A) Primary HOK cultures induced to differentiate using high calcium (0.15 mM) for 24 h and (B) Vero monkey kidney epithelial cell cultures were infected with KSHV. At 24 hpi, the cell cultures were fixed and stained for LANA (green) using the LN53 antibody with TSA enhancement. Cell nuclei were labeled using TO-PRO-3 (blue). The arrowheads indicate cells with punctate nuclear LANA fluorescence, and the arrows indicate cells with diffuse LANA fluorescence in the cytoplasm. Scale bar, 20 μm.

FIG 3

Differential localization of nuclear and cytoplasmic LANA in migrating Vero cells. (A and B) Vero cells plated in a 16-mm “spot” culture were infected with KSHV. At 24 hpi, the cultures were fixed and stained for LANA (green) and nuclei (blue). (A) Sparse edge of the spot culture, delineated with a dotted white line. The arrows indicate cells with diffuse LANA fluorescence in the cytoplasm located at the edge of the cell culture where the cells were expanding into the open area of the plate. The arrowheads indicate adjacent cells with punctate LANA fluorescence in the nucleus. (B) Center of the confluent spot culture. The arrowheads indicate examples of cells with punctate nuclear LANA fluorescence. (C) Confluent Vero cell cultures were scratch-wounded to stimulate cell migration and proliferation. After a 24-h migration period, the cultures were infected with KSHV. At 24 hpi, the cultures were fixed and stained as described above. The white line marks the edge of the confluent culture following wounding and represents the start point of cell migration. The arrows indicate cells in the migration area with diffuse LANA fluorescence in the cytoplasm (marked with a yellow dot in the nucleus). Adjacent cells and cells in the confluent area had punctate LANA fluorescence in the nucleus. (D) The positions of cells with cytoplasmic LANA relative to the wound edge are shown without the blue and green channels. Scale bars: A and B, 20 μm; C and D, 100 μm.

FIG 4

Cytoplasmic LANA correlates with the lytic cycle marker ORF59 after primary KSHV infection. (A to C) Subconfluent Vero cell cultures were infected with KSHV and fixed at 24 hpi. Cells were sequentially stained with mouse anti-ORF59 (red) and rat anti-LANA (LN53) (green) using TSA enhancement, as described in Materials and Methods. The cell nuclei were stained with TO-PRO-3 (blue). (A) Overlay of ORF59, LANA, and TO-PRO-3 fluorescence (red, green, and blue channels). The arrows point to cells with cytoplasmic LANA. All cells with cytoplasmic LANA also expressed nuclear ORF 59. (B) ORF59 fluorescence in panel A (red channel alone). (C) High-resolution image showing both nuclear ORF59 and cytoplasmic LANA fluorescence. (D and E) Confluent Vero cultures were scratch wounded, and cells on the edge of the wound migrated into the open space for 24 h. Cell cultures were infected with KSHV for 24 h, fixed, and sequentially stained for ORF59 and LANA, as described above. (D) Overlay of ORF59, LANA, and TO-PRO-3 fluorescence (red, green, and blue channels). The arrows point to cells with nuclear ORF59 and cytoplasmic LANA fluorescence in the cell migration area. (E) Overlay of ORF59 and TO-PRO-3 fluorescence (red and blue channels). The arrows point to cells with nuclear ORF59 fluorescence. The line demarcating the confluent and cell migration regions is shown. Scale bars: A and B, 20 μm; C and D, 50 μm.

