The high mobility group protein 1 is a coactivator of herpes simplex virus ICP4 in vitro

Abstract

ICP4 is an activator of herpes simplex virus early and late gene transcription during infection and in vitro can efficiently activate the transcription of a core promoter template containing only a TATA box and an initiator element. In this study, we noted that the extent of activation by ICP4 in vitro was highly dependent on the purity of TFIID when recombinant TFIIB, TFIIE, and TFIIF were used as sources of these factors. ICP4 efficiently activated transcription with a crude TFIID fraction. However, when immunoaffinity-purified TFIID was used in place of the less pure TFIID, ICP4 activated transcription to a significantly lesser extent. This finding indicated that the crude TFIID fraction may contain additional factors that serve as coactivators of ICP4. To test this hypothesis, the crude TFIID preparation was further fractionated by gel filtration chromatography. The TFIID that eluted from the column lacked the hypothesized coactivator activity. A fraction well separated from TFIID contained an activity that when added with the TFIID fraction resulted in higher levels of transcription in the presence ICP4. Further purification of the coactivator-containing fraction resulted in the isolation of a single 30-kDa polypeptide (p30). p30 was also shown to serve as a coactivator of ICP4 with immunoaffinity-purified TFIID; however, p30 had no effect on basal transcription. Amino acid sequence analysis revealed that p30 was the high mobility group protein 1, which has been shown to facilitate the formation of higher-order DNA-protein complexes.

FIG. 1

Abilities of different TFIID preparations to support ICP4-activated transcription. In vitro transcription reactions were performed with the Pol II fraction CC, rTFIIB, -E, and -F, and either immunoaffinity-purified eTFIID or the crude TFIID fraction, DB, in the absence and presence of purified ICP4. The quantity of each TFIID preparation was normalized as described in Materials and Methods. These conditions were tested on a gC core promoter template containing a TATA box and either a wt or a mut Inr. Shown are the primer extension products from reverse-transcribed RNA synthesized in the in vitro reactions.

FIG. 2

Purified TFIID lacks basal Inr activity and less efficiently supports ICP4 activation. The DB fraction was applied to a Superose 6 gel filtration column. The fractions containing TFIID were identified by slot blot analysis of column fractions with an antibody directed against TBP. The TFIID eluting from this column is designated Superose TFIID. In vitro transcriptions were performed with Pol II (CC), rTFIIB, -E, and -F, and either immunoaffinity-purified TFIID, DB fraction, or Superose TFIID. The quantity of each TFIID preparation was normalized as described in Materials and Methods. (A) Basal Inr activity of three different TFIID preparations. Each TFIID preparation was tested on the gC core promoter containing a TATA box and either a wt or a mut Inr. (B) ICP4-activated transcription using the three different TFIID preparations. Each reaction mixture received either 0, 50, or 100 ng of purified ICP4. Transcription reactions in lanes 1, 4, and 7 represent the basal levels of transcription with each of the three TFIID preparations.

FIG. 3

Gel filtration chromatography of the DB fraction further fractionates an ICP4 coactivator. (A) Transcription in the presence of ICP4 and gel filtration chromatography fractions. Superose 6 gel filtration fractions were assayed by adding a portion of each fraction to in vitro transcription reaction mixtures containing Pol II, rTFIIB, -E, and -F, Superose TFIID, and the wt gC core promoter template. ICP4 was included in each reaction except in lane B, which represents basal transcription levels with this combination of GTFs. The second lane indicates the level of ICP4-activated transcription without the addition of any column fractions. Only a subset (fractions 23 to 28) of the fractions tested are shown. A total of 30 fractions were collected from the column, with TFIID eluting in fraction 4. (B) Silver-stained gel after SDS-PAGE analysis of Superose 6 fractions. A portion of each fraction was loaded onto an SDS–10% polyacrylamide gel and silver stained. Shown are fractions 17 to 27.

FIG. 4

Ion-exchange chromatography of Superose 6 ICP4 coactivator fractions. Superose 6 fractions 24 and 25 were loaded onto a Mono Q ion-exchange column and eluted with a linear 0.1 M to 0.5 M KCl gradient. Fractions were assayed by adding a portion of each fraction to in vitro transcription reaction mixtures containing Pol II, rTFIIB, -E, and -F, Superose TFIID, and the wt gC core promoter template. ICP4 was included in each reaction except in lane B, which represents basal transcription levels with this combination of GTFs. The second lane indicates the level of ICP4 activated transcription without the addition of any column fractions. Flowthrough (FT) represents the material that did not bind the column at 0.1 M KCl. Shown are the results with fractions 18 to 24.

FIG. 5

A 30-kDa polypeptide possesses ICP4 coactivator activity. (A) In vitro transcription analysis of Mono Q fraction 23 with immunoaffinity-purified TFIID. In vitro transcription reactions were performed with Pol II, rTFIIB, -E, and -F, and either immunoaffinity-purified eTFIID or the crude TFIID fraction, DB, in the absence and presence of purified ICP4 and Mono Q fraction 23. The quantity of each TFIID preparation was normalized as described in Materials and Methods. Each condition was tested on a gC core promoter template containing a TATA box and either a wt or a mut Inr. (B) SDS-PAGE analysis of Mono Q fraction 23. A portion of Mono Q fraction 23 was loaded onto an SDS–15% polyacrylamide gel and stained with Coomassie blue. No additional bands were observed in this preparation when an overloaded gel was stained with silver (data not shown).

FIG. 6

p30 is HMG 1. p30 (Mono Q fraction 23) was subjected to amino-terminal sequence analysis through the first 39 amino acids. Shown is an amino acid sequence alignment comparing HMG 1 and p30.