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Protease of adenovirus type 2. Subcellular localization
Abstract:
The subcellular localization of the adenovirus type 2 core polypeptide specific protease activity was investigated using an in vitro assay system. The protease activity was recovered exclusively from infected cell nuclei and was insoluble, sedimenting with the membrane fraction. Endogenous activity could be demonstrated in young virions which contain precursor PVII molecules. This protease activity only became sensitive to L-1-tosylamide-2-phenylethylchloromethyl ketone- or phenylmethylsulfonyl fluoride-mediated inhibition after disruption of the virus particles by sonication, suggesting that the enzyme was internally located. The putative precursors to virus particles, referred to as top components, which do not contain a full complement of viral DNA, did not contain protease activity. The protease released from sonicated virions converted exogenous PVII substrate molecules to polypeptide VII. The noninfectious H2ts1 virus particles synthesized at the nonpermissive temperature phenotypically resemble young virions, but unlike their wild type counterparts, were devoid of protease activity. The results show that the protease enters the precursor particles concurrently with the viral chromosome and that its presence is a prerequisite for the processing and subsequent maturation of infectious adenovirions.
Insights
Adenovirus type 2 protease activity is found within infected cell nuclei and virions, essential for viral maturation. This internal protease processes precursor molecules, indicating its critical role in forming infectious adenovirions.
Area of Science:
- Virology
- Molecular Biology
- Biochemistry
Background:
- Adenovirus type 2 protease is crucial for viral maturation.
- Its subcellular localization and role in processing viral proteins were previously unclear.
Purpose of the Study:
- To investigate the subcellular localization of adenovirus type 2 protease activity.
- To determine the role of this protease in viral particle formation and maturation.
Main Methods:
- In vitro protease assay system using infected cell extracts and purified virions.
- Enzyme inhibition assays with L-1-tosylamide-2-phenylethylchloromethyl ketone and phenylmethylsulfonyl fluoride.
- Analysis of protease activity in precursor virus particles (top components) and temperature-sensitive mutants (H2ts1).
Main Results:
- Protease activity was exclusively localized to infected cell nuclei and insoluble fractions.
- Activity was detected in young virions containing precursor polypeptide VII (PVII).
- Protease was internally located within virions, becoming sensitive to inhibitors only after sonication.
- Protease processed exogenous PVII to polypeptide VII.
- Noninfectious H2ts1 virions lacked protease activity.
Conclusions:
- Adenovirus type 2 protease enters precursor particles with the viral chromosome.
- The protease is essential for processing PVII and subsequent maturation of infectious adenovirions.