The promoters of HSV-1 IE and E genes contain Sp1 binding sites upstream of the TATA box. In contrast, Sp1 binding sites are not found in true L promoters (54
). After viral DNA replication, IE and E gene transcription declines and L gene expression predominates. Considering that the abundance, modification state, or activity of cellular transcription factors can be modified as a consequence of viral infection, we entertained the hypothesis that Sp1, which activates IE and E promoters, is altered with respect to its activity and that this may contribute to the decrease in IE and E gene expression as infection proceeds.
To examine the abundance and modification state of Sp1 as a function of HSV infection, Vero cells were infected with wt HSV (strain KOS) and samples for Western blot analysis were obtained at different time points after infection. The Western blot probed with Sp1 antibody showed that there was no large change in the total amount of Sp1 throughout the infection (Fig. ). Sp1 was present in two major forms with different electrophoretic mobilities as reported previously (30
). Early in infection, most of the Sp1 was present as a faster-migrating form. However, the majority of Sp1 was modified to a slower-migrating form as infection proceeded. At approximately 4 h p.i., the slower-migrating form became the major species, and by 6 h p.i., the faster-migrating form of Sp1 was almost completely absent (Fig. ). When the kinetics of modification of Sp1 were compared to the kinetics of accumulation of an E gene (tk
) mRNA, the modification of Sp1 to the slower-migrating form occurred slightly prior to the decrease in the level of accumulated tk
message (Fig. ).
FIG. 1. Modification of Sp1 during HSV-1 infection. (A) Western blot analysis of cell extracts prepared from Vero cells infected with KOS in the presence and absence of PAA. At indicated times after infection, the infected cells were harvested directly into the (more ...)
One characteristic of most E gene expression is that it often decreases after DNA replication. When viral DNA replication was blocked by the addition of PAA, tk mRNA abundance persisted at a greater level than in the absence of PAA (Fig. ). Also shown in Fig. is that while there was an increase in the amount of the slower-migrating form of Sp1 as infection proceeded in the presence of PAA, the amount of the faster-migrating form of Sp1 persisted in greater quantities than that in the absence of PAA. Thus, the presence of the faster-migrating form of Sp1 correlates with increased tk mRNA abundance.
It has been shown that Sp1 is posttranslationally modified by glycosylation and phosphorylation on its Ser and Thr residues (4
). These modifications are indirectly or directly involved in regulating the activity of Sp1 (2
). To determine if the slower migration of Sp1 as a function of infection was due to phosphorylation, lysate from Vero cells infected for 8 h with KOS was treated with calf intestinal alkaline phosphatase (CIP). Within 10 min of incubation at 30°C, the slower-migrating form of Sp1 was completely changed to the faster-migrating form (Fig. ). Incubation at 4°C resulted in less than complete conversion. The mock reaction consisted of incubating the lysate at 30o
in the presence of phosphatase inhibitors and in the absence of CIP. The mobilities of the Sp1 forms in this sample resembled those of the 8-h sample in Fig. . These results suggest that Sp1 is phosphorylated during HSV-1 infection.
To investigate the genetic requirements for the phosphorylation of Sp1, the modification state of Sp1 was studied as a function of infection with several IE mutants (d120, d109, d106, and 5dl
1.2). These viruses possess mutations in different combinations of IE genes (12
). The modification state of Sp1 in cells infected with d120, d106, and d109 was similar to that in uninfected cells (Fig. ). A common feature of these mutants is that they do not produce ICP4. In cells infected with the ICP27 deletion mutant, 5dl
1.2, a notable amount of Sp1 was converted to the slower-migrating form (Fig. ). The pattern of Sp1 phosphorylation in 5dl
1.2-infected cells was similar to that seen in KOS-infected cells in the presence of PAA (Fig. and ) in that there was partial conversion to the phosphorylated form. Unlike d120, d106, and d109, ICP4 is expressed in 5dl
1.2-infected cells. As a consequence, E genes and some leaky L genes are expressed in this background (34
); however, DNA replication is severely reduced. This is similar to the gene expression profile of cells infected with wt HSV in the presence of PAA (11
). These results suggest that ICP4 is directly or indirectly involved in the phosphorylation of Sp1. It is possible that a viral or cellular gene induced by ICP4 is also involved in the modification of Sp1. Additionally, DNA replication may be required for the maximum modification of Sp1 seen during productive infection.
