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Foot-and-mouth disease virus particles contain replicase protein 3D
J F Newman1, P G Piatti, B M Gorman
1U.S. Department of Agriculture, Plum Island Animal Disease Center, Greenport, NY 11944.
Abstract:
An antibody against the Escherichia coli-expressed RNA polymerase of foot-and-mouth disease virus (FMDV) reacts with the virus in ELISA and radioimmunoprecipitation experiments and with a protein of the disrupted virus particle in an immunoblot analysis. Treatment of the virus with trypsin, which cleaves capsid protein VP1 and a 56-kDa polypeptide present in trace amount in the particles, reduces the level of the reaction in ELISA and radioimmunoprecipitation and eliminates the immunoblot reaction. Electron microscopy showed that only approximately 20% of the virus particles reacted with the anti-polymerase antibody, whereas most reacted with an antibody against the immunodominant G-H loop of the virus. In the presence of ammonium ions, the expressed polymerase degrades the RNA of the virus into molecules sedimenting at approximately 12 S, indicating that it can act as a hydrolytic as well as a polymerizing enzyme. Moreover, the RNA in trypsin-treated virus particles is degraded when incubated at 37 degrees C, suggesting that the cleaved 56-kDa protein still possesses hydrolytic activity. In addition, the anti-polymerase antibody, which inhibits the polymerase activity of the E. coli-expressed protein, also partially inhibits the hydrolytic activity of the previously described endonuclease of the virus particle, suggesting that this enzyme is identical with the polymerase or forms part of it.
Insights
An antibody targeting the foot-and-mouth disease virus (FMDV) RNA polymerase also identified a hydrolytic activity within the virus particle. This suggests the polymerase enzyme may also function as an endonuclease, impacting viral RNA.
Area of Science:
- Virology
- Molecular Biology
- Enzymology
Background:
- Foot-and-mouth disease virus (FMDV) is a significant pathogen affecting livestock.
- The FMDV RNA polymerase is crucial for viral replication.
- The FMDV particle contains proteins with potential enzymatic activities.
Purpose of the Study:
- To characterize the Escherichia coli-expressed FMDV RNA polymerase.
- To investigate the enzymatic activities associated with the FMDV particle.
- To determine if the FMDV RNA polymerase possesses hydrolytic activity.
Main Methods:
- ELISA, radioimmunoprecipitation, and immunoblot analysis using an anti-FMDV RNA polymerase antibody.
- Electron microscopy to assess antibody binding to viral particles.
- Enzymatic assays to evaluate RNA degradation and hydrolytic activity in the presence of ammonium ions and after trypsin treatment.
Main Results:
- The anti-FMDV RNA polymerase antibody reacted with the expressed protein and a 56-kDa polypeptide in disrupted virus particles.
- Trypsin treatment reduced antibody reactivity, suggesting cleavage of the 56-kDa polypeptide affects polymerase recognition.
- The expressed polymerase exhibited hydrolytic activity, degrading viral RNA, and the 56-kDa polypeptide retained hydrolytic activity after cleavage.
Conclusions:
- The FMDV RNA polymerase possesses both polymerizing and hydrolytic enzymatic functions.
- The 56-kDa polypeptide, likely related to the polymerase, exhibits endonuclease activity.
- The FMDV RNA polymerase may be identical to or part of the viral endonuclease.