Summary auto-generated
Researchers developed vtsT7, a hybrid vaccinia virus-bacteriophage T7 expression vector for studying late gene functions in vaccinia virus. The vector was constructed by inserting the T7 RNA polymerase gene into ts21, a temperature-sensitive mutant defective in late gene expression. At non-permissive temperatures (39°C), vtsT7 is conditionally defective for viral late genes but produces high levels of proteins under T7 promoter control. The virus accumulates unresolved DNA replicative intermediates at low multiplicity of infection. Comparisons with the standard vTF7.3 vector showed vtsT7 achieved comparable β-galactosidase expression and slightly higher HIV envelope protein expression, suggesting it may be preferable when vaccinia late genes interfere with target protein production. As a proof-of-concept, researchers expressed vaccinia virus topoisomerase via transfection into vtsT7-infected cells and confirmed its activity, though topoisomerase alone was insufficient for DNA replicative intermediate resolution. The system provides a tool for complementation analysis to identify genes and proteins required for vaccinia virus late processes, including DNA resolution, telomere processing, and virion assembly.
Key findings
- vtsT7 enables high-level expression of T7-promoter genes while blocking vaccinia late gene expression at 39°C, allowing complementation studies
- DNA replicative intermediates accumulate in vtsT7-infected cells at low multiplicity of infection, providing substrate for studying resolution mechanisms
- vtsT7 achieves comparable or superior protein expression compared to the standard vTF7.3 vector, particularly for proteins requiring post-translational modification
- Expressed vaccinia topoisomerase was enzymatically active but alone insufficient for resolving DNA replicative intermediates, indicating multiple factors are required for this process
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Abstract
A vaccinia virus--bacteriophage T7 RNA polymerase hybrid transient expression vector has been developed for complementation analysis of late gene functions in vaccinia virus. The conditionally defective virus ts21 was modified to express the bacteriophage T7 RNA polymerase. The derived virus, vtsT7, was conditionally defective in viral late gene expression but produced high levels of a target protein under the control of a T7 promoter at non-permissive temperatures. The level of beta-galactosidase expression under the control of a T7 promoter was slightly lower in vtsT7 infections than those with the prototypical T7 RNA polymerase vector vTF7.3. However, the levels of expression for the human immunodeficiency virus envelope gene, a protein which undergoes post-translational modification, was slightly higher in vtsT7 infections, suggesting that some proteins may be expressed better in the absence of vaccinia virus late gene expression. Infections using vtsT7 at a low m.o.i. at 39 degrees C resulted in the accumulation of high molecular mass, non-linear replicative intermediates of vaccinia virus DNA replication and high levels of expression of a transfected gene proximal to a T7 promoter. The virus vtsT7 provides a means for the analysis of potential trans-acting factors participating in vaccinia virus late processes such as resolution of DNA replicative intermediates.