Vaccinia virus-bacteriophage T7 expression vector for complementation analysis of late gene processes

Area of Science:

• Virology
• Molecular Biology
• Genetics

Background:

• Vaccinia virus (VACV) gene expression is complex, with distinct early, intermediate, and late phases.
• Complementation analysis is crucial for understanding the function of viral genes, particularly late genes.
• Transient expression systems are valuable for studying gene function without stable integration.

Purpose of the Study:

• To develop a novel vaccinia virus-based transient expression system for analyzing late gene functions.
• To create a recombinant vaccinia virus expressing bacteriophage T7 RNA polymerase for enhanced gene expression.
• To investigate the utility of this system for studying viral DNA replication and protein expression.

Main Methods:

• Construction of a recombinant vaccinia virus (vtsT7) by modifying the ts21 mutant to express bacteriophage T7 RNA polymerase.
• Infection of cells with vtsT7 at non-permissive temperatures to assess conditional defectiveness in late gene expression.
• Analysis of target protein expression (beta-galactosidase, HIV envelope gene) under the control of a T7 promoter.
• Investigation of vaccinia virus DNA replication intermediates and gene expression at low multiplicity of infection (m.o.i.) and elevated temperatures.

Main Results:

• The vtsT7 virus exhibited conditional defectiveness in viral late gene expression but allowed high-level T7 promoter-driven protein production at non-permissive temperatures.
• Beta-galactosidase expression levels were comparable to the vTF7.3 vector, while HIV envelope gene expression was slightly higher with vtsT7.
• Infections at 39°C with low m.o.i. led to the accumulation of high molecular mass, non-linear vaccinia virus DNA replication intermediates.
• High levels of transfected gene expression were observed proximal to a T7 promoter under these conditions.

Conclusions:

• The vtsT7 hybrid vector is a functional tool for complementation analysis of vaccinia virus late gene functions.
• This system facilitates the study of vaccinia virus DNA replication and the resolution of replicative intermediates.
• vtsT7 enables the investigation of trans-acting factors involved in vaccinia virus late gene expression and DNA processing.