Journal of General Virology, Vol 80, 1463-1469, Copyright © 1999 by Society for General Microbiology
Vaccinia virus--bacteriophage T7 expression vector for complementation analysis of late gene processes
D Eckert and M Merchlinsky
Laboratory of Viral Diseases, Center for Biologics Evaluation and Research, Food and Drug Administration, HFM-457, 1401 Rockville Pike, Bethesda, MD 20852-1448, USA
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.
