scholarly journals Autographa californica Multiple Nucleopolyhedrovirus LEF-2 Is a Capsid Protein Required for Amplification but Not Initiation of Viral DNA Replication

2010 ◽  
Vol 84 (10) ◽  
pp. 5015-5024 ◽  
Author(s):  
Carol P. Wu ◽  
Yi-Ju Huang ◽  
Jen-Yeu Wang ◽  
Yueh-Lung Wu ◽  
Huei-Ru Lo ◽  
...  
Keyword(s):  
Dna Replication ◽  
Autographa Californica ◽  
Host Cells ◽  
Growth Curve Analysis ◽  
Late Gene Expression ◽  
Viral Dna ◽  
Viral Dna Replication ◽  
Virus Particles ◽  
Multiple Nucleopolyhedrovirus ◽  
Infected Cells

ABSTRACT The late expression factor 2 gene (lef-2) of baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) has been identified as one of the factors essential for origin-dependent DNA replication in transient expression assays and has been shown to be involved in late/very late gene expression. To study the function of lef-2 in the life cycle of AcMNPV, lef-2 knockout and repair bacmids were generated by homologous recombination in Escherichia coli. Growth curve analysis showed that lef-2 was essential for virus production. Interestingly, a DNA replication assay indicated that lef-2 is not required for the initiation of viral DNA replication and that, rather, it is required for the amplification of DNA replication. lef-2 is also required for the expression of late and very late genes, as the expression of these genes was abolished by lef-2 deletion. Temporal and spatial distributions of LEF-2 protein in infected cells were also analyzed, and the data showed that LEF-2 protein was localized to the virogenic stroma in the nuclei of the infected cells. Analysis of purified virus particles revealed that LEF-2 is a viral protein component of both budded and occlusion-derived virions, predominantly in the nucleocapsids of the virus particles. This observation suggests that LEF-2 may be required immediately after virus entry into host cells for efficient viral DNA replication.

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

2010 ◽  
Vol 84 (12) ◽  
pp. 6153-6162 ◽  
Author(s):  
Mei Yu ◽  
Eric B. Carstens
Keyword(s):  
Gene Expression ◽  
Dna Replication ◽  
Viral Genome ◽  
Virus Production ◽  
Autographa Californica ◽  
Late Gene ◽  
Late Gene Expression ◽  
Viral Dna ◽  
Viral Dna Replication ◽  
Budded Virus

ABSTRACT Autographa californica multiple nucleopolyhedrovirus (AcMNPV) lef-3 is one of nine genes required for viral DNA replication in transient assays. LEF-3 is predicted to contain several domains related to its functions, including nuclear localization, single-strand DNA binding, oligomerization, interaction with P143 helicase, and interaction with a viral alkaline nuclease. To investigate the essential nature of LEF-3 and the roles it may play during baculovirus DNA replication, a lef-3 null bacmid (bKO-lef3) was constructed in Escherichia coli and characterized in Sf21 cells. The results showed that AcMNPV lef-3 is essential for DNA replication, budded virus production, and late gene expression in vivo. Cells transfected with the lef-3 knockout bacmid produced low levels of early proteins (P143, DNA polymerase, and early GP64) and no late proteins (P47, VP39, or late GP64). To investigate the functional role of domains within the LEF-3 open reading frame in the presence of the whole viral genome, plasmids expressing various LEF-3 truncations were transfected into Sf21 cells together with bKO-lef3 DNA. The results showed that expression of AcMNPV LEF-3 amino acids 1 to 125 was sufficient to stimulate viral DNA replication and to support late gene expression. Expression of Choristoneura fumiferana MNPV lef-3 did not rescue any LEF-3 functions. The construction of a LEF-3 amino acid 1 to 125 rescue bacmid revealed that this region of LEF-3, when expressed in the presence of the rest of the viral genome, stimulated viral DNA replication and late and very late protein expression, as well as budded virus production.

scholarly journals Analysis of an Autographa californica Multicapsid Nucleopolyhedrovirus lef-6-Null Virus: LEF-6 Is Not Essential for Viral Replication but Appears To Accelerate Late Gene Transcription

2002 ◽  
Vol 76 (11) ◽  
pp. 5503-5514 ◽  
Author(s):  
Guangyun Lin ◽  
Gary W. Blissard
Keyword(s):  
Dna Replication ◽  
Viral Replication ◽  
Gene Transcription ◽  
Cellular Localization ◽  
Autographa Californica ◽  
Late Gene ◽  
Infection Cycle ◽  
Viral Dna ◽  
Viral Dna Replication ◽  
Late Transcription

