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Replication activity of JC virus large T antigen phosphorylation and zinc finger domain mutants
J J Swenson1, P W Trowbridge, R J Frisque
1Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park 16802, USA.
Abstract:
The replication potential of the human polyomavirus JC virus (JCV) relative to that of the related monkey virus SV40 is limited, in part, by differences in the multifunctional T antigen (T Ag). Earlier genetic analyses of the SV40 T protein indicated that specific phosphorylation sites and a zinc finger motif are involved in the regulation of viral replication. The JCV and SV40 T Ags differ with respect to sequences encoding these functional domains, and in the present study mutational analysis of the JCV protein was conducted to assess the role that unconserved residues might play in the restricted lytic behavior of JCV. Amino acids Asn316 and His317 in the zinc finger domain and Thr664 and Glu666 in the carboxy-terminal phosphorylation domain were mutated to either an SV40-like residue or an alanine. Each of the mutant JCV genomes replicated with wild type efficiency suggesting that, unlike the case for SV40 T Ag, these amino acids are not critical to the regulation of viral replication. On the other hand, mutation of amino acid Thr125 within the amino-terminal phosphorylation domain abolished JCV DNA replication and viability. This site is conserved in the SV40 T Ag, and previous results have revealed that phosphorylation of this residue (Thr124) is required for T Ag replication function.
Insights
JC virus (JCV) T-antigen phosphorylation at Thr125 is crucial for viral replication. Mutations in other domains, unlike in SV40, did not affect JCV replication, highlighting unique regulatory mechanisms.
Area of Science:
- Virology
- Molecular Biology
- Genetics
Background:
- Human polyomavirus JC virus (JCV) replication is less efficient than simian virus 40 (SV40), partly due to differences in their T-antigens (T Ag).
- SV40 T Ag regulation involves specific phosphorylation sites and a zinc finger motif, which differ in sequence between JCV and SV40 T Ags.
Purpose of the Study:
- To investigate the role of unconserved residues in the JCV T Ag, specifically in the zinc finger and carboxy-terminal phosphorylation domains.
- To determine the importance of the amino-terminal phosphorylation domain in JCV replication.
Main Methods:
- Mutational analysis of the JCV T Ag protein was performed.
- Specific amino acids in the zinc finger (Asn316, His317) and carboxy-terminal phosphorylation domain (Thr664, Glu666) were mutated.
- The amino-terminal phosphorylation domain residue Thr125 was also mutated.
Main Results:
- Mutations in the zinc finger and carboxy-terminal phosphorylation domains did not affect JCV replication efficiency.
- Mutation of Thr125 in the amino-terminal phosphorylation domain abolished JCV DNA replication and viability.
- Thr125 is conserved in SV40 T Ag, where phosphorylation is essential for replication.
Conclusions:
- Unlike SV40, specific residues in the zinc finger and carboxy-terminal domains are not critical for JCV T Ag-mediated replication.
- Phosphorylation of Thr125 in the amino-terminal domain is essential for JCV DNA replication and viral viability, indicating a critical regulatory role for this site.