FIG 5

Cytoplasmic LANA correlates with ORF59 expression after reactivation of latent KSHV infections. (A) BCBL-1 cells carrying a long-term latent KSHV infection were treated with TPA (20 ng/ml) for 48 h to reactivate KSHV. The cells were fixed and sequentially stained for ORF59, LANA, and cell nuclei, as in Fig. 4. Cells with nuclear ORF59 and cytoplasmic LANA fluorescence (arrows) or punctate nuclear LANA fluorescence (arrowheads) are indicated. (B) Profile of the fluorescence staining across a BCBL-1 cell with cytoplasmic LANA (green-Ch3) and nuclear ORF59 (red-Ch2) in panel A, as well as TO-PRO-3 staining of DNA (blue-Ch1). Scale bars, 20 μm. (C) HPV-immortalized DMVEC cells were infected with KSHV and cultured for 35 days. The cells were fixed and stained sequentially for ORF59 (green) and LANA (orange). The ORF59 and LANA fluorescence are overlaid on a phase-contrast image. The cell with nuclear ORF59 (large arrow, green) also expressed cytoplasmic LANA (small arrow, orange). Punctate LANA and ORF59 fluorescence colocalized (yellow) in the nucleus of this cell. Punctate LANA fluorescence was observed alone in cells lacking ORF59 fluorescence (arrowheads).

FIG 6

Cytoplasmic LANA correlates with ORF59 expression after primary infection of differentiated gingival epithelial cells. (A) Primary HOK cultures were induced to differentiate using high calcium (0.15 mM) for 24 h. Cell nuclei were labeled with TO-PRO-3 (blue). The cell borders are indicated with dotted lines distinguishing large-diameter cells (40 to 70 μm), which have been previously shown to express markers of keratinocyte differentiation (labeled with an asterisk) (87) and small cells (15 to 25 μm) showing an undifferentiated phenotype (unlabeled). (B and C) A differentiated HOK culture was infected with KSHV and, at 24 hpi, the culture was fixed and sequentially stained for ORF59, LANA, and cell nuclei, as described in Fig. 4. Panel B shows an xy projection. The arrow identifies a cell with diffuse LANA fluorescence (green) in the cytoplasm and ORF59 fluorescence (red) in the nucleus. The arrowheads indicate cells with punctate LANA fluorescence in the nuclei lacking ORF59 fluorescence. Panel C is an xz projection showing that the cell with both ORF59 and cytoplasmic LANA fluorescence has migrated onto a basal layer of cells exhibiting punctate nuclear LANA fluorescence. Scale bars, 20 μm.

FIG 7

Overexpression of ORF50 RTA in KSHV-infected Vero cells induces cytoplasmic LANA and ORF59 expression. Vero cells were infected with KSHV for 24 h and then were either mock infected (A and B) or superinfected with BacK50 recombinant baculovirus expressing the KSHV ORF50 RTA (6 h) to activate KSHV replication (C and D); the cells were then cultured for an additional 24 h. The cells were fixed and sequentially stained for ORF59, LANA, and cell nuclei, as described in Fig. 4. Panels A and C show the TO-PRO-3 (blue) and ORF59 (red) fluorescence; panels B and D show the TO-PRO-3 (blue) and LANA (green) fluorescence, with the ORF59-positive nuclei rendered as red spheres. The arrowheads indicate cells with cytoplasmic LANA fluorescence and no ORF59 fluorescence. (E) Cell populations expressing cytoplasmic LANA and nuclear ORF59 were quantitated (number of cells analyzed = 375 [KSHV] and 339 [KSHV+BacK50]). Scale bar, 50 μm.

FIG 8

ORF50 RTA or sodium butyrate fails to induce cytoplasmic LANA or ORF59 expression in AGS.219 gastric epithelial cells latently infected with KSHV. The AGS.219 cell line latently infected with recombinant KSHV.219 was either left untreated (A to C) or activated with sodium butyrate (D to F) or BacK50 (G to I) for 6 h. The cells were cultured for an additional 24 h, fixed, and incubated with antibodies to LANA (A, D, and G), ORF59 (B, E, and H), or ORF50 (C, F, and I), followed by TSA enhancement. High levels of punctate, nuclear LANA were detected in both untreated (A) and activated (D and G) cells, but no cytoplasmic LANA was observed. No ORF59 or ORF50 fluorescence was detected in the untreated cultures (B and C, respectively) or the butyrate-activated cultures (E and F, respectively). The BacK50-treated cultured showed ORF50 fluorescence (I) but no ORF59 fluorescence (H). Scale bar, 20 μm.