ABSTRACT The Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) lef-6 gene was previously shown to be necessary for optimal transcription from an AcMNPV late promoter in transient late expression assays. In the present study, we examined the expression and cellular localization of lef-6 during the AcMNPV infection cycle and generated a lef-6-null virus for studies of the role of lef-6 in the infection cycle. Transcription of lef-6 was detected from 4 to 48 h postinfection, and the LEF-6 protein was identified in dense regions of infected cell nuclei, a finding consistent with its potential role as a late transcription factor. To examine lef-6 in the context of the AcMNPV infection cycle, we deleted the lef-6 gene from an AcMNPV genome propagated as an infectious BACmid in Escherichia coli. Unexpectedly, the resulting AcMNPV lef-6-null BACmid (vAclef6KO) was able to propagate in cell culture, although virus yields were substantially reduced. Thus, the lef-6 gene is not essential for viral replication in Sf9 cells. Two “repair” AcMNPV BACmids (vAclef6KO-REP-P and vAclef6KO-REP-ie1P) were generated by transposition of the lef-6 gene into the polyhedrin locus of the vAclef6KO BACmid. Virus yields from the two repair viruses were similar to those from wild-type AcMNPV or a control (BACmid-derived) virus. The lef-6-null BACmid (vAclef6KO) was further examined to determine whether the deletion of lef-6 affected DNA replication or late gene transcription in the context of an infection. The lef-6 deletion did not appear to affect viral DNA replication. Using Northern blot analysis, we found that although early transcription was apparently unaffected, both late and very late transcription were delayed in cells infected with the lef-6-null BACmid. This phenotype was rescued in viruses containing the lef-6 gene reinserted into the polyhedrin locus. Thus, the lef-6 gene was not essential for either viral DNA replication or late gene transcription, but the absence of lef-6 resulted in a substantial delay in the onset of late transcription. Therefore, lef-6 appears to accelerate the infection cycle of AcMNPV.

scholarly journals A Dominant-Negative Herpesvirus Protein Inhibits Intranuclear Targeting of Viral Proteins: Effects on DNA Replication and Late Gene Expression

2000 ◽  
Vol 74 (21) ◽  
pp. 10122-10131 ◽  
Author(s):  
Elizabeth E. McNamee ◽  
Travis J Taylor ◽  
David M. Knipe
Keyword(s):  
Gene Expression ◽  
Dna Replication ◽  
Viral Replication ◽  
Gene Transcription ◽  
Dominant Negative ◽  
Late Gene ◽  
Late Gene Expression ◽  
Viral Dna ◽  
Late Genes ◽  
Infected Cells

ABSTRACT The d105 dominant-negative mutant form of the herpes simplex virus 1 (HSV-1) single-stranded DNA-binding protein, ICP8 (d105 ICP8), inhibits wild-type viral replication, and it blocks both viral DNA replication and late gene transcription, although to different degrees (M. Gao and D. M. Knipe, J. Virol. 65:2666–2675, 1991; Y. M. Chen and D. M. Knipe, Virology 221:281–290, 1996). We demonstrate here that this protein is also capable of preventing the formation of intranuclear prereplicative sites and replication compartments during HSV infection. We defined three patterns of ICP8 localization using indirect immunofluorescence staining of HSV-1-infected cells: large replication compartments, small compartments, and no specific intranuclear localization of ICP8. Cells that form large replication compartments replicate viral DNA and express late genes. Cells that form small replication compartments replicate viral DNA but do not express late genes, while cells without viral replication compartments are incapable of both DNA replication and late gene expression. The d105 ICP8 protein blocks formation of prereplicative sites and large replication compartments in 80% of infected cells and formation of large replication compartments in the remaining 20% of infected cells. The phenotype ofd105 suggests a correlation between formation of large replication compartments and late gene expression and a role for intranuclear rearrangement of viral DNA and bound proteins in activation of late gene transcription. Thus, these results provide evidence for specialized machinery for late gene expression within replication compartments.

scholarly journals Analysis of an Autographa californica Nucleopolyhedrovirus lef-11 Knockout: LEF-11 Is Essential for Viral DNA Replication

2002 ◽  
Vol 76 (6) ◽  
pp. 2770-2779 ◽  
Author(s):  
Guangyun Lin ◽  
Gary W. Blissard
Keyword(s):  
Dna Replication ◽  
Viral Replication ◽  
Autographa Californica ◽  
Sf9 Cells ◽  
Infection Cycle ◽  
Wild Type ◽  
Viral Dna ◽  
Viral Dna Replication ◽  
Late Transcription ◽  
Late Expression

ABSTRACT The Autographa californica nucleopolyhedrovirus (AcMNPV) lef-11 gene was previously identified by transient late expression assays as a gene important for viral late gene expression. The lef-11 gene was not previously identified as necessary for DNA replication in transient origin-dependent plasmid DNA replication assays. To examine the role of lef-11 in the context of the infection cycle, we generated a deletion of the lef-11 gene by recombination in an AcMNPV genome propagated as a BACmid in Escherichia coli. The resulting AcMNPV lef-11-null BACmid (vAclef11KO) was unable to propagate in cell culture, although a “repair” AcMNPV BACmid (vAclef11KO-REP), which was generated by transposition of the lef-11 gene into the polyhedrin locus of the vAclef11KO BACmid, was able to replicate in a manner similar to wild-type or control AcMNPV viruses. Thus, the lef-11 gene is essential for viral replication in Sf9 cells. The vAclef11KO BACmid was examined to determine if the defect in viral replication resulted from a defect in DNA replication or from a defect in late transcription. The lef-11-null BACmid and control BACmids were transfected into Sf9 cells, and viral DNA replication was monitored. The viral DNA genome of the lef-11-null BACmid (vAclef11KO) was not amplified, whereas replication and amplification of the genomes of the repair BACmid (vAclef11KO-REP), wild-type AcMNPV, and a nonpropagating gp64-null control BACmid (vAcGUSgp64KO) were readily detected. Northern blot analysis of transcripts from selected early, late, and very late genes showed that late and very late transcription was absent in cells transfected with the lef-11-null BACmid. Thus, in contrast to prior studies using transient replication and late expression assays, studies of a lef-11-null BACmid indicate that LEF-11 is required for viral DNA replication during the infection cycle.

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