H-1 parvovirus-associated replication bodies: a distinct virus-induced nuclear structure

Abstract

We have identified a nuclear structure that is induced after infection with the autonomous parvovirus H-1. Using fluorescence microscopy, we observed that the major nonstructural protein (NS1) of H-1 virus which is essential for viral DNA amplification colocalized with virus-specific DNA sequences and sites of ongoing viral DNA replication in distinct nuclear bodies which we designated H-1 parvovirus-associated replication bodies (H-1 PAR-bodies). In addition, two cellular proteins were shown to accumulate in H1 PAR-bodies: (i) the proliferating cell nuclear antigen (PCNA) which is essential for chromosomal and parvoviral replication and (ii) the NS1-interacting small glutamine-rich TPR-containing protein (SGT), suggesting a role for the latter in parvoviral replication and/or gene expression. Since many DNA viruses target preexisting nuclear structures, known as PML-bodies, for viral replication and gene expression, we have determined the localization of H-1 PAR- and PML-bodies by double-fluorescence labeling and confocal microscopy and found them to be spatially unrelated. Furthermore, H-1 PAR-bodies did not colocalize with other prominent nuclear structures such as nucleoli, coiled bodies, and speckled domains. Electron microscopy analysis revealed that NS1, as detected by indirect immunogold labeling, was localized in ring-shaped electron-dense nuclear structures corresponding in size and frequency to H-1 PAR-bodies. These structures were also clearly visible without immunogold labeling and could be detected only in infected cells. Our results suggest that H-1 virus does not target known nuclear bodies for DNA replication but rather induces the formation of a novel structure in the nucleus of infected cells.

FIG. 1

NS1 localizes in the nucleoplasm, accumulates in nuclear bodies, and is absent from nucleoli. Immunofluorescence experiments were performed with synchronized and H-1-infected NBE cells at 12 h (a) and 16 h (b) after release of the aphidicolin block. Panels represent confocal sections showing NS1 immunolocalized with the monoclonal antibody 3D9 and detected via an FITC-conjugated secondary antibody. Bar, 20 μm in both panels.

FIG. 2

H-1 sequences colocalize with NS1 in nuclear bodies. Synchronized and infected NBE cells were simultaneously subjected to immunolocalization using the anti-NS1 polyclonal serum SP8 and FISH with the biotinylated probe pSR19. NS1 was detected via an FITC-conjugated secondary antibody (a), and H-1 genomes were visualized using streptavidin-Cy3 (b). The equivalence in size and shape of the signals present in both channels of one typical confocal section indicate a complete colocalization of NS1 and H-1 genomes. Bar, 20 μm in all panels.

FIG. 3

Parvoviral DNA replication colocalizes with NS1 in nuclear bodies. NS1 was localized via the polyclonal antiserum SP8 and an FITC-conjugated secondary antibody (a). Replication was monitored by incorporation of BrdU and indirect immunofluorescence using a TRITC-conjugated secondary antibody (b). The cell in the center of the confocal section (arrowhead) exhibits a colocalization of NS1 and viral DNA replication. Arrows point to cells in which chromosomal replication is still in process. Bar, 20 μm.

FIG. 4

The essential replication factor PCNA accumulates with NS1 in H-1 PAR-bodies. Double immunofluorescence was performed with synchronized and H-1-infected NBE cells 14 h after release from aphidicolin block. NS1 was immunolocalized with the anti-NS1 monoclonal antibody 3D9 and an FITC-conjugated secondary antibody (a), and PCNA was localized with a polyclonal anti-PCNA antibody and a TRITC-conjugated secondary antibody (b). Confocal images of both channels from the same confocal plane clearly indicate complete colocalization of the signals. Bar, 20 μm.

FIG. 5

NS1 and the cellular NS1-interacting protein SGT colocalize in H-1 PAR-bodies. Double immunofluorescence was performed with synchronized and H-1-infected NBE cells 14 h after release from aphidicolin block. NS1 detected with FITC results in a green signal (a). SGT was immunolocalized with the polyclonal anti-SGT serum AC1.2 and was stained using a TRITC-conjugated secondary antibody (b). (c) Merged confocal images of panels a and b, showing the colocalization of fractions of SGT and NS1. Bar, 20 μm.

FIG. 6

H-1 PAR-bodies form a distinct nuclear compartment. A set of different nuclear bodies were colocalized with NS1 in infected NBE cells 14 h after release from aphidicolin block. Shown are PML bodies recognized by an anti-PML antibody (a and c), coiled bodies recognized by an anti-p80/coilin antibody (d and f), speckled domains recognized by an anti-SC-35 antibody (g and i), and nucleoli recognized by an anti-No38/B23 antibody (j and l). (b, e, h, and k) Immunolocalization of NS1 in the same confocal plain as shown in the left column, detected with FITC. Overlays of the channels from the left and center columns (c, f, i, and l) exhibit differential staining of red and green signals, providing evidence for spatial separation of H-1 PAR-bodies from all nuclear bodies investigated in this study. Bars: a, 15 μm; d, g, and j, 20 μm.

FIG. 7

Electron micrographs of ultrathin sections through infected NBE cells showing the ultrastructure of H-1 PAR-bodies. (a and b) Immunogold localization of NS1 with the monoclonal antibody 3D9 (a) and the polyclonal antibody SP8 (b) to electron dense structures identified as H-1 PAR-bodies; (c and d) ultrastructure of H-1 PAR-bodies. Bars: 0.5 μm (a and c), 1 μm (b), and 0.1 μm (d and e).