Identification of the rubella virus nonstructural proteins

Area of Science:

• Virology
• Molecular Biology
• Protein Biochemistry

Background:

• Rubella virus (RUB) nonstructural protein open reading frame (NSP-ORF) encodes essential viral proteins.
• Understanding the synthesis and processing of RUB nonstructural proteins is crucial for comprehending viral replication.

Purpose of the Study:

• To identify and characterize the nonstructural polypeptides encoded by the RUB NSP-ORF.
• To determine the order and processing of these polypeptides during viral infection.

Main Methods:

• Cloning of RUB NSP-ORF segments into bacterial expression vectors.
• Generation of rabbit antisera against fusion proteins.
• Immunoprecipitation and immunofluorescence assays in infected and transfected cells.
• Synchronization of translation initiation using a hypertonic block.
• Pulse-chase experiments to analyze protein processing and stability.

Main Results:

• Three RUB nonstructural polypeptides were identified: a 200-kDa primary translation product (P200), a 150-kDa polypeptide (P150), and a 90-kDa polypeptide (P90).
• The order of polypeptides within the NSP-ORF was determined as NH2-P150-P90-COOH.
• P200 is proteolytically cleaved into P150 and P90, with P150 being stable and P90 showing turnover.
• P150 localizes to perinuclear and cytoplasmic structures, including the nuclear membrane.

Conclusions:

• The RUB NSP-ORF is translated as a P200 precursor that undergoes proteolytic cleavage.
• The processing and stability of nonstructural proteins are critical for viral function.
• The localization of P150 suggests its involvement in viral assembly or nuclear interactions.