Characterization of the Interaction between P143 and LEF-3 from Two Different Baculovirus Species: Choristoneura fumiferana Nucleopolyhedrovirus LEF-3 Can Complement Autographa californica Nucleopolyhedrovirus LEF-3 in Supporting DNA Replication

Abstract

The baculovirus protein P143 is essential for viral DNA replication in vivo, likely as a DNA helicase. We have demonstrated that another viral protein, LEF-3, first described as a single-stranded DNA binding protein, is required for transporting P143 into the nuclei of insect cells. Both of these proteins, along with several other early viral proteins, are also essential for DNA replication in transient assays. We now describe the identification, nucleotide sequences, and transcription patterns of the Choristoneura fumiferana nucleopolyhedrovirus (CfMNPV) homologues of p143 and lef-3 and demonstrate that CfMNPV LEF-3 is also responsible for P143 localization to the nucleus. We predicted that the interaction between P143 and LEF-3 might be critical for cross-species complementation of DNA replication. Support for this hypothesis was generated by substitution of heterologous P143 and LEF-3 between two different baculovirus species, Autographa californica nucleopolyhedrovirus and CfMNPV, in transient DNA replication assays. The results suggest that the P143-LEF-3 complex is an important baculovirus replication factor.The family Baculoviridae represents a unique group of large rod-shaped enveloped viruses carrying a double-stranded circular DNA genome and replicating only in invertebrates. Many of the advances in understanding the molecular biology of baculoviruses have resulted from studies of variants of the type species Autographa californica nucleopolyhedrovirus (AcMNPV). Nucleopolyhedroviruses (NPVs) replicate in cell nuclei and are characterized by the production of two virion phenotypes, the budded virions and the occlusion-derived virions. Both forms are produced following infection of cells in culture and are characteristic of late stages of the viral replication cycle following initiation of viral DNA replication at about 8 h postinfection (37). The early events prior to this time are characterized by the expression of several viral gene products, some of which have been shown to be essential for viral DNA replication. Nine viral genes (ie-1, ie-2, p143, dnapol, lef-1, lef-2, lef-3, pe38, and p35) are involved in directing replication of plasmids carrying viral DNA inserts in transfected cells (20,31,38). These data supported earlier genetic analysis of a conditional lethal AcMNPV mutant defective in DNA replication (13), which led to the description of the p143 gene: its nucleotide sequence and the identification of the lesion in the 1,221-amino-acid open reading frame (ORF) (143 kDa) responsible for the temperature-sensitive DNA negative phenotype (29). The p143 gene is essential for viral DNA replication in vivo since no replication occurs in cells infected at the nonpermissive temperature with ts8 (29).Biochemical characterization of extracts from AcMNPV-infected cells showed that P143 copurified through hydroxylapatite and coeluted from single-stranded DNA cellulose with another viral protein called LEF-3, suggesting a possible direct interaction between P143 and LEF-3 (22, 39). LEF-3, also demonstra...

Identification of Domains in Autographa californica Multiple Nucleopolyhedrovirus Late Expression Factor 3 Required for Nuclear Transport of P143

“…In addition, this construct could substitute for authenticate P143 in supporting DNA replication in transient assays but only in the presence of intact LEF-3. These results confirm previous experiments where fusing GFP to the CfMNPV P143 protein did not inhibit the ability of P143 to support DNA replication (2). From these results, we conclude that the presence of P143 in the nucleus is not sufficient to support DNA replication, in the absence of LEF-3.…”

“…Three closely related species (AcMNPV, Rachiplusia ou MNPV [RoMNPV], and Bombyx mori NPV [BmNPV]) appear to have an insert in this region consisting of nine amino acids (eight are identical in all these viruses), including flanking basic amino acids and a proline, compared with other group I NPVs. We have previously shown that Christoneura fumifurana MNPV (CfMNPV) LEF-3 self-localizes to the nucleus of infected cells (2), suggesting that this nine-amino-acid region is not essential for nuclear localization. Since all of our constructs in the vector pHSEH are His tagged, there is a string of eight basic amino acids fused immediately upstream of any sequences cloned into this vector.…”

“…Sf21cells (10 6 ), seeded into 35-mm dishes (with or without coverslips) were infected or transfected as previously described (2) and incubated at 28°C for 24 to 48 h. In some cases, the cells were heat shocked at 42°C for 30 min at 20 h posttransfection, and returned to 28°C for 3.5 h. Cells transfected with 2 g of plasmid DNA were harvested at 48 h postinfection and biochemically fractionated exactly as described (23).…”

“…Equal molar amounts of plasmids expressing all of the AcMNPV genes essential for viral DNA replication (AcMNPV replication library: pAcIE-1, pAcIE-2/PE38, pAcLEF-1, pAcLEF-2, pAcLEF-3, pAcP143, pAcDNAPOL, and pAcP35) were mixed and transfected into Sf21 cell as previously described (2). At 48 h postinfection, total infected cell DNA was purified, aliquots were digested with HindIII and/or DpnI for 6 h, and the DNA fragments were electrophoresed through 0.7% agarose gels.…”

Identification of a Domain of the Baculovirus Autographa californica Multiple Nucleopolyhedrovirus Single-Strand DNA-Binding Protein LEF-3 Essential for Viral DNA Replication

“…Sf21 cells were transfected with 1 g of purified bacmid DNA as previously described (6). At various times after transfection, supernatants were harvested and titers were determined in 50% tissue culture infective dose (TCID 50 ) endpoint dilution assays (25) to determine the replication properties of BV produced by the various bacmids.…”