Adenovirus replication and transcription sites are spatially separated in the nucleus of infected cells.

Abstract

We have visualized the intranuclear topography of adenovirus replication and transcription in infected HeLa cells. The results show that viral DNA replication occurs in multiple foci that are highly organized in the nucleoplasm. Pulse‐chase experiments indicate that newly synthesized viral double‐stranded DNA molecules are displaced from the replication foci and spread throughout the nucleoplasm, while the single‐stranded DNA replication intermediates accumulate in adjacent sites. Double‐labelling experiments and confocal microscopy show that replication occurs in foci localized at the periphery of the sites where single‐stranded DNA accumulates. The simultaneous visualization of viral replication and transcription reveals that the sites of transcription are predominantly separated from the sites of replication. Transcription is detected adjacent to the replication foci and extends around the sites of single‐stranded DNA accumulation. These data indicate that newly synthesized double‐stranded DNA molecules are displaced from the replication foci and spread in the surrounding nucleoplasm, where they are used as templates for transcription. Splicing snRNPs are shown to co‐localize with the sites of transcription and to be excluded from the sites of replication. This provides evidence that splicing of viral RNAs occurs co‐transcriptionally and that the sites of viral DNA replication are spatially distinct from the sites of RNA transcription and processing.

Spliced exons of adenovirus late RNAs colocalize with snRNP in a specific nuclear domain.

“…4 h). We and others have previously detected ad RNA at the site of transcription in intermediate phase cells (Pombo et al, 1994) and in cells limited to the early phase of gene expression (Zhang et al, 1994). Here we show that in late phase adinfected ceils, the interchromatin granule clusters that contain splicing factors also accumulate spliced RNA from the MLTU.…”

“…Thus, RNA that has undergone posttranscriptional processing events of polyadenylation and/or splicing accumulates in a specific nuclear structure during the late phase of viral gene expression. Although interchromatin granules are known to contain RNA in both infected and uninfected cells, the available data suggests that this structure is not a location for viral or cellular transcription (Pombo et al, 1994;Puvion-Dutilleul and Puvion, 1991;Fakan, 1994). Rather, RNA accumulates in the interchromatin granules posttranscriptionally.…”

“…In situ hybridizations and detection of biotin-labeled probes were done essentially as described (Pombo et al, 1994). However, it was necessary to adjust the hybridization and washing conditions for each probe to obtain the least background staining in uninfected cells.…”

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

“…In addition to RNA polymerase II, other host cellular factors are thought to be implicated in the HDV life cycle+ These include the polyadenylation machinery Hsieh & Taylor, 1991) and the cellular double-stranded RNA-specific adenosine deaminase (dsRAD/DRADA/ADAR1), which has been implicated in the editing of HDV RNA (Polson et al+, 1996)+ We therefore asked whether HDV induces any major reorganization of components of the polyadenylation machinery or ADAR1 in the nucleus of Huh7-D12 cells expressing dAg+ Because adenovirus requires the host cell cleavage and polyadenylation machinery for the 39 end processing of its mRNAs, we first studied the effect of this virus on the distribution of poly(A)-polymerase (PAP), cleavage and polyadenylation specificity factor (CPSF), and poly(A) binding protein II (PABII)+ When adenovirusinfected HeLa cells are probed with antibodies directed against these factors, the nuclear staining pattern changes significantly, depending on the stage of infection+ In noninfected cells, PAP and CPSF are distributed diffusely throughout the nucleoplasm with additional concentration of CPSF in "cleavage bodies" (Schul et al+, 1996), whereas polyA-binding protein is concentrated predominantly in clusters of interchromatin granules, as described previously (Krause et al+, 1994) (data not shown)+ During the early phase of infection (i+e+, before the onset of major viral DNA replication, which occurs at ;8 h postinfection), the staining pattern produced by each antibody is similar to that observed in noninfected cells (data not shown)+ Following the onset of viral replication (14-18 h postinfection), the normal nuclear architecture is grossly changed and the polyadenylation machinery reorganizes into ring-like structures (Fig+ 7A,B,C)+ Double-labeling experiments confirm that these rings surround the sites of viral DNA, as demonstrated previously for splicing snRNPs and the splicing factor U2AF 65 (Pombo et al+, 1994;GamaCarvalho et al+, 1997)+ In Huh7 cells, the distribution of PAP, CPSF, and PAB II is similar to that of noninfected HeLa cells, i+e+, PAP and CPSF are distributed diffusely throughout the nucleoplasm with additional concentration of CPSF in cleavage bodies, whereas PAB II is detected predominantly in nuclear speckles+ An identical distribution pattern is observed in Huh7-D12 cells and neither of these factors is detected in the viral foci (Fig+ 7D,G; E,H; F,I)+ Thus, in contrast with adenovirus, HDV does not alter the subnuclear distribution of the polyadenylation machinery+ In order to analyze the intranuclear distribution of ADAR1, immunofluorescence was performed using rabbit polyclonal antibodies raised and affinity purified against a recombinantly expressed ADAR1 fragment comprising amino acids 167-359 (S+ Krause, M+A+ O'Connell, & W+ Keller, unpubl+ data)+ As depicted in Figure 8, ADAR1 is detected throughout the nucleoplasm of Huh7 cells (Fig+ 8A) and, in Huh7-D12, the distribution remains largely unaltered (Fig+ 8B,C)+ Immunofluorescence was also performed using antibodies specific for ADAR2 (O'Connell et al+, 1997) and, similarly, no significant redistribution of this enzyme was observed in nuclei expressing HDV (data not shown)+ In summary, these data do not support the view that the delta foci represent major sites of HDV RNA synthesis or processing in the nucleus+…”

“…The delta foci do not correspond to sites of viral RNA synthesis or processing Previous work from several laboratories has demonstrated that viruses often subvert the host nuclear organization+ In particular, adenoviruses have been shown to recruit replication and splicing factors to the sites of active viral replication and transcription (Jiménez-Garcia & Spector, 1993;Pombo et al+, 1994)+ Here we have adapted and extended this approach to investigate the sites of active HDV RNA synthesis and processing in the nucleus of Huh7-D12 cells+ Br-UTP was microinjected into the cells, allowed to become incorporated in nascent RNA chains, and detected using an antibody that specifically reacts with brominated nucleotides+ The sites of RNA synthesis are visualized as a multitude of fine dots dispersed throughout the nucleoplasm, and no labeling is detected in the delta foci (Fig+ 6A,B, arrows)+ In agreement with this observation, we also do not detect RNA polymerase II in the viral foci (Fig+ 6C,D, arrow)+ Thus, it is unlikely that the foci correspond to sites of active HDV RNA synthesis+ This view is also consistent with the finding that the foci contain dAg-L (Fig+ 5D,E,F), which is a strong repressor of HDV replication+…”