LOCALIZATION OF HEPATITIS-DELTA VIRUS-RNA IN THE NUCLEUS OF HUMAN-CELLS

Hepatitis delta virus (HDV) is a human pathogen that can greatly incre ase the severity of liver damage caused by an hepatitis B infection. H DV contains a circular, single-stranded RNA genome that encodes a uniq ue protein, the delta antigen. Two forms of the delta antigen, delta A g-S and delta Ag-L, are derived from a single open reading frame by RN A editing. Here we analyze the subcellular distribution of HDV RNA and its spatial relationship to known intranuclear structures. The human hepatoma cell line Huh7 was stably transfected with wild-type HDV cDNA and the viral RNAs were localized by in situ hybridization and fluore scence confocal microscopy. HDV RNA is detected throughout the nucleop lasm, with additional concentration in focal structures closely associ ated with nuclear speckles or clusters of interchromatin granules. Bot h the smaller form of the delta antigen (delta Ag-S), which is require d for HDV genomic replication, and the larger form of the delta antige n (delta Ag-L), which represses replication, co-localize with delta RN A throughout the nucleoplasm and in the foci. However, the foci do not incorporate bromo-UTP and do not concentrate either RNA polymerase II or cleavage and polyadenylation factors required for viral RNA synthe sis and 3' end processing, respectively. Thus, it is unlikely that the delta foci represent major sites of viral transcription or replicatio n. In conclusion, the data show that viral RNA-protein complexes accum ulate in structures closely associated with interchromatin granules, a subnuclear domain highly enriched in small nuclear ribonucleoproteins , poly(A(+)) RNA, and RNA splicing protein factors. This implies a spe cific compartmentalization of ribonucleoproteins in the nucleus.