Specificity of the Endonuclease Activity of the Baculovirus Alkaline Nuclease for Single-stranded DNA

Abstract

Baculoviruses contain double-stranded (ds), 1 circular, supercoiled DNA genomes of 100 -180 kbp and belong to the family Baculoviridae, which includes two genera, the granuloviruses and the nucleopolyhedroviruses (NPVs) (1, 2). One NPV, Autographa californica multiple nucleocapsid NPV (AcMNPV), is widely used as a model for analysis of baculovirus replication in infected cells and for the generation of recombinant viruses for the expression of foreign genes. Replication of baculovirus genomes proceeds in discrete replication factories in the nuclei of infected cells (3, 4) presumably via a rolling-circle intermediates (5, 6), although the mechanisms of initiation, elongation, processing, and maturation have not been determined. Six viral factors including a transactivator of early gene transcription (IE-1), DNA polymerase, DNA helicase, DNA primase (LEF-1), an accessory factor (LEF-2), and ssDNA-binding protein (LEF-3) are necessary, and several other factors (P35, IE-2, PE38, and LEF-7) are stimulatory for replication of plasmids in the transient replication assays (7-12). The plasmid DNA synthesized in the presence of the essential and stimulatory replication factors in the transient assays is present as concatemeric molecules (5, 13) indicating that other viral products may be required for processing of nascent genomes. Two viral proteins, very late expression factor 1 (VLF-1) and alkaline nuclease (AN), have been predicted to participate in maturation of baculovirus genomes (14, 15). VLF-1 is required for expression of very late genes (16, 17) but also plays an unknown role in replication (14). VLF-1 is a member of the integrase/resolvase family of proteins (16), and it interacts preferentially with DNA crosses (18). This suggests that VLF-1 may function in the processing of branched intermediates in replication and recombination. The AN involvement in maturation of baculovirus genomes was predicted on the basis of structural homology of this enzyme and alkaline nuclease of viruses from the family Herpesviridae. Baculovirus AN contains five domains homologous to conserved motifs found in AN of alphaherpesviruses, although three other conserved motifs of the herpesvirus enzyme are not detected in the AcMNPV AN which is ϳ1.5-fold smaller than the herpes simplex virus type 1 (HSV-1) homolog (15). Although HSV-1 AN is not essential for viral DNA synthesis (19), deletion of the gene encoding AN results in the accumulation of complex branched concatemeric genomes indicating that AN either cleaves or prevents the generation of these structures (20,21). Because complex concatemeric intermediates are likely produced in DNA replication of baculoviruses (5, 6, 13), baculovirus AN may be also involved in the resolution of replication intermediates and genome maturation (15).Computer analyses reveal that AN of baculoviruses and herpesviruses belongs to a protein family typified by bacteriophage exonuclease (22, 23), a toroidal trimeric enzyme (24) that produces single-stranded DNA overhangs that serve as intermediates in...

Analysis of the Autographa californica Multiple Nucleopolyhedrovirus Overlapping Gene Pair lef3 and ac68 Reveals that AC68 Is a Per Os Infectivity Factor and that LEF3 Is Critical, but Not Essential, for Virus Replication

“…lef3 was originally identified as one of six genes required for viral DNA replication in transient assays (22). LEF3 has been shown to be an SSB protein and may regulate the viral protein alkaline nuclease (15,34). Recently, a lef3 knockout virus was constructed that deleted the lef3 ORF and the 5= end of ac68 (53).…”

“…lef3 was identified as one of the genes essential for baculovirus DNA replication by transient analyses and recently by construction of a KO mutant (22,53). LEF3 has also been shown to interact with the viral proteins P143 and alkaline exonuclease, which are involved in viral DNA replication and recombination (26,33,34). Using plasmid transfection experiments, it has also been shown that P143, a DNA helicase with ATPase and DNA binding activities, is dependent upon LEF3 for transport to the nucleus (15,32,50).…”

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

“…LEF-3 is also responsible for transporting P143, a predicted DNA unwinding (helicase) protein, into the nucleus, where it is required for viral DNA replication (26,29,45). LEF-3 may also regulate the activity of a viral alkaline nuclease (AN) during viral DNA replication (32). We have previously mapped the region carrying the nuclear localization signal of LEF-3 to residues 26 to 32 within the N-terminal 56-amino-acid domain (1,7).…”

Characterization of a Baculovirus Lacking the Alkaline Nuclease Gene

“…We have also found that it has an endonuclease activity and that this activity appears to be specific for singlestranded regions of DNA most likely caused by physical distortions that occur as a result of the negative superhelicity of the DNA (19). Furthermore, this endonucleolytic activity was associated with a high degree of specificity for secondary cleavage on the strand opposite the initial nick such that, rather than random endonucleolytic cleavage, the result is specific cleavage of both DNA strands at or very near the same position.…”