Altered E2 glycoprotein of Sindbis virus and its use in complementation studies

Area of Science:

• Virology
• Molecular Biology
• Protein Biochemistry

Background:

• Sindbis virus is an RNA virus with glycoproteins E1 and E2.
• Proteolytic cleavage of precursor PE2 to E2 is essential for viral maturation.
• Temperature-sensitive (ts) mutants are valuable tools for studying viral replication.

Purpose of the Study:

• To characterize a Sindbis virus variant with an altered E2 glycoprotein.
• To investigate the mechanism of E2 formation and its impact on viral complementation.
• To analyze the functional consequences of altered E2 on viral properties.

Main Methods:

• Detection and characterization of a smaller-molecular-weight E2 glycoprotein variant.
• Analysis of PE2 precursor processing and glycosylation patterns.
• Complementation assays using RNA+ and RNA- Sindbis virus mutants.
• Electrophoretic analysis of viral glycoproteins E1 and E2 under reducing and non-reducing conditions.

Main Results:

• A Sindbis virus variant with a smaller E2 glycoprotein was identified, resulting from aberrant proteolytic cleavage of PE2.
• The altered E2 formation was separable from temperature sensitivity and not preferential during viral maturation.
• Complementation studies revealed non-reciprocal interactions: RNA+ gene products corrected RNA- defects, but not vice versa.
• No significant changes in biological activity of virions with altered E2 were observed.
• Electrophoretic analysis showed E1 mobility was affected by reduction, while E2 mobility was not.

Conclusions:

• Aberrant proteolytic cleavage can generate Sindbis virus variants with altered E2 glycoproteins.
• Sindbis virus complementation between RNA+ and RNA- mutants is asymmetric.
• The altered E2 glycoprotein does not significantly affect viral infectivity or maturation.
• Further investigation into the non-reciprocal complementation mechanism is warranted.