Stable expression of hepatitis delta virus antigen in a eukaryotic cell line

Macnaughton, Thomas B.; Gowans, Eric J.; Reinboth, Betty; Jilbert, Allison R.; Burrell, Christopher J.

doi:10.1099/0022-1317-71-6-1339

Cited by 46 publications

(24 citation statements)

References 25 publications

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“…In the present work, we analyze the subcellular distribution of HDV RNA and its spatial relationship to known intranuclear structures+ Althought early studies have localized delta antigens in the nucleolus, more recent data argue against an involvement of this subnuclear compartment in HDV infection because dAg localizes to nucleoli predominantly in the absence of genome replication (Lai et al+, 1990;MacNaughton et al+, 1990aMacNaughton et al+, , 1990bBichko & Taylor, 1996)+ Consistent with this view, we show here that HDV RNA is exclusively localized in the nucleoplasm and that no viral RNA is detected in cells containing antigens confined to the nucleolus+ Furthermore, when either the small or the large form of dAg is transiently expressed in Huh7 cells (i+e+, cells devoid of HDV genomes), both forms of the protein are frequently localized in the nucleolus+ This strongly suggests that the nucleolar accumulation of dAg is caused by protein expression in the absence of appropriate levels of HDV RNA synthesis+ In the absence of its target RNA, the delta protein may concentrate in the nucleolus due to its ability to bind RNA nonspecifically at high concentrations and to the high density of RNA present in the nucleolus+ Within the nucleoplasm, HDV RNAs and antigens appear concentrated in discrete spherical nuclear inclusions that we refer to as delta foci+ A similar distribution pattern was described previously for dAg-S and proposed to correspond to sites where HDV RNA is transcribed or processed (Bichko & Taylor, 1996)+ However, the results presented here argue against that interpretation+ In fact, the HDV foci do not concentrate either nascent RNA chains, RNA polymerase II, or components of the 39 end processing machinery+ Furthermore, the foci are highly enriched in dAg-L, which represses replication Glenn & White, 1991)+ These results, taken together with the finding that HDV RNAs are dispersed throughout the entire nucleoplasm in addition to being concentrated in the foci, strongly suggest that synthesis and processing of viral RNAs are not confined to discrete sites+ This is in agreement with several previous reports demonstrating that RNA biogenesis occurs throughout the nucleus without an obligatory requirement for compartmentalization (reviewed in Fakan, 1994;Carmo-Fonseca et al+, 1996;Spector, 1996;Singer & Green, 1997)+ If the delta foci are not sites of viral transcription or replication, what may be their role in the nucleus? Based on the data obtained by transient expression of each form of the delta antigen in the absence of viral genome replication, we conclude that dAg-S on its own has the ability to assemble into focal aggregates+ Due to its tight interaction with HDV RNA and dAg-L, dAg-S could therefore be responsible for the appearance of the delta foci+ Because HDV depends on HBV to supply the envelope proteins required for completion of packaging, secretion, and infection (see Monjardino & Lai, 1993), mammalian cell systems such as Huh7-D12, which are solely transfected with HDV cDNA, fail to export viral particles to the cytoplasm+ Thus, the foci may represent depository sites for delta RNA and antigens trapped within the nucleus+ In this regard, we are ...…”

Section: Discussionmentioning

confidence: 98%

Localization of hepatitis delta virus RNA in the nucleus of human cells

Cunha

Monjardino

Cheng

et al. 1998

RNA

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Hepatitis delta virus (HDV) is a human pathogen that can greatly increase the severity of liver damage caused by an hepatitis B infection. HDV contains a circular, single-stranded RNA genome that encodes a unique protein, the delta antigen. Two forms of the delta antigen, dAg-S and dAg-L, are derived from a single open reading frame by RNA 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 fluorescence confocal microscopy. HDV RNA is detected throughout the nucleoplasm, with additional concentration in focal structures closely associated with nuclear speckles or clusters of interchromatin granules. Both the smaller form of the delta antigen (dAg-S), which is required for HDV genomic replication, and the larger form of the delta antigen (dAg-L), which represses replication, co-localize with delta RNA 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 synthesis and 39 end processing, respectively. Thus, it is unlikely that the delta foci represent major sites of viral transcription or replication. In conclusion, the data show that viral RNA-protein complexes accumulate 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 specific compartmentalization of ribonucleoproteins in the nucleus.

