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. 2000 Jun;74(11):5329-36.
doi: 10.1128/jvi.74.11.5329-5336.2000.

Hepatitis delta virus replication generates complexes of large hepatitis delta antigen and antigenomic RNA that affiliate with and alter nuclear domain 10

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Hepatitis delta virus replication generates complexes of large hepatitis delta antigen and antigenomic RNA that affiliate with and alter nuclear domain 10

P Bell et al. J Virol. 2000 Jun.

Abstract

Hepatitis delta virus (HDV), a single-stranded RNA virus, bears a single coding region whose product, the hepatitis delta antigen (HDAg), is expressed in two isoforms, small (S-HDAg) and large (L-HDAg). S-HDAg is required for replication of HDV, while L-HDAg inhibits viral replication and is required for the envelopment of the HDV genomic RNA by hepatitis B virus proteins. Here we have examined the spatial distribution of HDV RNA and proteins in infected nuclei, with particular reference to specific nuclear domains. We found that L-HDAg was aggregated in specific nuclear domains and that over half of these domains were localized beside nuclear domain 10 (ND10). At later times, ND10-associated proteins like PML were found in larger HDAg complexes that had developed into apparently hollow spheres. In these larger complexes, PML was found chiefly in the rims of the spheres, while the known ND10 components Sp100, Daxx, and NDP55 were found in the centers of the spheres. Thus, ND10 proteins that normally are closely linked separate within HDAg-associated complexes. Viral RNA of antigenomic polarity, whether expressed from genomic RNA or directly from introduced plasmids, colocalizes with L-HDAg and the transcriptional repressor PML. In contrast, HDV genomic RNA was distributed more uniformly throughout the nucleus. These results suggest that different host protein complexes may assemble on viral RNA strands of different polarities, and they also suggest that this RNA virus, like DNA viruses, can alter the distribution of ND10-associated proteins. The fact that viral components specifically linked to repression of replication can associate with one of the ND10-associated proteins (PML) raises the possibility that this host protein may play a role in the regulation of HDV RNA synthesis.

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Figures

FIG. 1
FIG. 1
Immunohistochemistry and in situ hybridization 4 days after HDV transfection of HEp-2 cells. The upper corners of each image show the color used for the respective protein or RNA: green, fluorescein; red, Texas red; blue, Hoechst stain for DNA. HDA denotes L-HDAg. (A) Single nucleus shown double labeled for DNA and L-HDAg. L-HDAg (top) is situated over an area devoid of DNA (see the same nucleus at the bottom). (B) Two nuclei double labeled for L-HDAg and SC35, showing that L-HDAg occupies a different space than SC35. (C) Mitotic cell labeled for SC35 and L-HDAg. SC35 is dispersed but excluded from chromosomes in the metaphase plate (vertical empty space in the cell). The L-HDAg aggregates are retained through mitosis. (D) Single nucleus double labeled for L-HDAg and PML, showing that many L-HDAg accumulations are found beside ND10. (E) Double-labeled single nucleus where Bcl6 aggregates do not have substantial association with ND10. (F) Double-labeled single nucleus showing that PML and L-HDAg occupy the rims of apparently hollow structures. (G) Single nucleus producing L-HDAg. L-HDAg and Sp100 labeling indicates that Sp100 is located inside the L-HDAg spheres. (H) Low magnification of cluster of cells producing L-HDAg, demonstrating that transfected cells formed a clone over 4 days and that Daxx locates beside L-HDAg. (I) Higher magnification of the same preparation as in panel G, showing two nuclei. In the lower one, Daxx is located in the inside of L-HDAg spheres and in small dots on their outsides. (J) SUMO-1 and L-HDAg labeling showing that SUMO-1 is present throughout the L-HDAg spheres. (K) In situ hybridization showing that the dominant distribution of genomic RNA is diffuse and not associated with ND10 4 days after transfection (lower cell; the upper cell is untransfected). (L) A small number of cells have some genomic RNA aggregates associated with ND10, as indicated by Sp100, but genomic RNA does not colocalize with ND10. (M) In situ hybridization to label antigenomic RNA and L-HDAg. The components colocalize. (N) Staining by in situ hybridization of antigenomic RNA and immunolabeling of Sp100. Sp100 is found in the center of the antigenomic RNA sphere. (O) Location of an as-yet-uncharacterized ND10-associated protein (ND10P) inside the antigenomic RNA sphere 4 days after transfection. (P) In situ hybridization to label antigenomic RNA transcribed directly from the transfected plasmid and L-HDAg. Antigenomic RNA also associates with L-HDAg if transcribed from DNA.

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