Intracellular localization of hepatitis delta virus proteins in the presence and absence of viral RNA accumulation

Abstract

Hepatitis delta virus (HDV) encodes one protein, hepatitis delta antigen (deltaAg), a 195-amino-acid RNA binding protein essential for the accumulation of HDV RNA-directed RNA transcripts. It has been accepted that deltaAg localizes predominantly to the nucleolus in the absence of HDV genome replication while in the presence of replication, deltaAg facilitates HDV RNA transport to the nucleoplasm and helps redirect host RNA polymerase II (Pol II) to achieve transcription and accumulation of processed HDV RNA species. This study used immunostaining and confocal microscopy to evaluate factors controlling the localization of deltaAg in the presence and absence of replicating and nonreplicating HDV RNAs. When deltaAg was expressed in the absence of full-length HDV RNAs, it colocalized with nucleolin, a predominant nucleolar protein. With time, or more quickly after induced cell stress, there was a redistribution of both deltaAg and nucleolin to the nucleoplasm. Following expression of nonreplicating HDV RNAs, deltaAg moved to the nucleoplasm, but nucleolin was unchanged. When deltaAg was expressed along with replicating HDV RNA, it was found predominantly in the nucleoplasm along with Pol II. This localization was insensitive to inhibitors of HDV replication, suggesting that the majority of deltaAg in the nucleoplasm reflects ribonucleoprotein accumulation rather than ongoing transcription. An additional approach was to reevaluate several forms of deltaAg altered at specific locations considered to be essential for protein function. These studies provide evidence that deltaAg does not interact directly with either Pol II or nucleolin and that forms of deltaAg which support replication are also capable of prior nucleolar transit.

FIG. 1.

δAg distribution during induction of 293-δAg and 293-HDV cells. Cells were induced by the addition of TET and studied at 16 and 40 h by immunostaining and confocal microscopy. Detection of δAg is indicated in red while detection of nucleolin or Pol II (as indicated) is in green; DAPI counterstaining is in blue.

FIG. 2.

Conditions of cell stress cause relocalization of δAg and nucleolin in 293-δAg cells. At 16 h after TET induction, cells were either untreated (A) or subjected to brief treatments (B to E) and then studied by immunostaining to detect δAg (red) or nucleolin (green); DAPI counterstaining is also shown (blue). The treatments were as follows: 10 ng/ml actinomycin for 1 h (actino; B), actinomycin followed by 2 h of recovery in the absence of actinomycin (C), 50 μM DRB for 1 h (D), and DRB followed by 2 h of recovery (E).

FIG. 3.

Movement of δAg from nucleoli to nucleoplasm following expression of nonreplicating HDV RNA sequences. Huh7 cells were transfected with a plasmid (pDL444) to express δAg, without or with a plasmid to express a chimera of HDV RNA (pDL482). After 24 h, immunostaining was used to detect δAg (red) and nucleolin (green). Panel A shows the pattern detected when cells expressed δAg only. Panels B and C show two new patterns detected when cells also expressed the nonreplicating HDV RNA. Quantitation for the patterns is summarized in Table 2.

FIG. 4.

Effect of amanitin and HMBA treatments on δAg distribution for induced 293-HDV cells. Panels A and B show 293-HDV cells at 16 h after TET induction, without and with an additional incubation with amanitin (10 μg/ml for 45 min). Panel C shows 293-HDV cells that were induced for 22 h in the presence of added HMBA (3 mM). Immunostaining was used to detect δAg (red) and Pol II or nucleolin (green); DAPI counterstaining is shown in blue.

FIG. 5.

Features of δAg. Shown is the localization on δAg of what are referred to as the CCD, core NLS, and the bipartite RBD. Also indicated are representations of the changes or deletions of five altered forms of δAg. These forms are as used in Fig. 6 and have been described previously (23, 28). WT, wild type.

FIG. 6.

Expression of altered forms of δAg in 293 cells. Plasmids encoding the wild type (WT; controls) and five altered forms, as indicated at left, of δAg were transfected into 293 cells. At 16 h cells were examined by immunostaining to detect δAg (red) and either Pol II or SC35 (green); DAPI counterstaining is shown in blue.