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. 2001 Dec;75(24):12402-11.
doi: 10.1128/JVI.75.24.12402-12411.2001.

Stabilization but not the transcriptional activity of herpes simplex virus VP16-induced complexes is evolutionarily conserved among HCF family members

S Lee  1 , W Herr
Affiliations

Stabilization but not the transcriptional activity of herpes simplex virus VP16-induced complexes is evolutionarily conserved among HCF family members

S Lee et al. J Virol. 2001 Dec.

Abstract

The human herpes simplex virus (HSV) protein VP16 induces formation of a transcriptional regulatory complex with two cellular factors-the POU homeodomain transcription factor Oct-1 and the cell proliferation factor HCF-1-to activate viral immediate-early-gene transcription. Although the cellular role of Oct-1 in transcription is relatively well understood, the cellular role of HCF-1 in cell proliferation is enigmatic. HCF-1 and the related protein HCF-2 form an HCF protein family in humans that is related to a Caenorhabditis elegans homolog called CeHCF. In this study, we show that all three proteins can promote VP16-induced-complex formation, indicating that VP16 targets a highly conserved function of HCF proteins. The resulting VP16-induced complexes, however, display different transcriptional activities. In contrast to HCF-1 and CeHCF, HCF-2 fails to support VP16 activation of transcription effectively. These results suggest that, along with HCF-1, HCF-2 could have a role, albeit probably a different role, in HSV infection. CeHCF can mimic HCF-1 for both association with viral and cellular proteins and transcriptional activation, suggesting that the function(s) of HCF-1 targeted by VP16 has been highly conserved throughout metazoan evolution.

