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. 2002 Oct 15;99(21):13403-8.
doi: 10.1073/pnas.202200399. Epub 2002 Sep 23.

An activation domain in the C-terminal subunit of HCF-1 is important for transactivation by VP16 and LZIP

Randy L Luciano  1 , Angus C Wilson
Affiliations

An activation domain in the C-terminal subunit of HCF-1 is important for transactivation by VP16 and LZIP

Randy L Luciano et al. Proc Natl Acad Sci U S A. .

Abstract

In herpes simplex virus, lytic replication is initiated by the viral transactivator VP16 acting with cellular cofactors Oct-1 and HCF-1. Although this activator complex has been studied in detail, the role of HCF-1 remains elusive. Here, we show that HCF-1 contains an activation domain (HCF-1(AD)) required for maximal transactivation by VP16 and its cellular counterpart LZIP. Expression of the VP16 cofactor p300 augments HCF-1(AD) activity, suggesting a mechanism of synergy. Infection of cells lacking the HCF-1(AD) leads to reduced viral immediate-early gene expression and lowered viral titers. These findings underscore the importance of HCF-1 to herpes simplex virus replication and VP16 transactivation.

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Figures

Figure 1
Figure 1
The C terminus of HCF-1 contains an activation domain. (A) 293T cells were transfected with expression plasmids (250 ng) encoding Gal4DBD, Gal4-HCF-1N, Gal4-HCF-1C, or the activation domains of Gal4-c-Jun, Gal4-CREB, and Gal4-Sp1, and assayed for transactivation of a 5xGal4-E1B-luc reporter (500 ng). Extracts were prepared after 40 h and assayed for luciferase activity. Values represent means and standard deviations of three independent transfections. (B) The structure of the HCF-1 precursor is shown. The boundaries of the activation domain were delineated using N-terminal, C-terminal, or internal deletions fused to the Gal4DBD. Luciferase activity is scored as fold activation above Gal4DBD alone. (C) Sequence of human HCF-1 (residues 1436–1756) encompassing the HCF-1AD (solid bar). Alternative splicing removes 44 amino acids (dashed underline). Secondary structure predictions [α (α-helix) and β (β-sheet)] were derived using the Frishman and Argos method (50). Arrows indicate the boundaries of truncations used in B.
Figure 2
Figure 2
Deletion of HCF-1AD does not affect association with the N terminus or nuclear localization. (A) HA-tagged HCF-1N450–1011 and T7-tagged HCF-1C WT or ΔAD were coexpressed in transfected 293T cells and association assayed by immunoprecipitation with αHA antibody followed by immunoblotting with the αT7 antibody. The HA-tagged HCF-1N450–1011 is in lanes 1–3 and the T7-tagged HCF-1C WT and ΔAD in lanes 1 and 4 and lanes 2 and 5, respectively. (B) HeLa cells were transfected with expression plasmids (500 ng) encoding GFP or GFP-tagged HCF-1C WT or ΔAD. DNA is visualized with Hoechst 33258 and GFP by fluorescence microscopy.
Figure 3
Figure 3
Characterization of R-tsBN67 HCF-1Δrep WT and ΔAD cells. (A) Immunoblot comparing tsBN67-derived cell lines stably expressing T7-tagged HCF-1Δrep WT and ΔAD. Protein extracts were prepared from equal numbers of R-tsBN67-HCF-1Δrep WT (lane 1) and ΔAD (lane 2) cells, resolved on a SDS-7% polyacrylamide gel and immunoblotted with the αT7 antibody. (B) Analysis of cell proliferation. R-tsBN67-HCF-1Δrep WT (■) and ΔAD (□) cells were seeded at a density of 1 × 105 cells per 10-cm dish and incubated at 39.5°C for 3 days. At the indicated times, three separate dishes were harvested and counted. (C) Protein extracts were prepared from an equal number of tsBN67, R-tsBN67-HCF-1ΔrepWT, and HCF-1ΔrepΔAD cells and assayed for the ability to support VP16-induced complex formation in a gel-mobility shift assay using a 32P-labeled ICP0 TAATGARAT element as a probe. Lane 1 contains probe alone, lanes 2–9 contain probe with Oct-1 POU domain, and lanes 3–9 contain probe, Oct-1 POU domain, and VP16FL fused to GST. Two amounts of each cell extract (a 3-fold difference) were incubated with VP16 and Oct-1 POU: WT extract (lanes 4 and 5), ΔAD extract (lanes 6 and 7), and tsBN67 extract (lanes 8 and 9). In lanes 10, 11, and 12, the probe was incubated with the highest amount of WT, ΔAD, and tsBN67 extracts.
Figure 4
Figure 4
Transactivation by VP16 and LZIP is severely compromised in tsBN67-derived cells expressing HCF-1ΔrepΔAD. (A) R-tsBN67-HCF-1ΔrepWT and R-tsBN67-HCF-1ΔrepΔAD cells were transiently transfected with 6xTAAT-tk-luc (500 ng) or 1xTAAT/GA-tk-luc (1 μg) reporters together with 0.1 or 1.0 μg of the VP16FL or VP16ΔC expression plasmids and assayed for luciferase activity after incubation at 39.5°C for 40 h. (B) As in A, except cells were transfected with: (Left) a CRE-luciferase reporter (CRE-luc, 500 ng) and an expression plasmid encoding LZIPN228; (Center) a Gal4-responsive reporter (5xGal4-E1B-luc reporter, 500 ng) and 500 ng of either Gal4DBD, Gal4-c-Jun, or Gal4-VP16AD; or (Right) an estrogen response element reporter (ERE-luc, 500 ng) and plasmid encoding the human estrogen receptor (ER).
Figure 5
Figure 5
The HCF-1AD synergizes with VP16 and p300. (A) 293T cells were transfected with 2xGal4-2xLexA-E1B-luc (200 ng) together with Gal4 activator (VP16AD or c-Jun, 100 ng) and LexA-HCF-1AD (10 ng). Extracts were prepared after 40 h and assayed for luciferase activity. (B) 293T cells were cotransfected with expression plasmids (500 ng) encoding Gal4-HCF-1CWT or ΔAD, Gal4-HCF-1C1436–1612, or Gal4-HCF-1AD with the 5xGal4-E1B-luc reporter gene (500 ng) and with (open bars) or without (filled bars) an expression plasmid (6 μg) encoding full-length p300.
Figure 6
Figure 6
The HCF-1AD is involved in HSV lytic replication. (A) Wild-type HSV-1 at moi of 0.001 was used to infect R-tsBN67-HCF-1Δrep WT and ΔAD cells maintained at 33.5°C and 39.5°C. After 5 days, cells were harvested, sonicated, and serial dilutions plated on Vero cells. After 4 days, cells were fixed and stained with crystal violet to visualize viral plaques. Graph shows the mean number of plaques at 10−6 the dilution based on four independent assays. (B) Wild-type HSV-1 at moi of 0.001 was used to infect R-tsBN67-HCF-1Δrep WT and ΔAD cells maintained at 39.5°C. RNA was isolated after 4 h and assayed by RT-PCR. Specific amplification products from hamster β-actin (lanes 1 and 4, amplification fragment, 360 bp), ICP0 (lanes 2 and 5, amplification fragment, 157 bp), and ICP4 (lanes 3 and 6 amplification fragment, 101 bp) are indicated.
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