Reconstructing the replication complex of AcMNPV

Abstract

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.

Characterization of a nucleopolyhedrovirus with a deletion of the baculovirus core gene Bm67

“…Although the mechanism of baculovirus genome replication is unclear, there is evidence that many viral replication factors are involved in the process (Hefferon & Miller, 2002;Mikhailov, 2003;Okano et al, 2006). Replication is associated with a specific structure known as virogenic stroma, in which many replication factors, such as IE1, IE2 and the DNA-binding protein (DBP), accumulate (Mainz et al, 2002;Okano et al, 1999).…”

Characterization of HCF‐1, a determinant of Autographa californica multiple nucleopolyhedrovirus host specificity

“…To construct the HA‐ and FLAG‐tagged expression plasmids, primers were designed that encompassed the lef‐1 , lef‐2 , lef‐3 , ie ‐ 1 , P143 , DNApol and hcf‐1 open reading frames. After PCR amplification, cDNAs were inserted into the Bgl II and Psp AI sites of the vectors phsp70HAHISVI+ and phsp70FLAGHISVI+ (Hefferon & Miller, 2002). A reporter plasmid, pCAPCAT, containing the CAT gene under the transcriptional control of the late vp39 promoter as well as a 473 Mlu I fragment containing a portion of the origin of replication hr5 was included in this study (Passarelli & Miller, 1993).…”

The Baculoviruses Occlusion‐Derived Virus: Virion Structure and Function

“…ODV nucleocapsids contain all of these proteins but for LEF-2 (Braunagel et al, 2003). The absence of LEF-2 is surprising given its oligomerization with LEF-1 (Evans et al, 1997;Hefferon and Miller, 2002) and its stabilization of LEF-1 binding to DNA (Mikhailov and Rohrmann, 2002a). BV nucleocapsids do not contain IE-1 or P143 (Braunagel et al, 2003).…”

“…IE-1 binds to replication origin DNA sequences called homologous repeats (hrs) (Kovacs et al, 1992;Pearson et al, 1992). LEF-3 binds to single-stranded DNA (Hang et al, 1995) and binds to P143 (Wu and Carstens, 1998) and to IE-1 (Hefferon and Miller, 2002). The baculovirus exonuclease AN has been shown to bind to DNA and to complex with LEF-3 (Mikhailov et al, 2003).…”