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Expression and functional analysis of a baculovirus gene encoding a truncated protein kinase homolog
1Department of Genetics, University of Georgia, Athens 30602.
Abstract:
Autographa californica nuclear polyhedrosis virus (AcMNPV) potentially encodes a 215-amino acid polypeptide containing 6 out of 11 motifs conserved among eukaryotic protein kinases (Morris et al., Virology 200, 360-369, 1994). We examined the expression of this gene, named pk2, at the transcriptional and translational levels and the possible role of the gene during baculovirus replication in cell culture and insect larvae. Northern (RNA) blot analysis revealed that pk2 was transcribed primarily as an early 1.2-kb RNA. Western blot analysis showed that pk2 was expressed as a 25-kDa protein, PK2, which was present both early and late during virus infection. To examine the function(s) of pk2, we constructed a mutant baculovirus, vKINdel, in which one-third of the PK2-coding region was deleted and then compared the characteristics of vKINdel with wild-type AcMNPV and a marker-rescued revertant. The pk2 deletion mutation had no discernable effect on the number, size, or appearance of plaques, the kinetics of protein synthesis or protein phosphorylation profiles during virus infection of cultured SF-21 cells. Deletion of pk2 also had no significant influence on the infectivity or virulence of the baculovirus in larval bioassays and the level of occluded virus production was normal. Thus, pk2 does not appear to have a significant influence on virus replication in the host systems examined.
Insights
The Autographa californica nuclear polyhedrosis virus (AcMNPV) pk2 gene encodes a protein kinase. Deleting this gene had no significant impact on viral replication in cell cultures or insect larvae, suggesting it is non-essential for AcMNPV infection.
Area of Science:
- Molecular virology
- Insect molecular biology
- Protein kinase research
Background:
- Autographa californica nuclear polyhedrosis virus (AcMNPV) is a baculovirus.
- AcMNPV potentially encodes a protein kinase (PK2) with conserved eukaryotic motifs.
- The role of PK2 in baculovirus replication was previously uncharacterized.
Purpose of the Study:
- To investigate the expression of the AcMNPV pk2 gene.
- To determine the function of the PK2 protein during baculovirus infection.
- To assess the impact of pk2 deletion on viral replication in cell culture and insect larvae.
Main Methods:
- Northern (RNA) blot analysis to examine pk2 transcription.
- Western blot analysis to detect PK2 protein expression.
- Construction of a pk2 deletion mutant (vKINdel) for functional analysis.
- Comparison of vKINdel with wild-type AcMNPV in cell culture and insect bioassays.
Main Results:
- pk2 was transcribed as an early 1.2-kb RNA and expressed as a 25-kDa protein (PK2) throughout infection.
- Deletion of pk2 did not affect plaque formation, protein synthesis, or phosphorylation in SF-21 cells.
- pk2 deletion had no significant effect on viral infectivity, virulence in larvae, or occluded virus production.
Conclusions:
- The AcMNPV pk2 gene is expressed during infection but is not essential for viral replication.
- PK2 does not play a significant role in the examined host systems (cell culture and insect larvae).
- Further research may be needed to identify potential subtle roles or alternative functions of PK2.