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 (␦Ag), a 195-amino-acid RNA binding protein essential for the accumulation of HDV RNA-directed RNA transcripts. It has been accepted that ␦Ag localizes predominantly to the nucleolus in the absence of HDV genome replication while in the presence of replication, ␦Ag 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 ␦Ag in the presence and absence of replicating and nonreplicating HDV RNAs. When ␦Ag 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 ␦Ag and nucleolin to the nucleoplasm. Following expression of nonreplicating HDV RNAs, ␦Ag moved to the nucleoplasm, but nucleolin was unchanged. When ␦Ag 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 ␦Ag in the nucleoplasm reflects ribonucleoprotein accumulation rather than ongoing transcription. An additional approach was to reevaluate several forms of ␦Ag altered at specific locations considered to be essential for protein function. These studies provide evidence that ␦Ag does not interact directly with either Pol II or nucleolin and that forms of ␦Ag which support replication are also capable of prior nucleolar transit.Hepatitis delta virus (HDV) is a subviral agent with a singlestranded circular RNA genome. As reviewed recently (6, 14, 37) this 1,679-nucleotide RNA is replicated by RNA-directed RNA synthesis dependent upon redirection of the host RNA polymerase II (Pol II). Replication involves the production of the antigenome, an exact complement of the genome. The small delta antigen (␦Ag) is translated from a third RNA, one with the same antigenomic polarity but with a linear rather than circular conformation, of less than full-length, and possessing a 5Ј cap and a 3Ј poly(A). ␦Ag is a 195-amino-acid-long RNA binding protein that is essential in one or more ways for the accumulation of HDV RNA-directed RNA transcripts. There is a second form of ␦Ag that is 19 amino acids longer at the C terminus; this species arises due to specific RNA editing that occurs on the antigenomic RNA during replication. Unlike the small form, the large does not support HDV genome replication and is a dominant-negative inhibitor of the replication driven by the small form. However, large ␦Ag does have an essential function in that in the presence of HBV envelope proteins, it facilitates the assembly of HDV genomic RNA into new virus particles.Our previous immunostaining studies have shown that in the absence of HDV genome replication, small ␦Ag localizes predominantly to th...

Hepatitis Delta Antigen Requires a Flexible Quasi-Double-Stranded RNA Structure To Bind and Condense Hepatitis Delta Virus RNA in a Ribonucleoprotein Complex

“…In this regard, Han et al observed that, in the absence of HDV RNA, HDAg accumulated in the nucleolus of transfected cells, perhaps by association with rRNA precursors (53). It remains to be determined whether such interactions occur during infection and whether they contribute to virus replication or pathogenesis.…”

Phosphorylation of Serine 177 of the Small Hepatitis Delta Antigen Regulates Viral Antigenomic RNA Replication by Interacting with the Processive RNA Polymerase II

“…However, one previous report showed that the speckles are unlikely to be the major sites for HDV viral transcription or replication, as 5-bromouridine triphosphate (BrUTP) was not incorporated into viral RNA in nuclear speckles (17). In addition, a recent study concluded that the majority of HDAg in the nucleoplasm colocalizes with cellular RNAP II and is an indication of HDV RNP accumulation rather than of ongoing viral transcription (28). Although several lines of evidence indicate that the replication of HDV RNA is carried out by different cellular machineries, there still is much controversy about whether HDV genomic and antigenomic RNA replication take place in the same cellular compartment.…”

Vectors for co-expression of two genes in Caenorhabditis elegans

“…1-3). However, the different patterns observed between RPS-16::RFP and DsRedLD could be due to different properties of the two proteins: RPS-16 is a ribosomal protein whereas the hepatitis D large antigen is a viral protein which has previously been shown to localize at the nucleolus, SC-35 speckles, or the Golgi (Tan et al, 2004;Han et al, 2009). Furthermore, 3'-UTRs of these two constructs were also different in context: rsp-16::rfp was followed by its own 3'-UTR while DsRedLD was followed by the 3'-UTR of unc-54.…”