Role for Centromeric Heterochromatin and PML Nuclear Bodies in the Cellular Response to Foreign DNA

Bishop, Cleo L.; Ramalho, Michal; Nadkarni, Nachiket A.; Kong, Wing May; Higgins, Christopher F.; Krauzewicz, Nina

doi:10.1128/mcb.26.7.2583-2594.2006

Cited by 29 publications

(7 citation statements)

References 64 publications

(75 reference statements)

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“…Likewise, it is conceivable that not all of the 240 kb HCMV BAC molecules remain intact during the DNA preparation and transfection procedure. Moreover, progression through the productive infection cycle may be impaired in the absence of tegument proteins that usually counteract the function of cellular restriction factors early in infection [ 34 , 35 ]. This may be overcome by simultaneously transfecting the cells with plasmids encoding the respective tegument proteins, such as pp71 which is known to enhance gene expression from HCMV DNA [ 36 ].…”

Section: Resultsmentioning

confidence: 99%

Analysis of Essential Viral Gene Functions after Highly Efficient Adenofection of Cells with Cloned Human Cytomegalovirus Genomes

Elbasani

Gabaev

Steinbrück

et al. 2014

Viruses

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Human cytomegalovirus (HCMV) has a large 240 kb genome that may encode more than 700 gene products with many of them remaining uncharacterized. Mutagenesis of bacterial artificial chromosome (BAC)-cloned CMV genomes has greatly facilitated the analysis of viral gene functions. However, the roles of essential proteins often remain particularly elusive because their investigation requires the cumbersome establishment of suitable complementation systems. Here, we show that HCMV genomes can be introduced into cells with unprecedented efficiency by applying a transfection protocol based on replication-defective, inactivated adenovirus particles (adenofection). Upon adenofection of several permissive cell types with HCMV genomes carrying mutations in essential genes, transfection rates of up to 60% were observed and viral proteins of all kinetic classes were found expressed. This enabled further analyses of the transfected cells by standard biochemical techniques. Remarkably, HCMV genomes lacking elements essential for viral DNA replication, such as the lytic origin of replication, still expressed several late proteins. In conclusion, adenofection allows the study of essential HCMV genes directly in BAC-transfected cells without the need for sophisticated complementation strategies.

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Section: Resultsmentioning

confidence: 99%

Analysis of Essential Viral Gene Functions after Highly Efficient Adenofection of Cells with Cloned Human Cytomegalovirus Genomes

Elbasani

Gabaev

Steinbrück

et al. 2014

Viruses

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“…In eukaryotic cells, PML protein has also been implicated in DNA repair responses by acting as sensor and regulator of p53 [ 39 , 59 ], both functions corrupted by several viruses to circumvent cellular antiviral measures [ 11 , 60 , 61 ]. Here, we show that disrupted PML-NBs share a close vicinity to p-p53 and pATR accumulations, suggesting that ASFV may exploit this scenario to increment the recognition of its genomes, corroborating the previously identified ATR pathway activation [ 6 ].…”

Section: Discussionmentioning

confidence: 99%

Alterations of Nuclear Architecture and Epigenetic Signatures during African Swine Fever Virus Infection

Simões

Rino

Pinheiro

et al. 2015

Viruses

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Viral interactions with host nucleus have been thoroughly studied, clarifying molecular mechanisms and providing new antiviral targets. Considering that African swine fever virus (ASFV) intranuclear phase of infection is poorly understood, viral interplay with subnuclear domains and chromatin architecture were addressed. Nuclear speckles, Cajal bodies, and promyelocytic leukaemia nuclear bodies (PML-NBs) were evaluated by immunofluorescence microscopy and Western blot. Further, efficient PML protein knockdown by shRNA lentiviral transduction was used to determine PML-NBs relevance during infection. Nuclear distribution of different histone H3 methylation marks at lysine’s 9, 27 and 36, heterochromatin protein 1 isoforms (HP1α, HPβ and HPγ) and several histone deacetylases (HDACs) were also evaluated to assess chromatin status of the host. Our results reveal morphological disruption of all studied subnuclear domains and severe reduction of viral progeny in PML-knockdown cells. ASFV promotes H3K9me3 and HP1β foci formation from early infection, followed by HP1α and HDAC2 nuclear enrichment, suggesting heterochromatinization of host genome. Finally, closeness between DNA damage response factors, disrupted PML-NBs, and virus-induced heterochromatic regions were identified. In sum, our results demonstrate that ASFV orchestrates spatio-temporal nuclear rearrangements, changing subnuclear domains, relocating Ataxia Telangiectasia Mutated Rad-3 related (ATR)-related factors and promoting heterochromatinization, probably controlling transcription, repressing host gene expression, and favouring viral replication.

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“…The MPyV-Gluc vector transduced 293TT cells and murine NIH-3T3 cells much less efficiently than the MCV-Gluc vector and it was therefore necessary to use a dose of 2 ng of MPyV VP1 per well. The MPyV neutralization assay was carried out in the presence of 100 nM trichostatin A (EMD Biosciences), a histone deacetylase inhibitor that has previously been shown to enhance MPyV vector-mediated transduction [45] .…”

Section: Methodsmentioning

confidence: 99%

Quantitation of Human Seroresponsiveness to Merkel Cell Polyomavirus

et al. 2009

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Merkel cell carcinoma (MCC) is a relatively uncommon but highly lethal form of skin cancer. A majority of MCC tumors carry DNA sequences derived from a newly identified virus called Merkel cell polyomavirus (MCV or MCPyV), a candidate etiologic agent underlying the development of MCC. To further investigate the role of MCV infection in the development of MCC, we developed a reporter vector-based neutralization assay to quantitate MCV-specific serum antibody responses in human subjects. Our results showed that 21 MCC patients whose tumors harbored MCV DNA all displayed vigorous MCV-specific antibody responses. Although 88% (42/48) of adult subjects without MCC were MCV seropositive, the geometric mean titer of the control group was 59-fold lower than the MCC patient group (p<0.0001). Only 4% (2/48) of control subjects displayed neutralizing titers greater than the mean titer of the MCV-positive MCC patient population. MCC tumors were found not to express detectable amounts of MCV VP1 capsid protein, suggesting that the strong humoral responses observed in MCC patients were primed by an unusually immunogenic MCV infection, and not by viral antigen expressed by the MCC tumor itself. The occurrence of highly immunogenic MCV infection in MCC patients is unlikely to reflect a failure to control polyomavirus infections in general, as seroreactivity to BK polyomavirus was similar among MCC patients and control subjects. The results support the concept that MCV infection is a causative factor in the development of most cases of MCC. Although MCC tumorigenesis can evidently proceed in the face of effective MCV-specific antibody responses, a small pilot animal immunization study revealed that a candidate vaccine based on MCV virus-like particles (VLPs) elicits antibody responses that robustly neutralize MCV reporter vectors in vitro. This suggests that a VLP-based vaccine could be effective for preventing the initial establishment of MCV infection.

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Role for Centromeric Heterochromatin and PML Nuclear Bodies in the Cellular Response to Foreign DNA

Cited by 29 publications

References 64 publications

Analysis of Essential Viral Gene Functions after Highly Efficient Adenofection of Cells with Cloned Human Cytomegalovirus Genomes

Analysis of Essential Viral Gene Functions after Highly Efficient Adenofection of Cells with Cloned Human Cytomegalovirus Genomes

Alterations of Nuclear Architecture and Epigenetic Signatures during African Swine Fever Virus Infection

Quantitation of Human Seroresponsiveness to Merkel Cell Polyomavirus

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