tailieunhanh - Báo cáo Y học: Reconstructing the replication complex of AcMNPV

Baculoviruses are well known for their large, circular, dou-ble-stranded DNA genomes. The type member, AcMNPV, is the best characterized andundergoes a successionof early, late and very late gene expression during its infection cycle. The viral genes involved inDNAreplicationhave previously been identified and their products are required for the acti-vation of late gene expression. In this study, we FLAG- and HA-tagged the replication late expression factors of AcMNPV, examined their expression and functional activities by CAT assay and Western blot analysis, and determined their subcellular localization in transfected cells by subcellular fractionation and immunofluorescent microscopy | Eur. J. Biochem. 269 6233-6240 2002 FEBS 2002 doi Reconstructing the replication complex of AcMNPV Kathleen L. Hefferon1 and Lois K. Miller2 1 Center for Virology Department of Botany University of Toronto Ontario Canada 2Department of Entomology University of Georgia Athens GA USA Baculoviruses are well known for their large circular double-stranded DNA genomes. The type member AcMNPV is the best characterized and undergoes a succession of early late and very late gene expression during its infection cycle. The viral genes involved in DNA replication have previously been identified and their products are required for the activation of late gene expression. In this study we FLAG- and HA-tagged the replication late expression factors of AcMNPV examined their expression and functional activities by CAT assay and Western blot analysis and determined their subcellular localization in transfected cells by subcellular fractionation and immunofluorescent microscopy. We found that all replication LEFs with the exception of P143 and P35 resided in the nucleus of transfected cells. We further investigated the interactions among various replication LEFs using both yeast two-hybrid and coprecipitation strategies. A summary of the interactive properties of the replication LEFs is presented and a model for a putative AcMNPV replication complex is offered. Keywords Autographica californica nucleopolyhedrosis virus coprecipitation DNA replication late expression factor yeast two-hybrid system. Autographa californica nucleopolyhedrosis virus or AcMNPV is the type member of the Baculoviridae a family of double-stranded DNA viruses with large circular genomes. The successive and concomitant expression of an assortment of early late and very late genes are instrumental for successful baculovirus infection and require a switch from early dependence on a host cell-derived RNA polymerase II to a novel virus-encoded RNA polymerase that is required for .