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The MultiBac Protein Complex Production Platform at the EMBL
Published on: July 11, 2013
Site-directed mutagenesis of the AcMNPV p143 gene: effects on baculovirus DNA replication
1Department of Microbiology and Immunology, Queen's University, Kingston, Ontario, K7L 3N6, Canada.
Abstract:
Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) encodes a 143-kDa protein (P143) required for viral DNA synthesis and involved in host range determination. The predicted amino acid sequence of P143 contains seven motifs (I, Ia, II-VI) shared with a superfamily of helicases involved in the unwinding of duplex nucleic acids; a putative DNA binding motif; a putative nuclear localization signal (NLS); and a demonstrated host range motif. In this study, the functional significance of these conserved P143 motifs was examined by site-specific mutation resulting in amino acid substitutions of conserved residues within each of them. An in vivo complementation replication assay was developed and each mutated P143 protein expressed from a transfected plasmid was tested for its ability to complement the replication-negative ts8 baculovirus mutant for the amplification of an origin-containing plasmid. Mutations in the helicase motifs I, Ia, and II and in a potential helix-turn-helix motif abolished the ability of P143 to complement the ts8 defect in DNA replication, suggesting that these conserved amino acid residues may be essential for the replication function of the protein. In contrast, mutation of conserved amino acid residues in the helicase motifs IV, V, and VI did not affect the ability of the P143 proteins to complement the replication defect of ts8. A mutation in motif III caused a reduction in the replication function of P143. Deletion of Gly552 in the host range region eliminated the replication function of P143. Mutations within a putative NLS had no effect on the ability of P143 to support DNA replication, suggesting that these residues are nonessential and that the putative P143 NLS sequence may not be responsible for the nuclear localization of the protein. The transient complementation system used in this study provides a simple method for functional analysis of essential baculovirus genes in infected cell cultures.
Insights
Specific helicase motifs within Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) P143 protein are crucial for viral DNA replication. Mutations in motifs I, Ia, and II, along with a helix-turn-helix motif, abolish replication, while others have minimal impact.
Area of Science:
- Molecular Virology
- Insect Pathology
Background:
- Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) encodes the P143 protein, essential for viral DNA synthesis and host range.
- P143 contains conserved helicase motifs, a DNA binding motif, a nuclear localization signal (NLS), and a host range motif.
Purpose of the Study:
- To investigate the functional significance of conserved motifs within the AcMNPV P143 protein.
- To determine which P143 motifs are essential for viral DNA replication.
Main Methods:
- Site-specific mutations were introduced into conserved residues of P143 motifs.
- An in vivo complementation replication assay was used, testing mutated P143 proteins for their ability to complement the replication-deficient ts8 baculovirus mutant.
- Analysis of origin-containing plasmid amplification in transfected cells.
Main Results:
- Mutations in helicase motifs I, Ia, and II, and a potential helix-turn-helix motif, abolished P143 replication function.
- Motifs IV, V, and VI mutations did not significantly affect replication.
- A mutation in motif III reduced replication function, and deletion in the host range region eliminated it.
- Mutations in the putative NLS did not impact DNA replication.
Conclusions:
- Conserved residues in helicase motifs I, Ia, and II, and the helix-turn-helix motif, are critical for AcMNPV P143 replication function.
- The putative NLS sequence is not essential for nuclear localization or P143 replication.
- A transient complementation system offers a straightforward method for analyzing essential baculovirus genes.
