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Isolation of Viral Replication Compartment-enriched Sub-nuclear Fractions from Adenovirus-infected Normal Human Cells
Published on: November 12, 2015
Early events in the infection process of adenovirus type 5 in HeLa cells
1Philips Research Laboratories, N. V. Philips' Gloeilampenfabrieken, Eindhoven, The Netherlands.
Abstract:
The fate of adenovirus type 5r after infection of HeLa cells has been studied with the help of purified labeled virus preparations. The infection process starts with the adsorption of virions on the cell surface followed by penetration into the cells. Shortly after penetration, the particles inside the cells have a similar appearance as intact virions. However, a small amount of protein must have been lost, as concluded from radioactivity measurements and from the change in buoyant density (1.35 g/cm(3) instead of 1.34 g/cm(3), the value for intact virions). Evidence is presented that antigen B is absent from the 1.35-particles. After the transformation of virions into 1.35-particles, further uncoating leads to the formation of DNase-sensitive DNA-protein complexes. The proteins released during the uncoating show a buoyant density of 1.31 g/cm(3) and are almost completely insoluble in acid. No extensive breakdown of protein can be detected. The processes of attachment, penetration, and uncoating are not inhibited by prevention of de novo protein synthesis, as could be shown with cycloheximide.
Insights
Adenovirus type 5r undergoes initial uncoating in HeLa cells, losing some protein and antigen B. Further uncoating yields DNA-protein complexes, unaffected by inhibited protein synthesis.
Area of Science:
- Virology
- Cell Biology
- Molecular Biology
Background:
- Adenovirus infections are significant in human health.
- Understanding viral entry and uncoating is crucial for antiviral development.
Purpose of the Study:
- To elucidate the fate of adenovirus type 5r (Ad5r) within HeLa cells post-infection.
- To characterize the sequential changes in viral particles during the early stages of infection.
Main Methods:
- Infection of HeLa cells with purified, radiolabeled Ad5r.
- Analysis of viral particle properties including buoyant density and DNase sensitivity.
- Assessment of protein synthesis inhibition using cycloheximide.
Main Results:
- Ad5r virions adsorb to and penetrate HeLa cells.
- Intracellular particles exhibit altered buoyant density (1.35 g/cm(3)) and absence of antigen B, indicating initial protein loss.
- Further uncoating generates DNase-sensitive DNA-protein complexes.
- Released proteins have a buoyant density of 1.31 g/cm(3) and are acid-insoluble.
- Early infection stages (attachment, penetration, uncoating) are independent of de novo protein synthesis.
Conclusions:
- Ad5r undergoes a multi-step uncoating process in HeLa cells.
- Initial uncoating involves protein loss and antigen B removal, preceding DNA-protein complex formation.
- Viral entry and early uncoating mechanisms are robust and do not require host cell protein synthesis.
