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Physicochemical characterization and specificity of the murine leukaemia virus Pr65gag proteolytic factor
Abstract:
The Pr65gag proteolytic factor obtained from Moloney (MoLV) or Rauscher (RLV) leukaemia virus has been characterized. We found that it was present in small amounts in virions and was extremely unstable. Although it eluted at the trailing edge of p12 on Sephadex G-75 columns, it could clearly be separated from p12 on DEAE-Sephadex A-50M columns, making it unlikely that the factor is p12 or any other major murine leukaemia virus (MuLV) protein. This fact also distinguishes the murine factor from the avian tumour viruses and is stable to column purification methods (von der Helm, 1977; Dittmar & Moelling, 1978). We further observed that: (i) the murine proteolytic factor had an estimated mol. wt. of 20,000 to 22,000, relative to MuLV p12, which eluted as a dimer on Sephadex G-75 columns in the presence of 0.1% NP-40; and (ii) in vitro cleavage of an iodinated Pr65gag-rich, p30-deficient substrate yielded a clear increase in both p30 and p12, which suggests that the in vitro cleavage of Pr65gag is similar to its processing in vivo.
Insights
A newly identified proteolytic factor from murine leukemia viruses (MuLV) is unstable and distinct from major viral proteins. Its in vitro activity mimics in vivo processing of the Pr65gag precursor protein.
Area of Science:
- Virology
- Molecular Biology
- Biochemistry
Background:
- Murine leukemia viruses (MuLV) possess a Pr65gag precursor polyprotein.
- The processing of Pr65gag is crucial for viral maturation and infectivity.
- Previous studies have identified various viral and cellular proteases involved in polyprotein cleavage.
Purpose of the Study:
- To characterize a proteolytic factor associated with Moloney (MoLV) and Rauscher (RLV) leukemia viruses.
- To determine the relationship between this factor and known viral proteins like p12 and p30.
- To investigate the in vitro cleavage activity of the murine proteolytic factor on Pr65gag.
Main Methods:
- Purification of the proteolytic factor using Sephadex G-75 and DEAE-Sephadex A-50M chromatography.
- Molecular weight estimation using gel filtration.
- In vitro cleavage assays using iodinated Pr65gag substrate.
Main Results:
- The proteolytic factor was found in low amounts in virions and was highly unstable.
- The factor eluted separately from p12 on DEAE-Sephadex A-50M, suggesting it is not p12 or another major MuLV protein.
- The estimated molecular weight of the murine factor was 20,000–22,000.
- In vitro cleavage of Pr65gag resulted in the generation of p30 and p12, mimicking in vivo processing.
Conclusions:
- A distinct proteolytic factor, separate from major MuLV proteins, is involved in Pr65gag processing.
- This factor's in vitro activity suggests a role in the in vivo maturation of MuLV.
- Further characterization is needed to identify the precise nature and origin of this factor.