Binding of ICP4, TATA-binding protein, and RNA polymerase II to herpes simplex virus type 1 immediate-early, early, and late promoters in virus-infected cells - PubMed (original) (raw)
Binding of ICP4, TATA-binding protein, and RNA polymerase II to herpes simplex virus type 1 immediate-early, early, and late promoters in virus-infected cells
Padmavathi Sampath et al. J Virol. 2008 Mar.
Abstract
The binding of herpes simplex virus type 1 ICP4, TATA-binding protein (TBP), and RNA polymerase II (polII) to the promoter regions of representative immediate-early (IE) (ICP0), early (E) (thymidine kinase [tk]), and late (L) (glycoprotein C [gC]) genes on the viral genome was examined as a function of time postinfection, viral DNA replication, cis-acting sites for TFIID in the tk and gC promoters, and genetic background of ICP4. The binding of TBP and polII to the IE ICP0 promoter was independent of the presence of ICP4, whereas the binding of TBP and polII to the tk and gC promoters occurred only when ICP4 also bound to the promoters, suggesting that the presence of ICP4 at the promoters of E and L genes in virus-infected cells is crucial for the formation of transcription complexes on these promoters. When the TATA box of the tk promoter or the initiator element (INR) of the gC promoter was mutated, a reduction in the amount of TBP and polII binding was observed. However, a reduction in the amount of ICP4 binding to the promoters was also observed, suggesting that the binding of TBP-containing complexes and ICP4 is cooperative. The binding of ICP4, TBP, and polII was also observed on the gC promoter at early times postinfection or when DNA synthesis was inhibited, suggesting that transcription complexes may be formed early on L promoters and that additional events or proteins are required for expression. The ability to form these early complexes on the gC promoter required the DNA-binding domain but in addition required the carboxyl-terminal 524 amino acids of ICP4, which is missing the virus n208. This region was not required to form TBP- and polII-containing complexes on the tk promoter. n208 activates E but not L genes during viral infection. These data suggest that a region of ICP4 may differentiate between forming TBP- and polII-containing complexes on E and L promoters.
Figures
FIG. 1.
Standard real-time PCR curve generated with each primer pair in each experiment. The results are plotted on a semilogarithmic graph. Ct, threshold cycle. The KOS DNA standard described in Materials and Methods are shown as open circle. The software package SDS 2.3 from Applied BioSystems generates the curves and places on it the values for the experimental unknowns (closed circles).
FIG. 2.
Association of ICP4 and polII with the ICP0 and tk promoters in n12- and KOS-infected cells. ChIP experiments were performed as described in Materials and Methods. The graphs give the number of genomes bound by the tk and ICP0 promoters on the n12 and KOS genomes to ICP4 and polII. In this experiment we compare wt (KOS) to the ICP4 mutant n12 at 4 and 8 h postinfection.
FIG. 3.
Binding of ICP4, polII, and TBP to the tk promoters of KOS and LS-29-18. (A) ChIP experiments were done to determine the numbers of genomes bound to ICP4, polII, and TBP at 4 and 8 h postinfection in the presence and absence of PAA. (B) Representation of results in panel A as a percentage of genomes bound.
FIG. 4.
Binding of ICP4, polII, and TBP to different regions of the tk gene. The top panel shows a schematic of the tk gene and promoter and the regions which are amplified by the designated primers, which are listed in Table 1. The bottom panels give the percentages of genomes bound by ICP4, TBP, and polII at the promoter and different regions of the tk gene in KOS- and LS-29-18-infected cells. The results are for 4 h postinfection.
FIG. 5.
Binding of ICP4, polII, and TBP to the gC promoters of KOS and gCInr. (A) ChIP experiments were done to determine the numbers of genomes bound to ICP4, polII, and TBP at 4 and 8 h postinfection in the presence and absence of PAA. (B) Representation of results in panel as a percentage of genomes bound.
FIG. 6.
Similar binding pattern of ICP4, polII, and TBP to the ICP8 promoter in KOS-, LS-29-18-, and gCInr-infected cells. ChIP analysis was conducted on KOS-, LS-19-18-, and gCInr-infected cells at 4 and 8 h postinfection to examine ICP4, TBP, and polII binding to the ICP8 promoter. (A) Percentage of genomes bound at 4 and 8 h postinfection in the absence of PAA. (B) Percentage of genomes bound at 4 and 8 h postinfection in the presence of PAA.
FIG. 7.
Binding of ICP4, TBP, and polII to the ICP0, tk, and gC promoters in KOS-, i13-, and n208-infected cells. ChIP analysis was conducted on KOS-, i13-, and n208-infected cells to examine the binding of ICP4, TBP, and polII to the ICP0, tk, and gC promoters on the viral genomes. The graphs give percentages of genomes bound at 4 h postinfection (solid bars) and 8 h postinfection (hatched bars).
References
- Alwine, J. C., W. L. Steinhart, and C. W. Hill. 1974. Transcription of herpes simplex type 1 DNA in nuclei isolated from infected HEp-2 and KB cells. Virology 60302-307. - PubMed
- Campbell, M. E., J. W. Palfreyman, and C. M. Preston. 1984. Identification of herpes simplex virus DNA sequences which encode a trans-acting polypeptide responsible for stimulation of immediate early transcription. J. Mol. Biol. 1801-19. - PubMed
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