Dynamic Nuclear Localization of the Baculovirus Proteins IE2 and PE38 during the Infection Cycle: The Promyelocytic Leukemia Protein Colocalizes with IE2

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Knebel-Mörsdorf

2007

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Baculovirus DNA binding protein (DBP) binds preferentially single-stranded DNA in vitro and colocalizes with viral DNA replication sites. Here, its putative role as viral replication factor has been addressed by RNA interference. Silencing of DBP in Autographa californica multiple nucleopolyhedrovirus-infected cells increased expression of LEF-3, LEF-4, and P35. In contrast, expression of the structural genes coding for P39 and polyhedrin was suppressed while expression of genes coding for P10 and GP64 was unaffected. In the absence of DBP, viral DNA replication sites were formed, indicating replication of viral DNA. Electron microscopy studies, however, revealed a loss of formation of polyhedra and virus envelopment, suggesting that the primary role of DBP is viral formation rather than viral DNA replication.

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confidence: 99%

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confidence: 99%

Studies of the Silencing of Baculovirus DNA Binding Protein

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Knebel-Mörsdorf

2007

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“…Hence, we investigated the time course of TBP expression during AcMNPV infection. The insect cell lines Trichoplusia ni TN-368 and S. frugiperda IPLB21 were infected with AcMNPV plaque isolate E at a multiplicity of 10 to 20 PFU per cell, and detergentbased nuclear protein extracts were prepared as previously described (15). The analysis was performed by Western blotting with a polyclonal antibody directed against S. frugiperda TBP.…”

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Baculovirus Infection Raises the Level of TATA-Binding Protein That Colocalizes with Viral DNA Replication Sites

Mainz

Mans

et al. 2002

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During the infection cycle of Autographa californica multicapsid nuclear polyhedrosis virus, the TATAbinding protein (TBP) of the insect host cell likely participates in early viral transcription, which is mediated by the host RNA polymerase II. However, the role of TBP in late and very late viral transcription, which is accomplished by an alpha-amanitin-resistant RNA polymerase, is unclear. We observed a dramatic increase of TBP protein during the late phases of infection. TBP mRNA levels, however, were not coordinately increased. Indirect-immunofluorescence studies revealed a nuclear redistribution of TBP during infection. After labeling of viral replication centers with bromodeoxyuridine (BrdU), costaining of TBP and BrdU showed that TBP localized to viral DNA replication centers. These results suggest a putative role of TBP during late viral transcription, which may occur in close proximity to viral DNA replication.The TATA-binding protein (TBP) is a universal transcription factor that is required for initiation by all three eukaryotic RNA polymerases. TBP was identified as a subunit of TFIID, a multisubunit complex composed of TBP and TBP-associated factors (for a review, see reference 7). As part of the preinitiation complex at both TATA box-containing and TATA-less promoters, TBP resembles a target for transcriptional activators and repressors (for a review, see reference 19). One unique feature of TBP is its high level of conservation among all eukaryotes and archaea. In most cases, TBP is encoded by a single gene. The carboxy-terminal core domains of all characterized TBPs are more than 75% identical to the human TBP, while the amino-terminal domain is poorly conserved (7). In insects, TBP-encoding cDNAs have been cloned from Drosophila melanogaster cells (8) and from Spodoptera frugiperda cells, which resemble a permissive cell line of the baculovirus Autographa californica multicapsid nuclear polyhedrosis virus (AcMNPV) (20). An amino acid sequence comparison reveals that the C terminus of S. frugiperda TBP is 93% identical to that of Drosophila TBP and about 75% identical to the C termini of all other known TBPs (20).The gene expression cascade of AcMNPV is temporally regulated and characterized by the sequential involvement of two different RNA polymerases. Host RNA polymerase II recognizes early viral, TATA box-containing promoters; thus, TBP is thought to participate in early viral transcription. Late genes and the hyperexpressed very late genes encoding p10 and polyhedrin are transcribed by an alpha-amanitin-resistant RNA polymerase that has been reported to be a complex of four virus-encoded proteins (3, 4). The purified RNA polymerase recognizes late and very late viral promoters, although the burst of very late transcription was not observed by in vitro experiments, suggesting that factors contributing to the hyperexpression of the very late promoters are still unknown (4). Only when in vitro transcription assays were performed with protein extracts of purified cell nuclei was the burst in ver...

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“…(D) Expression of the very late protein P10 was detectable with rabbit anti-P10 serum (29). Expression of LEF-4, IE2, LEF-3, DBP, P39, and P10 was analyzed on samples of nuclear protein fractions, and P35 and GP64 expression was detected in cytoplasmic fractions as described previously (21). Protein size markers are shown on the left, and the identities of the viral proteins are indicated on the right.…”

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“…S. frugiperda cells were infected with AcMNPV at 10 PFU/cell, and detergent-based nuclear extracts were prepared from infected cells 4,8,16,24,48, and 72 h postinfection (p.i.). As previously described, the cell pellet was lysed in 0.5% NP-40 and centrifuged and the supernatant (cytoplasmic fraction) was adjusted to 0.1 N NaOH (21). The pelleted nuclei were resuspended in 1% NP-40 and adjusted to 0.1 N NaOH.…”

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Expression of Baculovirus Late and Very Late Genes Depends on LEF-4, a Component of the Viral RNA Polymerase Whose Guanyltransferase Function Is Essential

Knebel-Mörsdorf

et al. 2006

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