HHV-8 encoded LANA-1 alters the higher organization of the cell nucleus

Abstract

The latency-associated nuclear antigen (LANA-1) of Human Herpes Virus 8 (HHV-8), alternatively called Kaposi Sarcoma Herpes Virus (KSHV) is constitutively expressed in all HHV-8 infected cells. LANA-1 accumulates in well-defined foci that co-localize with the viral episomes. We have previously shown that these foci are tightly associated with the borders of heterochromatin 1. We have also shown that exogenously expressed LANA-1 causes an extensive re-organization of Hoechst 33248 DNA staining patterns of the nuclei in non-HHV-8 infected cells 2. Here we show that this effect includes the release of the bulk of DNA from heterochromatic areas, in both human and mouse cells, without affecting the overall levels of heterochromatin associated histone H3 lysine 9 tri-methylation (3MK9H3). The release of DNA from the heterochromatic chromocenters in LANA-1 transfected mouse cells co-incides with the dispersion of the chromocenter associated methylcytosin binding protein 2 (MECP2). The localization of 3MK9H3 to the remnants of the chromocenters remains unaltered. Moreover, exogeneously expressed LANA-1 leads to the relocation of the chromocenters to the nuclear periphery, indicating extensive changes in the positioning of the chromosomal domains in the LANA-1 harboring interphase nucleus. Using a series of deletion mutants we have shown that the chromatin rearranging effects of LANA-1 require the presence of a short (57 amino acid) region that is located immediately upstream of the internal acidic repeats. This sequence lies within the previously mapped binding site to histone methyltransferase SUV39H1. We suggest that the highly concentrated LANA-1, anchored to the host genome in the nuclear foci of latently infected cells and replicated through each cell generation, may function as "epigenetic modifier". The induction of histone modification in adjacent host genes may lead to altered gene expression, thereby contributing to the viral oncogenesis.

Figure 1

Comparison of expression levels of virus encoded and exogeneously introduced LANA-1 in the nucleus of HHV-8 positive BCBL-1 body cavity lymphoma (left) and HHV-8 negative MCF7 breast carcinoma cell (middle). Immunofluorescence staining (green) using human anti-LANA-1 serum detected by FITC conjugated mouse anti-human immunoglobulins. The images are Z axis projections of stacks of 10 optical sections, 0.5 micrometer apart, captured from identically stained and processed nuclei using an automated wide field fluorescence microscope. The counterstaining of BCBL-1 DNA with Hoechst 33258 (blue) is shown for easier orientation. The 3D projection of the plot of the measured staining intensity (right) illustrates that the nuclear foci of latently infected cells contain comparable amount of LANA-1 to the transiently transfected ones.

Figure 2

LANA-1 (green) dissolves DNA (blue) from perinucleolar heterochromatin in transfected MCF7 cells. Increasing amount of transfected LANA-1 (compare top to the middle or bottom panels) leads to the elimination of the DNA staining (blue) in the perinucleolar heterochromatic rings. Adjacent non-transfected cells serve as controls. Right panel shows magnified pictures of chromatin organization of the selected nuclear areas selected by red border boxes in the left panel. The middle and bottom panels represent the two distinct types of chromatin effects: The smoothing out of the chromatin staining (middle panel) versus induction of newly condensed chromatin cords (bottom panel). Importantly both changes lead to the elimination of perinucleolar heterochromatin.

Figure 3

Dissolution of DNA from perinucleolar heterochromatin is not accompanied by the release of trimethylated lysine 9 histone H3 (3MK9H3) – green immunofluorescence staining in LANA-1 transfected cells (red). 3MK9H3 staining clearly identifies perinucleolar areas (white arrows) with diminished heterochromatic DNA staining in the transfected cells.

