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Cellular integrity is required for inhibition of initiation of cellular DNA synthesis by reovirus type 3
Abstract:
Synchronized HeLa cells, primed for entry into the synthesis phase by amethopterin, were prevented from initiating DNA synthesis 9 h after infection with reovirus type 3. However, nuclei isolated from synchronized cells infected with reovirus for 9 or 16 h demonstrated a restored ability to synthesize DNA. The addition of enucleated cytoplasmic extracts from infected or uninfected cells did not affect this restored capacity for synthesis. The addition of ribonucleotide triphosphates to nuclei isolated from infected cells stimulated additional DNA synthesis, suggesting that these nuclei were competent to initiate new rounds of DNA replication. Permeabilization of infected cells did not restore the ability of these cells to synthesize DNA. Nucleoids isolated from intact or permeabilized cells, infected for 9 or 16 h displayed an increased rate of sedimentation when compared with nucleoids isolated from uninfected cells. Nucleoids isolated from the nuclei of infected cells demonstrated a rate of sedimentation similar to that of nucleoids isolated from the nuclei of uninfected cells. The inhibition of initiation of cellular DNA synthesis by reovirus type 3 appears not to have been due to a permanent alteration of the replication complex, but this inhibition could be reversed by the removal of that complex from factors unique to the structural or metabolic integrity of the infected cell.
Insights
Reovirus type 3 inhibits cellular DNA synthesis initiation in HeLa cells. However, isolated nuclei retain DNA synthesis capacity, suggesting the inhibition is reversible and not due to permanent replication complex damage.
Area of Science:
- Cell Biology
- Virology
- Molecular Biology
Background:
- Reovirus type 3 is known to affect host cell processes.
- Understanding viral interference with host DNA replication is crucial.
Purpose of the Study:
- To investigate the mechanism by which reovirus type 3 inhibits cellular DNA synthesis.
- To determine if the inhibition involves permanent damage to the DNA replication machinery.
Main Methods:
- Synchronized HeLa cells were infected with reovirus type 3.
- DNA synthesis was measured in intact cells and isolated nuclei.
- Nucleoids were analyzed for sedimentation rates.
- Enucleated cytoplasmic extracts were used to assess cytoplasmic factors.
Main Results:
- Reovirus type 3 infection inhibited DNA synthesis initiation in intact HeLa cells.
- Isolated nuclei from infected cells showed restored DNA synthesis capacity.
- Addition of ribonucleotide triphosphates stimulated DNA synthesis in isolated nuclei.
- Nucleoid sedimentation rates increased in infected cells but not in isolated nuclei.
Conclusions:
- Reovirus type 3-mediated inhibition of DNA synthesis is not due to permanent alteration of the replication complex.
- The inhibition can be reversed upon removal of the replication complex from the infected cell environment.
- Reovirus likely interferes with cellular DNA synthesis through factors associated with the infected cell's integrity.