The replication of viral DNA genomes within host cell nuclei takes place in discrete regions, which can be visualized as distinctive structures specific to virus-infected cells. Centers of viral DNA replication represent an accumulation of replication proteins and other viral and cellular components in addition to newly synthesized viral DNA. This has been best characterized in herpes simplex virus type 1 (HSV-1)-infected cells (24). Viral replication centers have been proposed to assemble nonrandomly but appear to be determined by preexisting host nuclear structures (3). There is evidence that viral DNA replication sites are located adjacent to promyelocytic leukemia protein (PML) bodies. PML is the defining component of PML bodies, PML oncogenic domains (PODs) or nuclear domain 10, which are thought to be involved in the modulation of cell growth and proliferation (for a review see reference 17).We demonstrated a dynamic nuclear localization pattern for early baculovirus proteins IE2 and PE38 during the infection cycle of Autographa californica multicapsid nuclear polyhedrosis virus (AcMNPV) (22). The genes encoding both factors belong to a set of 10 viral genes involved in baculovirus DNA replication. Based on transient complementation assays using origin-containing plasmids, IE2 and PE38 are not essential but rather have a stimulatory effect on DNA replication (12, 16). How they contribute to the replication of baculovirus DNA in infected cells is still speculative. It has been suggested that other factors, such as late expression factor 3 (LEF-3), a homotrimeric single-stranded DNA binding protein (SSB) which improves the strand displacement ability of the viral DNA polymerase, are essential in viral DNA replication (5,8,12,19). In addition, LEF-3 may be involved in the transport of putative helicase P143 to the nucleus, which is in line with the observation that LEF-3 interacts directly with P143 (6, 30).Our study of the functional role of IE2 and PE38 is based on experiments in which their localization within infected cells was determined. We previously demonstrated that IE2 is localized in nuclear domains that changed to threadlike structures late in infection, while PE38 was only partly present in nuclear dots very early in infection (22). After transient expression both proteins were found to be associated with PODs in mammalian cells; however, during the early phase of infection only IE2 and human PML colocalized in insect cells (22). If both PE38 and IE2 were directly involved in viral DNA replication, their localization would be expected to be close to that of replicating DNA. Since PE38 shows mainly a sandy nuclear staining pattern, its spatial distribution hardly contributes to a better understanding of whether PE38 is associated with sites of viral DNA replication. Therefore, we focused on the spatial and temporal distribution of IE2 in the context of viral DNA replication.Here, we present the first evidence that AcMNPV replica-* Corresponding author. Mailing address:
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...
The TATA-box-binding protein (TBP) plays a key role in initiating eukaryotic transcription and is used by many viruses for viral transcription. We previously reported increased TBP levels during infection with the baculovirus Autographa californica multicapsid nuclear polyhedrovirus (AcMNPV). The TBP antiserum used in that study, however, cross-reacted with a baculoviral protein. Here, we reported that increased amounts of nuclear TBP were detected upon infection of Spodoptera frugiperda and TN-368 cells with a TBP-specific antiserum. TBP levels increased until 72 h post-infection (p.i.), whilst tbp transcripts decreased by 16 h p.i., which suggested a virus-induced influence on the TBP protein levels. To address a potential modification of the TBP degradation pathway during infection, we investigated the possible role of viral ubiquitin. Infection studies with AcMNPV recombinants carrying a mutated viral ubiquitin gene revealed that the TBP increase during infection was not altered. In addition, pulse-chase experiments indicated a high TBP half-life of~60 h in uninfected cells, suggesting that a virus-induced increase of TBP stability was unlikely. This increase in TBP correlated with a redistribution to nuclear domains resembling sites of viral DNA synthesis. Furthermore, we observed colocalization of TBP with host RNA polymerase (RNAP) II, but only until 8 h p.i., whilst TBP, but not RNAPII, was present in the enlarged replication domains late during infection. Thus, we suggested that AcMNPV adapted a mechanism to accumulate the highly stable cellular TBP at sites of viral DNA replication and transcription.
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