Related Experiment Videos
Walleye dermal sarcoma virus cyclin interacts with components of the mediator complex and the RNA polymerase II
Joel Rovnak1, Sandra L Quackenbush
1Department of Molecular Biosciences, The University of Kansas, Lawrence 66045, USA.
Abstract:
Walleye dermal sarcoma virus (WDSV) encodes an accessory protein, OrfA, with sequence homology to cyclins (retrovirus cyclin). In cells transfected with an expression construct, OrfA was localized to the nucleus and was concentrated in interchromatin granule clusters (IGCs), sites where splicing factors are concentrated. Other proteins identified in IGCs include transcription factors, the large subunit of RNA polymerase II (Pol II), and cyclin-dependent kinase 8 (cdk8). cdk8 is the kinase partner of cyclin C and a component of the mediator complex, associated with the Pol II holoenzyme. cdk8 and cyclin C can regulate transcription via phosphorylation of cyclin H and the carboxy-terminal domain of Pol II. OrfA in transfected HeLa cells was found to colocalize and copurify with hyperphosphorylated forms of Pol II (Pol IIO) in IGCs, and OrfA was coimmunoprecipitated from lysates of transfected cells with an antibody against Pol IIO. Likewise, Pol IIO could be coprecipitated with an antibody against OrfA. A survey with antibodies against several different cdks resulted in coimmunoprecipitation of OrfA with anti-cdk8, and antiserum against OrfA was able to coprecipitate cdk8 from lysates of cells that express OrfA. Coprecipitation of OrfA with anti-cyclin C demonstrated that it was included in complexes with OrfA and cdk8. OrfA has sequence and structural similarities to cyclin C, and, functionally, OrfA appears to have the capacity to both enhance and inhibit the activity of promoters in a cell-specific manner, similar to functions of the mediator complex. These data suggest that WDSV OrfA functions through its interactions with these large, transcription complexes. Further investigations will clarify the role of the retrovirus cyclin in control of virus expression and transformation.
Insights
Walleye dermal sarcoma virus OrfA protein interacts with transcription machinery, including RNA polymerase II and cdk8. This retrovirus cyclin may regulate viral gene expression and cell transformation.
Area of Science:
- Virology
- Molecular Biology
- Cell Biology
Background:
- Walleye dermal sarcoma virus (WDSV) encodes OrfA, a protein with similarities to cyclins.
- OrfA localizes to the nucleus, specifically in interchromatin granule clusters (IGCs) where splicing factors assemble.
- IGCs are also known to contain transcription factors, RNA polymerase II (Pol II), and cyclin-dependent kinase 8 (cdk8).
Purpose of the Study:
- To investigate the cellular localization and protein interactions of the WDSV OrfA protein.
- To determine if OrfA interacts with components of the transcription and RNA polymerase II machinery.
- To elucidate the functional role of OrfA in relation to cellular transcription complexes.
Main Methods:
- Transfection of HeLa cells with an OrfA expression construct.
- Immunofluorescence microscopy to determine OrfA localization.
- Co-immunoprecipitation assays to identify OrfA interacting proteins, including Pol II and cdk8.
- Copurification studies to confirm protein complexes.
Main Results:
- OrfA was found to localize to IGCs and colocalize with hyperphosphorylated Pol II (Pol IIO).
- OrfA was co-immunoprecipitated with antibodies against Pol IIO and cdk8, and vice versa.
- OrfA was found in complexes with cdk8 and cyclin C, suggesting it functions similarly to a cyclin.
- OrfA demonstrated the ability to enhance or inhibit promoter activity in a cell-specific manner.
Conclusions:
- WDSV OrfA interacts with key components of the cellular transcription machinery, including Pol II and cdk8.
- OrfA's interactions suggest a role in regulating viral gene expression and potentially cellular transformation.
- OrfA functions as a retrovirus cyclin, influencing transcription through large transcription complexes.