FIG. 2. Effect of IE gene mutations on the phosphorylation of Sp1. Vero cells were infected with indicated viruses, lysed at 2 and 6 h after infection, and then subjected to Western blot analysis as described for Fig. . The IE genes not expressed (more ...)
Sp1 can be phosphorylated by variety of cellular kinases, such as protein kinase A (PKA), DNA-dependant protein kinase (DNA-PK), and casein kinase II (CKII), and its activity is affected by several different mechanisms (2
). In some cases phosphorylation of Sp1 results in a change in its DNA binding affinity (2
). Therefore, we compared the abilities of infected-cell Sp1 and uninfected-cell Sp1 to bind to DNA in mobility shift assays. Whole-cell lysates were prepared from uninfected and KOS-infected Vero cells at 2 and 8 h p.i. Purified baculovirus-produced Sp1 was used as a control. Purified Sp1 showed a single DNA-protein complex (a), which was supershifted in the presence of an Sp1-specific antibody (c) (Fig. ). Several DNA-protein complexes were seen by using samples from uninfected cells. One of these (a) had the same mobility as that seen with the purified Sp1 and was supershifted by the addition of antibody (c). Another complex migrating closely with the a complex (b) was not supershifted by the antibody. With the infected-cell lysates, there were no apparent differences in the amounts of Sp1-DNA complexes formed between the mock and 2-h-p.i. samples, or 2- and 8-h-p.i. samples. These results suggest that phosphorylation of Sp1 during HSV-1 infection does not cause significant changes in the DNA binding ability of Sp1 (Fig. ). This was also confirmed indirectly during purification of Sp1 using DNA affinity chromatography. Both forms of Sp1 were purified to very similar yields (Fig. ).
FIG. 3. DNA binding of uninfected and infected cell Sp1. Electrophoretic mobility shift assay using KOS-infected Vero cell lysates. Cell lysates were prepared from uninfected and infected cells at the indicated times p.i. The cell lysate was incubated with a (more ...)
FIG. 4. Activity of purified Sp1. (A) Silver-stained gel of purified uninfected-cell (M) and infected-cell Sp1. Nuclear extracts were prepared from 6 liters of KOS-infected (12 h) and uninfected HeLa cells. Sp1 was purified from the nuclear extracts by passage (more ...)
To more directly investigate the effect of phosphorylation on Sp1 activity, Sp1 was purified from uninfected and infected HeLa cells. Using wheat germ agglutinin and DNA affinity columns (26
), both forms of Sp1 were purified with similar yields and to near-homogeneity (Fig. ). The majority of the uninfected-cell Sp1 was of the higher-mobility species, while most of the infected-cell Sp1 was comprised of the lower-mobility species. While it is difficult to determine the absolute concentration of Sp1 in these preparations, the silver-stained gel of Fig. demonstrates that the two preparations of Sp1 were of similar concentrations.
To test the abilities of the two forms of Sp1 to activate a promoter containing Sp1 binding sites, the ICP4 promoter was used in reconstituted in vitro transcription reactions with and without purified Sp1 from infected and uninfected cells. The ICP4 promoter was used because it contains Sp1 binding sites and because the tk promoter is not sufficiently active in this system to produce unambiguous results. The specificity of activation was first established by adding uninfected-cell Sp1 to reactions, comparing templates driven by the entire ICP4 promoter and by an ICP4 promoter with a deletion for the upstream region. As shown in Fig. , the purified Sp1 activated transcription from the ICP4 promoter. When assayed on a similar template with the Sp1 binding sites deleted (pTP4/LSWT), activation of transcription by Sp1 was not observed (Fig. ). These results demonstrated that the purified Sp1 was functional and specific.
Subsequently, both the infected- and uninfected-Sp1 preparations were tested at different concentrations for the ability to activate the intact ICP4 promoter (Fig. ). Figure represents data averaged from three experiments. At all the concentrations of Sp1 tested, the uninfected-cell Sp1 was more efficient in activation of the ICP4 promoter than the infected-cell Sp1. Only after the addition of 10 μl of infected-cell Sp1 did the level of activation exceed that seen with 2 μl of uninfected-cell Sp1. The level of activation with the infected-cell Sp1 may be expected to increase at the higher concentrations if the low level of high-mobility Sp1 in this preparation (Fig. ) has the same activity as uninfected-cell Sp1. Therefore, we conclude that the form of Sp1 phosphorylated during infection is less functional for activation in vitro.