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Evidence for dissimilar properties of comoviral and picornaviral RNA polymerases
H van Bokhoven1, M Mulders, J Wellink
1Department of Molecular Biology, Agricultural University, Wageningen, The Netherlands.
Abstract:
The poliovirus RNA polymerase has been synthesized in Spodoptera frugiperda cells by using the baculovirus expression system. Crude sonicates of these cells exhibited an RNA-elongating activity of a synthetic oligo(U) primer with poly(A) or cowpea mosaic virus (CPMV) RNA as a template. A similar polymerase activity was found in extracts of insect cells in which foot-and-mouth disease virus (FMDV) proteins, including the putative polymerase, were produced. The analogous CPMV 87K protein and several of its precursors, synthesized in S. frugiperda cells, did not show any detectable polymerase activity in the same assay under a variety of conditions. The results indicate that, in contrast to the picornaviral polymerases, the CPMV polymerase is unable to function in an oligo(U)-primed polymerase assay.
Insights
Poliovirus RNA polymerase expressed in insect cells showed RNA-elongating activity. However, cowpea mosaic virus (CPMV) polymerase did not function in the same assay, unlike other picornaviruses.
Area of Science:
- Molecular Biology
- Virology
- Biochemistry
Background:
- Picornaviruses, including poliovirus and foot-and-mouth disease virus (FMDV), possess RNA-dependent RNA polymerases crucial for replication.
- The expression and characterization of viral polymerases in heterologous systems like insect cells are vital for understanding their enzymatic functions.
Purpose of the Study:
- To investigate the RNA polymerase activity of poliovirus and foot-and-mouth disease virus (FMDV) expressed in Spodoptera frugiperda insect cells.
- To compare the enzymatic function of picornaviral polymerases with the analogous CPMV 87K protein.
Main Methods:
- Utilized the baculovirus expression system for synthesizing poliovirus and FMDV proteins in Spodoptera frugiperda cells.
- Assessed RNA-elongating activity using a synthetic oligo(U) primer with poly(A) or CPMV RNA as a template in crude cell sonicates and extracts.
Main Results:
- Sonicates of cells expressing poliovirus RNA polymerase demonstrated RNA-elongating activity.
- Extracts from cells producing FMDV proteins also exhibited similar polymerase activity.
- The analogous CPMV 87K protein and its precursors, synthesized in insect cells, showed no detectable polymerase activity under tested conditions.
Conclusions:
- Picornaviral RNA polymerases, such as poliovirus and FMDV, can be functionally expressed in insect cells and display RNA-elongating activity.
- In contrast to picornaviral polymerases, the CPMV polymerase is not active in the oligo(U)-primed assay, suggesting differences in substrate recognition or catalytic mechanisms.