The lef-3 gene of Autographa californica nuclear polyhedrosis virus encodes a single-stranded DNA-binding protein

Hang, Xin; Dong, Weijia; Guarino, Linda A.

doi:10.1128/jvi.69.6.3924-3928.1995

Cited by 79 publications

(35 citation statements)

References 27 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…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).…”

Section: Dna Replication Proteins In the Nucleocapsidmentioning

confidence: 99%

The Baculoviruses Occlusion‐Derived Virus: Virion Structure and Function

Slack

Arif

2006

Advances in Virus Research

260

240

View full text Add to dashboard Cite

Section: Dna Replication Proteins In the Nucleocapsidmentioning

confidence: 99%

The Baculoviruses Occlusion‐Derived Virus: Virion Structure and Function

Slack

Arif

2006

Advances in Virus Research

260

240

View full text Add to dashboard Cite

“…Determination of the oligomeric structure of LEF-3 required isolation of the protein in a purified form. To accomplish this, 16-h-postinfection nuclear extracts were prepared (12) and subjected to single-stranded DNA agarose chromatography by using a modification of the method of Hang et al (16). LEF-3 eluted from the single-stranded DNA agarose column in 800 mM to 1 M NaCl, resulting in 80 to 90% purification of LEF-3 ( Fig.…”

Section: Lef-3 Interaction Experimentsmentioning

confidence: 99%

The baculovirus single-stranded DNA binding protein, LEF-3, forms a homotrimer in solution

Evans

Rohrmann

1997

J Virol

View full text Add to dashboard Cite

LEF-3 is one of six proteins from Autographa californica multinucleocapsid polyhedrosis virus required for transient DNA replication and has the properties of a single-stranded DNA binding protein. In this report we demonstrate that LEF-3 interacts with itself in both yeast two-hybrid assays and glutathione S-transferase fusion affinity assays. LEF-3 deletion clones which were unable to interact with full-length LEF-3 also failed to support transient DNA replication, suggesting that this interaction is required for the proper function of LEF-3. LEF-3 was purified to homogeneity and characterized by analytical ultracentrifugation and native polyacrylamide gel electrophoresis. These studies revealed that LEF-3 was present as a 132-kDa complex, indicating that its native conformation is that of a homotrimer. This result was confirmed by cross-linking with glutaraldehyde followed by matrix-assisted laser desorption/ionization mass spectrometry.Autographa californica multinucleocapsid nuclear polyhedrosis virus (AcMNPV) has a genome of 134 kb that encodes approximately 150 genes (1). Transient replication assays have been used to identify six essential and three stimulatory genes involved in baculovirus DNA replication (23, 32). The six genes required for DNA replication encode the following proteins: DNA polymerase and helicase, whose functions are implied by DNA sequence homology (31, 42); LEF-3, a single-stranded DNA binding protein (16); IE-1, a transcriptional activator (15) which also binds to putative replication origins (4,14,27,30,38); and two proteins, LEF-1 and LEF-2, neither of which have been assigned specific functions but which have been shown to interact with one another (10). The three gene products which are stimulatory for DNA replication include two additional transcriptional activators, IE-2 (2) and PE-38 (28), and P35, which blocks apoptosis (5, 18) and therefore may not function directly in DNA replication.Characterization of the interactions between the products of these nine genes by using the yeast two-hybrid system revealed several interactions. We recently described the interaction of LEF-1 and LEF-2 and demonstrated that mutants which failed to interact also failed to support transient DNA replication (10). In this report, we describe the interaction between LEF-3 and itself and provide evidence indicating that LEF-3 forms a homotrimer in solution. MATERIALS AND METHODSInsect cells. Spodoptera frugiperda (Sf-9) cell (44) monolayers were cultured in TNM-FH medium (20) supplemented with 10% fetal bovine serum. Suspension cultures were maintained in serum-free SF900 II SFM medium (GIBCO-BRL) supplemented with penicillin G (50 U/ml), streptomycin (950 g/ml) (both from BioWhittaker Inc.), and fungisone (375 ng/ml) (GIBCO-BRL). Cell culture maintenance was carried out according to published procedures (41).Bacterial and yeast cells. All bacterial plasmids were maintained in Escherichia coli DH5␣. Saccharomyces cerevisiae Y166 (MATa ura3-52 leu2-3,112 his3⌬200 ade2-101 trp1-901 gal4⌬ gal80⌬ RNR::...

show abstract

“…The six essential genes were predicted to encode an origin binding protein, a priming factor to provide a free 3Ј hydroxyl, a DNA polymerase, a helicase to unwind the DNA duplex, and a single-stranded DNA (ssDNA) binding protein (SSB) to maintain the ssDNA exposed by the helicase. The AcNPV proteins DNApol and LEF-3 were shown to exhibit DNA polymerase and ssDNA binding activity, respectively (8,23). IE-1 may function as the origin binding protein, as it is the only essential replication protein shown to bind specifically to a putative origin of replication (2).…”

Section: Autographa Californica Nuclear Polyhedrosis Virus (Acnpv)mentioning

confidence: 99%

The Autographa californica Nuclear Polyhedrosis Virus p143 Gene Encodes a DNA Helicase

McDougal¹,

Guarino

2000

J. Virol.

View full text Add to dashboard Cite

The P143 protein of Autographa californica nuclear polyhedrosis virus is essential for replication of viral DNA. To determine the function of P143, the protein was purified to near homogeneity from recombinant baculovirus-infected cells that overexpress P143. ATPase activity copurified with P143 protein during purification and also during gel filtration at a high salt concentration. The ATPase activity did not require the presence of single-stranded DNA, but was stimulated fourfold by the addition of single-stranded DNA. The ATPase activity of P143 had a K m of 60 M and a turnover of 4.5 molecules of ATP hydrolyzed/s/molecule of enzyme, indicating moderate affinity for ATP and high catalytic efficiency. P143 unwound a 40-nucleotide primer in an ATP-dependent manner, indicating that the enzyme possesses in vitro DNA helicase activity. Based on this result, it seems likely that P143 functions as a helicase in viral DNA replication.

show abstract

The lef-3 gene of Autographa californica nuclear polyhedrosis virus encodes a single-stranded DNA-binding protein

Cited by 79 publications

References 27 publications

The Baculoviruses Occlusion‐Derived Virus: Virion Structure and Function

The Baculoviruses Occlusion‐Derived Virus: Virion Structure and Function

The baculovirus single-stranded DNA binding protein, LEF-3, forms a homotrimer in solution

The Autographa californica Nuclear Polyhedrosis Virus p143 Gene Encodes a DNA Helicase

Contact Info

Product

Resources

About