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Intracellular targeting of walleye dermal sarcoma virus Orf A (rv-cyclin)
J Rovnak1, J W Casey, S L Quackenbush
1Department of Molecular Biosciences, University of Kansas, Lawrence, Kansas 66045, USA.
Abstract:
Walleye dermal sarcoma virus (WDSV) induces tumors and allows or possibly directs tumor regression. WDSV encodes a putative cyclin homologue, Orf A, and six variant Orf A transcripts have been identified. Northern analysis indicated that a 3.3-kb transcript, encoding full-length Orf A, is the predominant transcript in developing, but not regressing, tumors. Three Orf A proteins, one full-length and two amino-truncated forms, were expressed in mammalian and piscine cells, and their intracellular locations were determined. The full-length form was nuclear and concentrated in interchromatin granule clusters, defined by colocalization with SC-35. The amino-truncated forms were cytoplasmic. Fusion of amino-terminal portions of Orf A to a heterologous protein demonstrated that residues 1-112 were necessary for nuclear localization. Mutation of aa K80 and/or E110 disrupted nuclear localization, suggesting a mechanism similar to that of cellular A- and D-type cyclins for its nuclear import.
Insights
Walleye dermal sarcoma virus (WDSV) Orf A protein drives tumor development. Nuclear localization of full-length Orf A is crucial for its function in WDSV-induced tumors.
Area of Science:
- Virology
- Oncology
- Molecular Biology
Background:
- Walleye dermal sarcoma virus (WDSV) is known to induce tumors and facilitate tumor regression.
- WDSV encodes a protein, Orf A, with homology to cyclins, and multiple transcripts exist.
- The predominant 3.3-kb transcript for full-length Orf A is found in developing tumors, but not regressing ones.
Purpose of the Study:
- To investigate the role of WDSV Orf A protein in tumor development and regression.
- To determine the subcellular localization of Orf A proteins and identify regions responsible for nuclear import.
Main Methods:
- Northern blot analysis to identify Orf A transcripts.
- Expression of Orf A proteins in mammalian and piscine cells.
- Immunofluorescence and co-localization studies with SC-35 to determine protein localization.
- Fusion protein experiments and site-directed mutagenesis to map nuclear localization signals.
Main Results:
- A 3.3-kb transcript encoding full-length Orf A was predominant in developing tumors.
- Three Orf A protein variants (one full-length, two truncated) were expressed.
- Full-length Orf A localized to the nucleus, specifically interchromatin granule clusters, while truncated forms were cytoplasmic.
- Residues 1-112 of Orf A were necessary for nuclear localization, with specific amino acids (K80, E110) playing a role in import.
Conclusions:
- The nuclear localization of full-length WDSV Orf A is critical for its function in WDSV-induced tumor formation.
- The nuclear import mechanism of Orf A shares similarities with cellular cyclins.
- Understanding Orf A's role provides insights into viral oncogenesis and potential therapeutic targets.