FIG 9

Cytoplasmic LANA is sensitive to mild detergent extraction. Vero cells were infected with KSHV and induced to lytically replicate by superinfection with BacK50 as in Fig. 7 and were either left untreated (A to C) or briefly treated with 0.1% NP-40 (D to F), or the ProteoExtract fraction I extraction buffer (G to I) to solubilize cytoplasmic proteins. The cells were fixed and sequentially stained for LANA, ORF59, and cell nuclei, as in Fig. 4. In the untreated culture (A to C), the arrowheads indicate one of many cells with cytoplasmic LANA (A) and nuclear ORF59 fluorescence (B), merged in panel C. The arrow indicates one of many cells with punctate nuclear LANA (A) and no ORF59 fluorescence (B). After extraction with 0.1% NP-40 or fraction I buffer, the arrowhead indicates one of many cells, in which cytoplasmic LANA was removed (D and G), yet the nuclear ORF59 and LANA remain (E and H; merged in panels F and I). The arrows indicate cells with punctate nuclear LANA (D and G) or no nuclear ORF59 (E and H). Scale bar, 50 μm.

FIG 10

LANA isoforms migrating with high and low relative molecular masses are isolated from the cytosolic fraction of KSHV-infected Vero cells treated with BacK50. Vero cells undergoing a primary KSHV infection (24 hpi) (A to C) and puromycin-selected AGS.219 cells undergoing a long-term latent infection (D) were superinfected with BacK50, as described in Fig. 7. The ProteoExtract fractionation system was used to purify subcellular fractions from the cytosol (lanes I), membrane/organelles (lanes II), nucleus (lanes III), and cytoskeleton (lanes IV) of the infected cells. These fractions were screened by Western blotting for LANA isoforms (A and D) and nuclear lamin β1 (C), as described in Materials and Methods. Dots indicate multiple LANA isoforms ranging from 118- to 230-kDa relative molecular masses in the cytosol fraction I of the induced Vero cells (A) and in the nuclear and cytoskeleton fractions III and IV of the latent AGS.219 cells (D). An asterisk indicates nuclear lamin β1 isoforms in the nuclear and cytoskeleton fractions III and IV of the induced Vero cells (C). (B) Proteins in the cytosol fraction I of the infected Vero cells were further purified on a LANA affinity column for mass spectrometry analysis and screened by Western blotting for LANA isoforms. M, molecular mass standards.

FIG 11

Full-length LANA isoforms containing the N-terminal NLS motif are identified in the cytosolic fraction of BacK50-induced KSHV-infected Vero cells. (A) The LANA affinity-purified proteins from the cytosol fraction I of the BacK50-induced, KSHV-infected Vero cells (Fig. 10B) were separated by SDS-gel electrophoresis, and gel regions 1 and 2 containing the different LANA isoforms were analyzed separately by mass spectrometry. (B) A schematic of the BCBL-1 LANA structure (1,117 aa) is shown with N-terminal domain containing the major bipartite NLS (58) and the C-terminal domain separated by a large region of different repeated motifs. The trypsin peptides identified by mass spectrometry in the gel regions 1 and 2 are labeled A-U and are mapped onto the structure. Peptide derivatives are labeled with a prime designation (′) and peptides with modifications are indicated with superscript letter (m, methylation; p, phosphorylation). The number of peptide spectrum matches identified in multiple spectrometry analyses is shown below each peptide. No peptides were detected in the “edd,” “deqq,” and “eqel” repeat regions, which lack obvious trypsin cleavage sites. (C) The positions and sequences of the peptides matching the ORF73 LANA sequence in the BCBL-1 strain of KSHV (NCBI accession no. ADQ57959) used for infection are shown, with the positions of modified amino acids. The confidence level of the spectral calls is indicated (H, high; M, medium; L, low).