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Section: Discussionmentioning

confidence: 98%

Localization of hepatitis delta virus RNA in the nucleus of human cells

Cunha

Monjardino

Cheng

et al. 1998

RNA

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“…Unlike the viroids, HDV produces a single protein, the hepatitis 8 antigen (HDAg), which is translated from an antigenomic sense mRNA (9,10). HDAg is an RNA binding protein (11)(12)(13) and is found as a mixture of 24-and 27-kDa species in both infected cells and virions (14,15). The two forms of HDAg play central roles in the HDV replication cycle.…”

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confidence: 99%

Structural requirements for RNA editing in hepatitis delta virus: evidence for a uridine-to-cytidine editing mechanism.

Casey

Bergmann

Brown

et al. 1992

Proc. Natl. Acad. Sci. U.S.A.

128

132

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Hepatitis 6 virus (HDV) nucleotide 1012 is edited from uridine to cytidine in 10-40% of the RNA gonomes during replication. This editing event is an important control point in the HDV life cycle because it results in both the packaging of viral RNA and the inhibition of HDV replication. We find that the editing event is highly specific for both the sequences neighboring nucleotide 1012 and the base-paired context of position 1012 within the unbranched rod structure of HDV RNA. Prior studies identified the base transition at nucleotide 1012 but were unable to distinguish between editing of the genomic versus the antigenomic strands [Luo, G. X., Chao, M., Hsieh, S. Y., Sureau, C., Nishikura, K. & Taylor, J. (1990) J. Virol. 64, 1021-1027. In this study, comparisons of mutations that differentiate between base pairing in genomic and antigenomic RNAs indicate that the genomic strand of HDV is the actual editing substrate. We conclude that the virus uses a uridine to cytidine editing mechanism, which is provided by the host cell.Hepatitis 6 virus (HDV) is a subviral pathogen of humans. It requires concurrent infection with hepatitis B, which provides the viral coat protein (1, 2). Compared to infection with hepatitis B virus alone, coinfection or superinfection with HDV significantly increases the risk of more severe liver disease, including fulminant hepatitis (3). The HDV genome is an -1.7-kilobase, single-stranded, circular RNA molecule, which shares some unusual features with plant viroid RNAs (reviewed in ref. 4). The circular RNA molecule possesses significant intramolecular complementarity such that 70%o of the nucleotides can form base pairs in an unbranched rod structure (5, 6). Consistent with a rolling circle replication mechanism similar to that of the plant viroid agents, monomers and multimers of both genomic and antigenomic RNAs are found in infected and transfected cells (7-9). Unlike the viroids, HDV produces a single protein, the hepatitis 8 antigen (HDAg), which is translated from an antigenomic sense mRNA (9, 10). HDAg is an RNA binding protein (11-13) and is found as a mixture of 24-and 27-kDa species in both infected cells and virions (14, 15). The two forms of HDAg play central roles in the HDV replication cycle. p24 is necessary for HDV RNA replication in transfected cells in culture (8), while p27 both inhibits replication (16, 17) and enables packaging of the HDV RNA genome (18). As shown by others (19), the heterogeneity of HDAg likely arises via an RNA editing mechanism wherein the antigenomically encoded stop codon for p24 is changed from UAG to UGG; the resultant translation product, p27, contains an additional 19 amino acids at the C terminus. Because of the circular replication cycle of HDV RNA, the base change was found in both the genomic and antigenomic RNAs (19); it was therefore unclear whether the genomic or the antigenomic RNA is the actual substrate for editing. An A-to-G transition in the antigenomic strand, possibly involving a host doublestranded RNA modifying activi...

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“…The 22K protein is probably a degraded 24K protein still retaining antigenic sites recognized by the polyclonal anti-HD serum. Several groups have presented data on HDAg-specific degradation products found in the liver (Bergmann & Gerin, 1986;Saldanha et al, 1990a), in bacteria expressing recombinant HDAg (Weiner et al, 1988) and in a hepatoma cell line (Macnaughton et al, 1990). They all found one or more degradation products which were reduced in size by 2K to 4K compared to the smaller HDAg protein found in serum.…”

Section: Discussionmentioning

confidence: 99%