Site-directed mutagenesis of the AcMNPV p143 gene: effects on baculovirus DNA replication.
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.
Liu
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Department of Microbiology and Immunology, Queen's University, Kingston, Ontario, K7L 3N6, Canada.
Carstens
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D000255:Adenosine Triphosphate / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D004261:DNA Replication; D004279:DNA, Viral / Q000096:biosynthesis*; D005814:Genes, Viral; D019077:Helix-Turn-Helix Motifs / Q000235:genetics; D017874:Immediate-Early Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017924:Nucleopolyhedroviruses / Q000235:genetics*; D018411:Spodoptera; D015534:Trans-Activators / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D014158:Transcription, Genetic; D014764:Viral Proteins / Q000235:genetics* / Q000502:physiology; D014779:Virus Replication
C091746:Ac-bro protein, Autographa californica nucleopolyhedrovirus; D004279:DNA, Viral; D017874:Immediate-Early Proteins; D015534:Trans-Activators; D014764:Viral Proteins; C072773:IE2 protein, polyhedrosis virus; D000255:Adenosine Triphosphate