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Three active forms of aspartic proteinase from Mason-Pfizer monkey virus
A Zábranský1, M Andreánsky, O Hrusková-Heidingsfeldová
1Department of Biochemistry, Academy of Sciences of the Czech Republic, Prague, Czech Republic.
Abstract:
Mason-Pfizer monkey virus (M-PMV) proteinase, released by the autocatalytic cleavage of Gag-Pro and Gag-Pro-Pol polypeptide precursors, catalyzes the processing of viral precursors to yield the structural proteins and enzymes of the virion. In retroviruses, usually only one proteolytically active form of proteinase exists. Here, we describe an unusual feature of M-PMV, the existence of three active forms of a retroviral proteinase with molecular masses of 17, 13, and 12 kDa as determined by mass spectroscopy. These forms arise in vitro by self-processing of a 26-kDa proteinase precursor. We have developed a process for isolation of each truncated product and demonstrate that all three forms display proteolytic activity. Amino acid analyses, as well as the determination of N- and C-terminal sequences, revealed that the N-termini of all three forms are identical, confirming that in vitro autoprocessing of the 17-kDa form occurs at the C-terminus to yield the truncated forms. The 17-kDa form and the newly described 13-kDa form of proteinase were identified in virions collected from the rhesus monkey CMMT cell line chronically infected with M-PMV, confirming that multiple forms exist in vivo.
Insights
Mason-Pfizer monkey virus (M-PMV) proteinase exhibits an unusual three active forms (17, 13, and 12 kDa) generated through self-processing. These multiple M-PMV proteinase forms were confirmed both in vitro and in vivo within infected cells.
Area of Science:
- Virology
- Molecular Biology
- Biochemistry
Background:
- Mason-Pfizer monkey virus (M-PMV) proteinase is crucial for viral maturation, processing Gag-Pro and Gag-Pro-Pol precursors.
- Typically, retroviruses possess a single active proteinase form.
Purpose of the Study:
- To investigate the unusual existence of multiple active forms of M-PMV proteinase.
- To characterize the processing and activity of these M-PMV proteinase forms.
Main Methods:
- Mass spectrometry was used to determine the molecular masses of M-PMV proteinase forms.
- In vitro self-processing of a 26-kDa precursor was analyzed.
- Isolation and characterization of truncated proteinase products.
- Amino acid and sequence analyses (N- and C-terminal) were performed.
- Western blot or similar techniques to detect proteinase forms in virions.
Main Results:
- Three active M-PMV proteinase forms (17, 13, and 12 kDa) were identified via mass spectrometry.
- These forms result from the in vitro self-processing of a 26-kDa precursor.
- All three isolated forms demonstrated proteolytic activity.
- N-terminal sequences were identical, indicating C-terminal truncation.
- The 17-kDa and 13-kDa forms were detected in M-PMV virions from infected cells, confirming in vivo existence.
Conclusions:
- M-PMV proteinase exists in multiple active forms (17, 13, and 12 kDa), unlike most retroviruses.
- Autoprocessing occurs at the C-terminus of the 17-kDa form to generate smaller active species.
- These multiple proteinase forms are biologically relevant, existing both in vitro and in vivo.