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Isolation of Viral Replication Compartment-enriched Sub-nuclear Fractions from Adenovirus-infected Normal Human Cells
Published on: November 12, 2015
Adenovirus chromatin structure at different stages of infection
Abstract:
We investigated the structure of adenovirus deoxyribonucleic acid (DNA)-protein complexes in nuclei of infected cells by using micrococcal nuclease. Parental (infecting) DNA was digested into multimers which had a unit fragment size that was indistinguishable from the size of the nucleosomal repeat of cellular chromatin. This pattern was maintained in parenteral DNA throughout infection. Similar repeating units were detected in hamster cells that were nonpermissive for human adenovirus and in cells pretreated with n-butyrate. Late in infection, the pattern of digestion of viral DNA was determined by two different experimental approaches. Nuclear DNA was electrophoresed, blotted, and hybridized with labeled viral sequences; in this procedure all virus-specific DNA was detected. This technique revealed a diffuse protected band of viral DNA that was smaller than 160 base pairs, but no discrete multimers. All regions of the genome were represented in the protected DNA. To examine the nuclease protection of newly replicated viral DNA, infected cells were labeled with [3H]thymidine after blocking of cellular DNA synthesis but not viral DNA synthesis. With this procedure we identified a repeating unit which was distinctly different from the cellular nucleosomal repeat. We found broad bands with midpoints at 200, 400, and 600 base pairs, as well as the limit digest material revealed by blotting. High-resolution acrylamide gel electrophoresis revealed that the viral species comprised a series of closely spaced bands ranging in size from less than 30 to 250 base pairs.
Insights
Adenovirus deoxyribonucleic acid (DNA)-protein complexes show distinct structural patterns. Parental DNA resembles cellular chromatin, while newly replicated viral DNA exhibits unique repeating units, differing from host cell nucleosomes.
Area of Science:
- Molecular Biology
- Virology
- Structural Biology
Background:
- Adenovirus infection involves complex interactions between viral DNA and host cell proteins.
- Understanding the structural organization of viral DNA-protein complexes is crucial for deciphering viral replication and pathogenesis.
Purpose of the Study:
- To investigate the structural organization of adenovirus deoxyribonucleic acid (DNA)-protein complexes within infected cell nuclei.
- To compare the DNA-protein complex structure of parental and newly replicated viral DNA with cellular chromatin.
Main Methods:
- Micrococcal nuclease digestion of DNA-protein complexes in infected cell nuclei.
- Electrophoresis, blotting, and hybridization techniques to analyze viral DNA.
- [3H]thymidine labeling to track newly synthesized viral DNA.
- High-resolution acrylamide gel electrophoresis.
Main Results:
- Parental adenovirus DNA digestion yielded fragments similar in size to cellular chromatin nucleosomal repeats.
- Newly replicated viral DNA showed distinct repeating units, different from cellular nucleosomes, with protected fragments ranging from <30 to 250 base pairs.
- Analysis revealed diffuse protected viral DNA bands smaller than 160 base pairs, with all viral genome regions represented.
Conclusions:
- Adenovirus DNA-protein complexes exhibit distinct structural characteristics depending on whether the DNA is parental or newly replicated.
- Newly replicated viral DNA forms complexes with a unique repeating structure, differing from the host cell's nucleosomal organization.
- These findings provide insights into the packaging and structural dynamics of viral DNA during adenovirus infection.
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