Related Experiment Videos
Polo-like kinase 1 as a target for human cytomegalovirus pp65 lower matrix protein
A Gallina1, L Simoncini, S Garbelli
1Istituto di Genetica Biochimica ed Evoluzionistica, Consiglio Nazionale delle Ricerche, Pavia, Italy.
Abstract:
Human cytomegalovirus (HCMV) pp65 protein is the major constituent of viral dense bodies but is dispensable for viral growth in vitro. pp65 copurifies with a S/T kinase activity and has been implicated in phosphorylation of HCMV IE1 immediate-early protein and its escape from major histocompatibility complex 1 presentation. Furthermore, the presence of pp65 correlates with a virion-associated kinase activity. To clarify the role of pp65, yeast two-hybrid system (THS) screening was performed to identify pp65 cellular partners. A total of 18 out of 48 yeast clones harboring cDNAs for putative pp65 binding proteins encoded the Polo-like kinase 1 (Plk1) C-terminal domain. Plk1 behaved as a bona fide pp65 partner in THS control crosses, and the interaction was confirmed by in vitro binding experiments. Endogenous Plk1 was coimmunoprecipitated with pp65 from transiently transfected COS7 cells. In infected fibroblasts, Plk1 was coimmunoprecipitated with pp65 at late infection stages. Furthermore, Plk1 was detected within wild-type HCMV particles but not within the particles of a pp65-negative mutant (RVAd65). The hydrophilic region of pp65 was phosphorylated in vitro by Plk1. These results suggest that one function of pp65 may be to capture a cell kinase, perhaps in order to alter its activity, nucleotide preference, substrate specificity, or subcellular localization to the advantage of HCMV.
Insights
Human cytomegalovirus (HCMV) pp65 protein interacts with Polo-like kinase 1 (Plk1). This interaction, found in HCMV particles, suggests pp65 may recruit host kinases to benefit viral replication.
Area of Science:
- Virology
- Molecular Biology
- Cellular Biology
Background:
- Human cytomegalovirus (HCMV) pp65 protein is a major component of viral dense bodies.
- pp65 is associated with kinase activity and implicated in HCMV IE1 protein phosphorylation and immune evasion.
- The precise function of pp65, particularly its kinase interactions, remains incompletely understood.
Purpose of the Study:
- To identify cellular partners of the HCMV pp65 protein using a yeast two-hybrid system.
- To investigate the interaction between pp65 and Polo-like kinase 1 (Plk1).
- To determine the role of this interaction in the context of HCMV infection.
Main Methods:
- Yeast two-hybrid system (THS) screening to identify pp65 binding proteins.
- In vitro binding assays and co-immunoprecipitation to confirm protein interactions.
- Detection of Plk1 in HCMV particles using wild-type and mutant viruses.
- In vitro phosphorylation assays.
Main Results:
- Yeast two-hybrid screening identified Plk1 as a pp65 binding partner.
- The interaction between pp65 and Plk1 was confirmed by in vitro binding and co-immunoprecipitation experiments.
- Plk1 was detected in HCMV particles, specifically within those produced by wild-type virus but not a pp65-negative mutant.
- Plk1 was shown to phosphorylate the hydrophilic region of pp65 in vitro.
Conclusions:
- HCMV pp65 protein interacts with the host cell kinase Plk1.
- This interaction is present within HCMV virions, suggesting a role in viral particle composition or function.
- pp65 may function to recruit or modulate Plk1 activity to the advantage of HCMV, potentially influencing viral replication or pathogenesis.