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Figures

FIG. 1
FIG. 1
HCF proteins share sequence similarity in the amino- and carboxy-terminal regions. (A) Schematic diagram of the human HCF proteins HCF-1 and HCF-2 and the C. elegans HCF protein CeHCF. Above the diagram of HCF-1 are shown the positions of (i) functional regions of HCF-1 (e.g., VP16-induced complex [VIC] formation and HCF-1 subunit association [SAS1N and SAS1C]) and (ii) structural features of HCF-1 (e.g., HCF-1KEL repeats and basic region). The solid and open triangles indicate active and inactive HCF-1PRO repeats. The tandem solid arrowheads indicate fibronectin type 3 (Fn3) repeats. Below the diagram of HCF-1 are shown the schematic structures of HCF-2 and CeHCF. Regions of similarity are aligned by the lines connecting the diagrams. (B) Sequence identity of HCF proteins. The percentage of identical amino acid residues between each pair of proteins is shown. The sequence used in each comparison was as follows: HCFKEL repeats, HCF-1 (amino acids [aa] 18–360), HCF-2 (aa 8–353), and CeHCF (aa 29–375); SAS1N, HCF-1 (aa 361–401), HCF-2 (aa 354–393), and CeHCF (aa 376–416); and SAS1C, HCF-1 (aa 1812–2002), HCF-2 (aa 598–784), and CeHCF (aa 553–749).
FIG. 2
FIG. 2
All three HCF proteins can stabilize the VP16-induced complex. (A) VP16-induced-complex formation. COS cell extracts containing different HCF proteins were analyzed for VP16-induced-complex formation activity with VP16ΔC, the Oct-1 POU domain, and radiolabeled VP16 response element DNA probe, as described in Materials and Methods. Protein-DNA complexes were resolved in an electrophoretic mobility retardation assay. The positions of the free probe, the Oct-1 POU domain-bound probe, and the VP16-induced complexes (VICs) formed by different HCF proteins are indicated on the left. Lane 1, probe alone; lane 2, probe with Oct-1 POU domain; lane 3, probe with VP16ΔC. Lanes 4, 5, 7 to 9, 11 to 13, 15 to 17, 19 to 21, 23 to 25, 27 to 29, and 31 to 33 contain the Oct-1 POU domain and VP16ΔC. Lane 5 contains in addition unprogrammed COS cell extract, and lanes 6 to 33 contain the COS cell extract with HCF protein as indicated. Each set of three titration lanes contains a twofold titration, and the non-VP16-containing sample contains the most concentrated extract. The position of each VP16-induced complex is indicated with a dot. +, present; −, absent. (B) Immunoblot analysis of the COS cell extracts used in the electrophoretic mobility retardation assay. Extracts containing HA-tagged HCF proteins were resolved by sodium dodecyl sulfate–8% polyacrylamide gel electrophoresis, transferred to a nitrocellulose membrane, and blotted with the 12CA5 anti-HA antibody. The asterisk on the right indicates a nonspecific band. The multiple species smaller than 175 kDa in lane 2 are unrelated to HCF-1 but instead reflect 12CA5-specific cross-reacting cellular proteins that appear only in this sample because it contains the most cell extract after sample normalization.
FIG. 3
FIG. 3
VP16 activates transcription in association with HCF-1 and CeHCF but only weakly in association with HCF-2. (A) Transcriptional activation by VP16 in tsBN67 cells grown at 33.5°C. The cells were transfected with an HCF expression construct, a VP16 expression construct, and reporter constructs, and the resulting β-globin and α-globin reporter RNAs were probed by RNase protection analysis as described in Materials and Methods. Odd-numbered samples contained no cotransfected VP16 expression vector. The samples shown here were normalized to the level of internal control α-globin transcript. The positions of the RNase-protected fragments corresponding to α-globin (α), correctly initiated β-globin (β), and read-through (RT) β-globin transcripts are indicated on the left. +, present; −, absent. (B) Quantitation of relative β-globin transcript levels. The intensity of each band corresponding to the β-globin transcript in the samples containing VP16 was measured by phosphorimager analysis. The transcript levels are shown relative to the HCF-1N1011 sample (panel A, lane 4). The results represent the average of two complete experiments; the CeHCFN395 was uncharacteristically high in the experiment not shown here (high error bars), and its apparently higher activity than CeHCFFL shown here is not a true representation of its activity. (C) Immunoblot analysis of the tsBN67 cell extracts used in the in vivo transcription assay shown in panel A. Extracts were resolved by sodium dodecyl sulfate–8% polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane, and the HA-tagged HCF and VP16 proteins were detected with the 12CA5 anti-HA antibody. Only the VP16-containing samples are shown. The position of each relevant HCF species is indicated with a black dot to the right of each lane. A long exposure of lane 4 is shown on the right (lane 4′). Relative levels of HCF-protein synthesis are indicated above each lane. Lane 1, no ectopic HCF protein synthesized.
FIG. 4
FIG. 4
The HCF-2KEL repeat region can participate in rescue of the tsBN67 cell proliferation defect. (A) Colony formation assay. tsBN67 cells were transfected with different HCF expression plasmids as indicated. After transfection, the plates were incubated at 40°C for 14 days to permit colony formation. The colonies were visualized by staining them with crystal violet. (B) Schematic diagrams of HCF proteins analyzed and relative levels of rescue of tsBN67 cell proliferation at 40°C. See the legend to Fig. 1A for a description of the diagrams. Asterisk, P134S tsBN67 point mutation. (C) Levels of HCF proteins in transfected tsBN67 cells at nonpermissive temperature. Transfected cells (E plates [see Materials and Methods]) were collected 48 h after transfection and temperature shift to the nonpermissive temperature (40°C) and were used to make protein extracts. The extracts were resolved by sodium dodecyl sulfate–8% polyacrylamide gel electrophoresis, transferred to a nitrocellulose membrane, and probed with the 12CA5 anti-HA epitope tag antibody to monitor the levels of HA-tagged HCF protein synthesis. The position of each relevant band is indicated with a black dot. A long exposure of the immunoblot is shown for lanes 2 to 5 (lanes 2′ to 5′).
FIG. 5
FIG. 5
C. elegans HCF interacts with human LZIP in a yeast two-hybrid assay. A yeast GAL1-HIS3 reporter strain was transformed with expression plasmids encoding different HCF proteins fused to the GAL4 DBD together with an expression plasmid encoding either one of two known HCF-1 binding proteins (VP16 and LZIP) or nonbinding proteins (VP16ΔC and SNF-4) fused to a GAL4 AD. (A) Key for GAL4-AD fusion protein samples shown in panel B. (B) Yeast two-hybrid assays. The DBD fusion protein used is indicated above each plate. Successful growth with histidine (+His; left half of each plate) shows that each expression plasmid has no lethal effect. The interaction between a DBD fusion protein and an AD fusion protein is demonstrated by successful growth in the absence of histidine (−His; right half of each plate).
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