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. 2005 Nov 25;342(2):240-51.
doi: 10.1016/j.virol.2005.08.011. Epub 2005 Sep 15.

An activation domain within the walleye dermal sarcoma virus retroviral cyclin protein is essential for inhibition of the viral promoter

Joel Rovnak  1 , Brett W Hronek, Sean O Ryan, Sumin Cai, Sandra L Quackenbush
Affiliations

An activation domain within the walleye dermal sarcoma virus retroviral cyclin protein is essential for inhibition of the viral promoter

Joel Rovnak et al. Virology. .

Abstract

Walleye dermal sarcoma virus (WDSV) is a complex retrovirus associated with seasonal dermal sarcomas. Developing tumors have low levels of accessory gene transcripts, A1 and B, and regressing tumors have high levels of full-length and spliced transcripts. Transcript A1 encodes a retroviral cyclin (rv-cyclin) with limited homology to host cyclins. The rv-cyclin is physically linked to components of the transcriptional co-activator complex, Mediator, and regulates transcription. In walleye fibroblasts, it inhibits the WDSV promoter independently of cis-acting DNA sequences. The rv-cyclin activates transcription from GAL4 promoters when fused to the GAL4 DNA binding domain. A 30 a.a. activation domain in the carboxy region can be inactivated by single point mutations, and these mutations diminish the ability of the rv-cyclin to inhibit the WDSV promoter. When fused to glutathione S-transferase, the rv-cyclin, its carboxy region, and the activation domain pull down components of transcription complexes from nuclear extracts, and pull down is lost by mutation of the activation domain.

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Figures

Fig. 1
Fig. 1
(A) Consecutive 50 bp deletions from the 5′ end of the WDSV U3 region (d1–d7) were tested for the ability to drive luciferase expression in walleye fibroblast W12 cells without (white bars) or with expressed rv-cyclin (black bars). (B and C) Assays, as in panel A, of the WDSV U3 region with specific mutations in either the enhancer (B) or core promoter region (C). Functional mutations within the indicated sites were defined previously by DNA footprinting, electrophoretic mobility shift assays, and luciferase assays (Hronek et al., 2004).
Fig. 2
Fig. 2
Assay of transcription activation by rv-cyclin and rv-cyclin fragments in HeLa cells. Indicated portions of the rv-cyclin protein were fused to the GAL4 DBD, and their ability to activate luciferase expression from the pFR-luc GAL4 reporter construct measured after 48 h. Boundaries of each segment are indicated by a.a. position at the top. The outlines of the predicted cyclin box folds, here designated ‘Cyclin box A’ and ‘Cyclin box B’ are indicated. ‘CC’ represents the putative coiled-coil region. The smallest active region, a.a. 228 – 283, is indicated.
Fig. 3
Fig. 3
(A) Individual alignments of overlapping, rv-cyclin 31 a.a. segments with a prototype acidic activator, VP16 (a.a. 425 – 451; Cress and Triezenberg, 1991). Segments span the rv-cyclin activator region, a.a. 228–283, identified in Fig. 2. Similar and identical residues are shaded. φ designates hydrophobic side chains of the VP16 sequence. Asterisks mark a.a.s L257, D259, and V260 of the rv-cyclin, which were subject to mutation. (B) Luciferase assay of GAL4-DBD vector, pFA, and GAL4-DBD GAL4 fusion proteins containing the full-length rv-cyclin, 1–297, or the wild type (wt) and mutated versions (L257A, D259A, V260A, V260S, and V260F) of the 240–270 a.a. segment in HeLa cells. (C) Luciferase assay of GAL4 fusion proteins as in panel B, but performed in the walleye fibroblast line, W12. (D) Luciferase assay of GAL4 fusions of the entire carboxy region of VP16 (a.a. 413–490) and double mutant VP16 F442S/ F475A, and subdomains, VP16N (413– 452) and VP16C (453–490) and their corresponding individual mutations of F442S and F475A in HeLa cells. (E) Assays of the wild type GAL4 DBD-VP16 fusion constructs in the W12 cell line. Results from the wild type rv-cyclin AD, 240–270, are shown for comparison.
Fig. 4
Fig. 4
Effects of wild type (wt) and mutated full-length rv-cyclin on transcription. Luciferase expression in W12 cells from the WDSV U3-luciferase construct, pGL3WDSVU3, co-transfected with control vector pKH3 or an rv-cyclin expression vector, pKH3OrfA (wild type (wt) or mutated forms L257A, D259A, V260A, V260S, and V260F).
Fig. 5
Fig. 5
Western blot analysis of coIPs from 150 μg HeLa cell lysates. Antibodies were used to precipitate proteins either from control, pKH3-transfected cell lysates (left lane of each pair) or from lysates of HA-tagged, rv-cyclin-transfected cells (pKH3OrfA vector; right lane of each pair). The first two samples were immunoprecipitated with preimmune rabbit sera (NRS), and the second two with rabbit sera reactive to the rv-cyclin protein (rv-cyc). Subsequent pairs were precipitated with antibody reactive to the indicated cellular protein. All Westerns were probed with anti-HA MAb (12CA5). Cdk8, cycC, Sur2, Med6, CRSP70, and TRAP proteins are components of the Mediator co-activator complex. The IP antibodies used for CBP and p300 are specific for these proteins and do not crossreact.
Fig. 6
Fig. 6
Pulldown of proteins from 75 μg of HeLa nuclear extracts by GST fusion proteins and glutathione sepharose. (A) Western blot of pulldown by GST-rv-cyclin (1–297) probed and reprobed with the indicated antibodies specific for p300, Sur2, and TBP. (B) Pulldown by GST fused to the carboxy region of the rv-cyclin (219–297) and probed successively for p300, CBP, TBP, TFIIB, cdk8, and Sur2. Antibodies for the detection of CBP and p300 detect unique regions and are not crossreactive. (C) Pulldowns by wild type (wt) GST-rv-cyclin AD (240–270) and mutated forms, V260S and V260F, and by VP16 subdomain fusions, GST-VP16N and GST-VP16C and GST-VP16 AD and corresponding mutated forms, F442S, F475A, and double mutation, 442S/475A, in the VP16 AD. The input GST fusion proteins were detected with anti-GST (αGST; bottom panel). ‘GST’ indicates pulldown with purified GST only. Input represents 5% of total extract used for each pulldown.
Fig. 7
Fig. 7
Pulldown of proteins from 150 μg of walleye cell nuclear extracts by GST fusion proteins and glutathione sepharose. (A) Western blot of pulldown from W12 cell nuclear extract by wild type (wt) and mutated (V260S) GST-rv-cyclin AD (240–270) probed with antibodies that crossreact with walleye homologs of p300 and Sur2. (B) Pulldowns from nuclear extracts prepared from regressing walleye dermal sarcomas by wild type (wt) GST-rv-cyclin AD (240–270) and mutated forms, V260S and V260F and by VP16 subdomain fusions, GST-VP16N and GST-VP16C and GST-VP16 AD and corresponding mutated forms, F442S, F475A, and double mutation, 442S/475A, in the VP16 AD. ‘GST’ indicates pulldown with purified GST only. Input represents 2.5% of total extract used for each pulldown. 2 μg each of nuclear extracts from W12 cells (WNE) and HeLa cells (HNE) was loaded in the last two lanes and demonstrates crossreactivity of antisera to walleye proteins, p300 and Sur2.
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