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Characterization of a human cytomegalovirus with phosphorylation site mutations in the immediate-early 2 protein
Julie A Heider1, Yongjun Yu, Thomas Shenk
1Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544-1014, USA.
Abstract:
A human cytomegalovirus mutant (TNsubIE2P) was constructed with alanine substitutions of four residues (T27, S144, T233, and S234) previously shown to be phosphorylated in the immediate-early 2 (IE2) protein. This mutant grew as well as the wild type at both low and high multiplicities of infection. The mutant activated the major immediate-early, UL4, and UL44 promoters to similar levels, and with similar kinetics, as wild-type virus. However, the TNsubIE2P mutant virus transactivated an endogenous simian virus 40 early promoter 4 h earlier and to higher levels than the wild-type virus in infected human fibroblasts. The modification of the IE2 protein by SUMO-1 (i.e., its sumoylated state) was also examined.
Insights
A novel human cytomegalovirus mutant with altered IE2 protein phosphorylation showed no growth defects but enhanced transactivation of the simian virus 40 early promoter in human cells.
Area of Science:
- Virology
- Molecular Biology
- Cellular Biology
Background:
- Human cytomegalovirus (HCMV) immediate-early 2 (IE2) protein plays a crucial role in viral gene regulation.
- Phosphorylation of IE2 protein residues is known to influence its function.
- Understanding IE2 protein modifications is key to deciphering HCMV replication and pathogenesis.
Purpose of the Study:
- To investigate the functional impact of specific IE2 protein phosphorylation sites on HCMV replication and promoter transactivation.
- To characterize a novel HCMV mutant (TNsubIE2P) with alanine substitutions at four key phosphorylation sites in the IE2 protein.
- To assess the effect of these modifications on viral growth, viral promoter activation, and heterologous promoter transactivation.
Main Methods:
- Construction of a recombinant HCMV mutant (TNsubIE2P) by site-directed mutagenesis, replacing four phosphorylated residues in the IE2 protein with alanine.
- Assessment of viral growth kinetics at various multiplicities of infection (MOI).
- Analysis of viral promoter activation (major immediate-early, UL4, UL44) and transactivation of an endogenous simian virus 40 early promoter in infected human fibroblasts.
- Examination of IE2 protein modification, specifically sumoylation.
Main Results:
- The TNsubIE2P mutant exhibited growth kinetics comparable to wild-type HCMV at both low and high MOI.
- Mutant virus demonstrated similar activation levels and kinetics for HCMV major immediate-early, UL4, and UL44 promoters compared to wild-type.
- The TNsubIE2P mutant showed accelerated and enhanced transactivation of the simian virus 40 early promoter in infected human fibroblasts.
- The study also explored the sumoylated state of the modified IE2 protein.
Conclusions:
- Specific phosphorylation sites in the HCMV IE2 protein are not essential for viral replication or the activation of viral promoters.
- Modification of these phosphorylation sites can enhance the IE2 protein's ability to transactivate heterologous promoters, suggesting a regulatory role in viral gene expression.
- These findings provide insights into the complex post-translational modifications of the IE2 protein and their impact on HCMV transcriptional regulation.