Transcription releases protein VII from adenovirus chromatin

The association of host histones with parvoviral DNA is poorly understood. We analyzed the chromatinization and histone acetylation of canine parvovirus DNA during infection by confocal imaging and in situ proximity ligation assay combined with chromatin immunoprecipitation and high-throughput sequencing. We found that during late infection, parvovirus replication bodies were rich in histones bearing modifications characteristic of transcriptionally active chromatin, i.e., histone H3 lysine 27 acetylation (H3K27ac). H3K27ac, in particular, was located in close proximity to the viral DNA-binding protein NS1. Importantly, our results show for the first time that in the chromatinized parvoviral genome, the two viral promoters in particular were rich in H3K27ac. Histone acetyltransferase (HAT) inhibitors efficiently interfered with the expression of viral proteins and infection progress. Altogether, our data suggest that the acetylation of histones on parvoviral DNA is essential for viral gene expression and the completion of the viral life cycle. IMPORTANCEViral DNA introduced into cell nuclei is exposed to cellular responses to foreign DNA, including chromatinization and epigenetic silencing, both of which determine the outcome of infection. How the incoming parvovirus resists cellular epigenetic downregulation of its genes is not understood. Here, the critical role of epigenetic modifications in the regulation of parvovirus infection was demonstrated. We showed for the first time that a successful parvovirus infection is characterized by the deposition of nucleosomes with active histone acetylation on the viral promoter areas. The results provide new insights into the regulation of parvoviral gene expression, which is an important aspect of the development of parvovirus-based virotherapy. N uclear chromatin is composed of DNA and histone proteins (1). The histone proteins assemble DNA into nucleosomes, the composition and spacing of which contribute to higher-order chromatin packing. The chromatin is organized into regions of less-condensed actively transcribed chromatin (euchromatin) and highly condensed transcriptionally repressed chromatin (heterochromatin). Epigenetic modifications of histone proteins have been shown to correlate with the spatial distribution of active and repressed chromatin (2, 3). The acetylation of lysine 9 or 27 of histone H3 (H3K9ac and H3K27ac, respectively) and trimethylation of lysine 4 (H3K4me3) correlate with transcriptional activity, while repressed chromatin is characterized by, e.g., trimethylation or dimethylation of the same H3 lysine residues (H3K9me3 and H3K27me3 as well as H3K9me2 and H3K27me2) (4-8).Foreign DNA introduced into mammalian cells can be recognized as a threat by the host cell. The cellular responses to the foreign DNA, such as viral DNA, include the chromatinization of the entering DNA, leading to its transcriptional silencing (9-11). How viruses resist cellular chromatinization and silencing is known for only a few viruses (9, 12, 13). These include h...

Section: Discussionmentioning

confidence: 78%

Promoter-Targeted Histone Acetylation of Chromatinized Parvoviral Genome Is Essential for the Progress of Infection

Mäntylä

Salokas

Oittinen

et al. 2016

“…Data from immunofluorescence-based experiments suggested that core VII remains stably associated with wild-type Ad DNA throughout the early phase of infection, until the onset of late gene expression (ϳ10 to 12 hpi), and that transcription is necessary to evict core VII (10). Using hdAd which contained only noncoding DNA (i.e., lacked a transgene), VII was shown to remain stably associated with the vector DNA until at least 18 hpi by immunofluorescence analysis.…”

Section: Resultsmentioning

confidence: 99%

“…However, even up to 24 hpi, a small proportion of VII does persist in the nucleus and may be associated with the vector DNA. Previous work by Chen and coworkers demonstrated that the level of VII within cells changed very little between 4 and 12 hpi and that foci of VII (presumably VII-associated viral genomes) could be detected in the nucleus by immunofluorescence for up to ϳ12 hpi (10). However, that report did not examine the levels of VII at earlier time points (i.e., between 0 and 4 hpi) or whether the nuclear foci of VII contained Ad DNA completely or only partially wrapped in VII.…”

Section: Discussionmentioning

confidence: 99%

“…During wild-type Ad infection, the major DNA-binding protein, protein VII, enters the nucleus with the Ad DNA (9). However, conflicting data suggest that VII either stably associates with Ad DNA throughout the early phase of infection (9,77) or is evicted within a few hours (70), and eviction of VII may require active transcription (10). Few studies have addressed whether Ad DNA in the nucleus directly interacts with histones or assembles into chromatin; indeed, conflicting data suggest that Ad DNA is (5,13,14,66) or is not (76) assembled into chromatin.…”

mentioning

confidence: 99%

See 1 more Smart Citation

Assembly of Helper-Dependent Adenovirus DNA into Chromatin Promotes Efficient Gene Expression

Ross

Kennedy

Christou

et al. 2011

Helper-dependent adenovirus (hdAd) vectors have shown tremendous potential in animal models of human disease in numerous preclinical studies. Expression of a therapeutic transgene can be maintained for several years after a single administration of the hdAd vector. However, despite the long-term persistence of hdAd DNA in the transduced cell, little is known of the fate and structure of hdAd DNA within the host nucleus. In this study, we have characterized the assembly of hdAd DNA into chromatin in tissue culture. Eviction of the Ad DNA-packaging protein VII, histone deposition, and vector-associated gene expression all began within 2 to 6 h of host cell transduction. Inhibition of transcription elongation through the vector DNA template had no effect on the loss of VII, suggesting that transcription was not necessary for removal of the majority of protein VII. Vector DNA assembled into physiologically spaced nucleosomes within 6 h. hdAd vectors incorporated the histone H3 variant H3.3, which was dependent on the histone chaperone HIRA. Knockdown of HIRA reduced hdAd association with histones and reduced expression of the vector-carried transgene by 2-to 3-fold. Our study elucidates an essential role for hdAd DNA chromatinization for optimal vector gene expression.

“…Initial work on the adenovirus genome showed transcriptiondependent chromatinization of the adenoviral genome (29). Since this discovery, the importance of the chromatinization of the viral genome in DNA damage (30) and transcription control (31,32) has become increasingly apparent.…”

Section: Discussionmentioning

confidence: 99%

Adenovirus E1A Recruits the Human Paf1 Complex To Enhance Transcriptional Elongation

Fonseca

Cohen

Mymryk

2014

During infection by human adenovirus (HAdV), the proteins encoded by the early region 1A (E1A) gene bind and appropriate components of the cellular transcriptional machinery to activate the transcription of viral early genes. Previously, we identified roles for the human Bre1 (hBre1) and hPaf1 complexes in E1A-mediated transcriptional activation of HAdV early genes. Here we show that E1A binds hBre1 directly and that this complex targets the hPaf1 complex via the Rtf1 subunit. Depletion of hPaf1 reduces E1A-dependent activation of transcription from the E2e, E3, and E4 viral transcription units, and this does not result from a reduced ability of RNA polymerase II to be recruited to the promoter-proximal regions of these genes. In contrast, depletion of hPaf1 reduces the occupancy of RNA polymerase II across these transcription units. This is accompanied by reductions in the level of H3K36 trimethylation, a posttranslational histone modification associated with efficient transcriptional elongation, and the number of full-length transcripts from these genes. Together, these results indicate that E1A uses hBre1 to recruit the hPaf1 complex in order to optimally activate viral early transcription by enhancing transcriptional elongation. IMPORTANCEThis work provides the mechanism by which the hPaf1 complex contributes to E1A-dependent activation of early gene transcription. The work also demonstrates that E1A induces gene expression by stimulating transcriptional elongation, in addition to its better-characterized effects on transcriptional initiation.