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Separation of a murine leukaemia virus protein kinase activity from its Pr65gag polyprotein substrate after
Abstract:
We have recently found that, in vitro, the murine leukaemia virus (MuLV)-associated protein kinase activity predominantly phosphorylates Pr65gag, a virus protein present in relatively small amounts in partially purified virus preparations. Other virus proteins, such as p10, Pr27gag and Pr40gag, are also phosphorylated in vitro, but to a lesser degree. Furthermore, when immature core subparticles which are enriched in Pr65gag are prepared from virions by Sepharose 6B exclusion column chromatography, about 50% of the kinase activity (as assayed with the exogenous substrate phosvitin) remains associated with the cores. We report here that this core-associated activity is distinct from Pr65gag since it can be separated from Pr65gag by chromatography on denatured DNA--cellulose columns followed by centrifugation of the 0.2 M-NaCl-eluted fraction. Under these conditions, Pr65gag is pelleted while the kinase activity, which can phosphorylate both endogenous (MuLV Pr65gag and p10) as well as exogenous (phosvitin) substrates, remains in the supernatant. Interestingly, when the amount of Pr65gag is reduced, as in such preparations, p10 then becomes more heavily phosphorylated.
Insights
Murine leukemia virus (MuLV) protein kinase phosphorylates Pr65gag. This core-associated kinase activity is distinct from Pr65gag and can phosphorylate other viral proteins like p10.
Area of Science:
- Virology
- Molecular Biology
- Biochemistry
Background:
- Murine leukemia virus (MuLV) possesses associated protein kinase activity.
- This kinase predominantly phosphorylates the Pr65gag protein, a component of immature viral cores.
Purpose of the Study:
- To characterize the MuLV-associated protein kinase activity.
- To determine if the kinase activity is associated with Pr65gag or a separate entity.
Main Methods:
- In vitro kinase assays using viral proteins as substrates.
- Sepharose 6B chromatography to isolate immature core subparticles.
- Denatured DNA-cellulose chromatography to separate kinase activity from Pr65gag.
Main Results:
- The MuLV-associated kinase primarily phosphorylates Pr65gag in vitro.
- Approximately 50% of the kinase activity remains with immature core subparticles.
- Core-associated kinase activity is separable from Pr65gag using DNA-cellulose chromatography.
- The kinase phosphorylates both endogenous (Pr65gag, p10) and exogenous (phosvitin) substrates.
- Reduced Pr65gag levels lead to increased phosphorylation of p10.
Conclusions:
- The MuLV-associated protein kinase activity is distinct from the Pr65gag protein.
- This kinase plays a role in the phosphorylation of viral proteins, potentially including p10.
- Further characterization of this kinase is warranted for understanding MuLV replication.