trans processing of vaccinia virus core proteins

Area of Science:

• Virology
• Molecular Biology
• Biochemistry

Background:

• Vaccinia virus (VV) virion proteins undergo proteolytic maturation from precursors during assembly.
• Putative cleavage sites within these precursors contain Ala-Gly-X motifs, suggesting a specific proteolytic mechanism.

Purpose of the Study:

• To identify sequence and structural parameters governing efficient cleavage site recognition in VV protein maturation.
• To investigate the role of the Ala-Gly-X motif in the proteolytic processing of VV precursor proteins.

Main Methods:

• Development of a trans-processing assay system using a FLAG-tagged P25K precursor (P25K:FLAG).
• Monitoring proteolytic processing via immunoblotting and pulse-chase experiments in coinfected cells.
• Site-directed mutagenesis to alter the Ala-Gly-X motif and assess its impact on cleavage efficiency.

Main Results:

• The Ala-Gly-Ala motif at residues 31-33 of P25K:FLAG is essential for producing the 25K:FLAG product; mutation to Ile-Asp-Ile blocked cleavage.
• Cleavage of P25K:FLAG was inhibited by rifampin, indicating a pathway shared with VV core proteins.
• The intermediate cleavage site (Ala-Gly-Ser at residues 17-19) was identified, with mutations blocking the formation of the 25K' intermediate.

Conclusions:

• The Ala-Gly-X motif is a critical determinant for VV virion protein proteolytic maturation.
• Proteolytic processing of VV proteins occurs during virion assembly, requiring proper localization to virosomes.
• Biochemical and genetic evidence supports the functional significance of the Ala-Gly-X motif in VV maturation.