Figure 4

LANA-1 expression does not effect 3MK9H3 (green) levels as measured using extended field laser scanning microscopy (EFLCM) that automatically captured 300 adjacent fields as a mosaic of Z projected images of 12 optical sections each. The total fluorescence intensity measurement for 3MK9H3, DNA (blue) and LANA-1 (red) of the individual nuclei is plotted in the order of increasing amount of LANA-1. The amount of 3MK9H3 staining is also compared on population levels of LANA-1 positive and negative nuclei on a box chart.

Figure 5

Effect of LANA-1 on mouse pericentromeric heterochromatin organized as chromocenters. Increasing amount of LANA-1 (green) leads to gradual disappearance of chromocenters in the transfected nuclei of murine L-cells. DNA stained with Hoechst 33258 (blue).

Figure 6

High resolution comparison of LANA-1 (red) positive L-cell nucleus with an adjacent non-transfected cell in a single optical section that slices both nuclei in the middle level. The intensity plot is recorded along the white line and demonstrate a massive release of the bulk DNA (blue) from the chromocenters in the transfected cell without effecting the 3MK9H3 (green) levels in the remnants of the chromocenters (white staple on the right side of the line plot).

Figure 7

Although single optical sections (left) of LANA-1-transfected nuclei may suggest extensive decrease in 3MK9H3 staining, reconstitution of the summarized staining signal from the entire stack of 15 images (right) shows no detectable alteration in the total levels of 3MK9H3 in mouse L cell nuclei. (LANA-1 – red, 3MK9H3 – green, DNA – blue).

Figure 8

Careful examination of the series of sixteen individual optical sections along the Z axis reveals that LANA-1 induces a major rearrangement in the positioning of 3MK9H3 positive chromocenter remnants from the inside of the nucleus to the periphery of the nucleus. (LANA-1 – red, 3MK9H3 – green, DNA – blue).

Figure 9

3D reconstitution of the triple-stained nuclei shows that LANA-1 staining and the 3MK9H3 positive remnants of the heterochromatic chromocenters are non-overlapping. See the full rotational image sequences in the supplementary material [see Additional file 1]. The selected middle sections (5–9) demonstrate the extensive peripheral localization of the chromocenters. High magnification representation of a single section shows mutual avoidance of 3MK9H3 staining and LANA-1 distribution. 3D reconstitution of LANA-1 transfected nuclei also reveals well-defined nuclear areas that are devoid of LANA-1 staining. The identity and molecular content of these areas are currently unknown but they apparently include the nucleoli and the chromocentric remnants. The 3 dimensional digital cast of the LANA-1 negative areas in relation to 3mK9H3 and DNA staining is shown as image sequence in the supplementary material [see Additional file 2]. (LANA-1 – red, 3MK9H3 – green, DNA – blue).

Figure 10

Unlike 3MK9H3, the heterochromatin associated methyl cytosine binding protein 2 (MECP2) shows an extensive release from the chromocenters of LANA-1 transfected L cells both at moderate (left) and high expression levels (right). (LANA-1 – red, MECP2 – green, DNA – blue).

Figure 11

Importantly however MECP2 is absent from the LANA bodies in BCBL-1 cells that harbor latent HHV-8 genomes. (LANA-1 – red, MECP2 – green, DNA – blue).

Figure 12

Series of deletion mutants affecting both the C terminus and the central acidic repeats identify the N terminal area immediately preceding the central acidic repeats as an area that is required for the chromatin modifying effects. This area closely overlaps with the previously identified site of histone methyl transferase SUV39H1.

Figure 13

Deletion mutant (del 275–972) lacking the central acidic repeats and the adjacent N terminal region show effective targeting to the surface of heterochromatin both in human MCF7 and in mouse L-cells without any visible effect on the chromatin organization or positioning of chromocenters. (ΔLANA-1 – green, DNA – blue).

Figure 14

Chromatin dissolution effect is preserved in the deletion mutant LANA (ΔLANA-1 – green, DNA – blue). (del 332–972) as shown in four adjacent confocal sections of transfected L cell nuclei.