Expression of bacteriophage T7 RNA polymerase in avian and mammalian cells by a recombinant fowlpox virus

Abstract

The bacteriophage T7 RNA polymerase gene was integrated into the fowlpox virus genome under the control of the vaccinia virus early/late promoter, P7.5-The recombinant fowlpox virus, fpEFLT7pol, stably expressed T7 RNA polymerase in avian and mammalian cells, allowing transient expression of transfected genes under the control of the T7 promoter. The recombinant fowlpox virus expressing T7 RNA polymerase offers an alternative to the widely used vaccinia virus vTF7-3, or the recently developed modified vaccinia virus Ankara (MVA) T7 RNA polymerase recombinant, a highly attenuated strain with restricted host-range. Recombinant fowlpox viruses have the advantage that as no infectious virus are produced from mammalian cells they do not have to be used under stringent microbiological safety conditions. Recombinant vaccinia viruses (rVV) have been produced expressing a variety of bacteriophage DNA-dependent RNA polymerases: T7 RNA polymerase (Fuerst et al., 1986), T3 RNA polymerase (Rodriguez et al., 1990) and SP6 RNA polymerase (Usdin et al., 1993). Cells infected with these rVVs can express biologically active products transiently from genes under the control of the appropriate RNA polymerase promoter. The most utilized system is an rVV expressing T7 RNA polymerase, vTF7-3. Translation of the T7 transcripts was inefficient, as only 5-10 % were capped, but inclusion of an internal ribosome entry signal (IRES) sequence, from encephalomyocarditis virus, at the 5' end of the T7-derived RNA transcripts, resulted in mRNAs that were CAP-independent and efficiently translated (Elroy-Stein et al., 1989).In addition to transient expression, a gene under the control of the T7 RNA polymerase promoter can also be integrated into the genome of a second rVV allowing high level expression of the gene product in cells dually infected with vTF7-3 and the second rVV (Fuerst et al., 1987). The vTF7-3 system has been utilized for the expression of RNA per se, e.g. a defective RNA (D-

Recombinant Newcastle disease virus as a viral vector: effect of genomic location of foreign gene on gene expression and virus replication

“…The fowlpox recombinant virus fpEFLT7pol (Britton et al, 1996) (hereafter called FPV-T7), which expresses T7 RNA polymerase, was grown on primary chicken embryo liver cells.…”

Virulence of pigeon paramyxovirus type 1 does not always correlate with the cleavability of its fusion protein

“…Details of this construction are available upon request. To rescue virus from the cDNA, QM5 cells were infected with recombinant fowlpox virus FPV-T7 (Britton et al, 1996) and subsequently cotransfected with full-length cDNA constructs and helper plasmids expressing P and L as described previously (de Leeuw et al, 2005;Peeters et al, 1999); inclusion of the NP-expressing helper plasmid was not essential. Virus designated rgAV324 could be rescued and was completely non-virulent in chickens, similar to the parental virus AV324/96p4pp, as evidenced by an ICPI of 0.00.…”

Expression of target genes by coinfection with replication-deficient viral vectors.

“…Furthermore, the inability of fowlpox virus to replicate in the mammalian cells may result in lower copy number of the HCV minigene for transcription by T7 RNA polymerase. A fowlpox virus expressing T7 RNA polymerase has been established by Britton et al (1996) and it offers an alternative to another hybrid T7 system for transient expression of foreign genes. However, overall the susceptibility of mammalian cells, especially MT-2 cells, to fowlpox virus infection was lower than that to adenovirus infection (Fig.…”

“…There are various eukaryotic transient-expression systems based on recombinant viruses synthesizing T7 RNA polymerase in mammalian cells available today. For instance, vaccinia virus (VacT7) (Fuerst et al, 1986), modified vaccinia virus Ankara (MVAT7) (Wyatt et al, 1995), fowlpox virus (FPVT7) (Britton et al, 1996), baculovirus (AcCAT7) (Yap et al, 1997) and adenovirus (AdexCAT7) (Y. Aoki, H. Aizaki, T. Shimoike, H. Tani, K. Ishii, I. Saito, Y. Matsuura & T. Miyamura, unpublished results).…”