A highly recombinogenic system for the recovery of infectious Sendai paramyxovirus from cDNA: generation of a novel copy-back nondefective interfering virus.

Abstract

We have recovered infectious Sendai virus (SeV) from full‐length cDNA (FL‐3) by transfecting this cDNA and pGEM plasmids expressing the nucleocapsid protein (NP), phosphoprotein and large proteins into cells infected with a vaccinia virus which expresses T7 RNA polymerase. These cells were then injected into chicken eggs, in which SeV grows to very high titers. FL‐3 was marked with a BglII site in the leader region and an NsiI site (ATGCAT) in the 5′ nontranslated region of the NP gene, creating a new, out‐of‐frame, 5′ proximal AUG. All the virus stocks generated eventually removed this impediment to NP expression, by either point mutation or recombination between FL‐3 and pGEM‐NP. The recovery system was found to be highly recombinogenic. Even in the absence of selective pressure, one in 20 of the recombinant SeV generated had exchanged the NP gene of FL‐3 with that of pGEM‐NP. When a fifth plasmid containing a new genomic 3′ end without the presumably deleterious BglII site was included as another target for recombination, the new genomic 3′ end was found in the recombinant SeV in 12 out of 12 recoveries. Using this approach, a novel copy‐back nondefective virus was generated which interferes with wild‐type virus replication.

Initiation of Sendai virus multiplication from transfected cDNA or RNA with negative or positive sense

“…In contrast to previous attempts with SeV and other mononegaviruses Garcin et al 1995;Lawson et al 1994;Radecke et al 1995;Schnell et al 1994;Whelan et al 1995), our system consistently recovered infectious virus even from pSeV(ÿ) that generates (ÿ)RNA (Table 1). Although the frequency was lower than that from pSeV( ) (1/50-1/100), an additional passage in eggs was sufficient to generate maximal amounts of virus (Table 1).…”

“…We constructed the cDNA to generate a viral authentic RNA sequence with the correct 5 0 end. In contrast, Garcin et al (1995) genetically tagged the cDNA by introducing two nucleotide changes upstream of the NP initiation codon. Furthermore, their cDNA generated three G residues templated by a T7 promoter, which were attached to the 5 0 end of transcript.…”

“…Furthermore, their cDNA generated three G residues templated by a T7 promoter, which were attached to the 5 0 end of transcript. These sequence changes and extra residues were obviously impeding for virus recovery because they had to be corrected or removed by deletion, point mutation and/ or recombination during the passages in eggs, ultimately leading to the selection of the most suitable authentic viral sequence (Garcin et al 1995). Also to be noted is that to minimize the deleterious effect of VV by a combination of ara C and rifampicin and to supply the supporting plasmids at an optimal ratio are critical for virus recovery as well as minigenome replication.…”

“…The recovery of infectious viruses from cloned and transfected cDNA will be an innovative technology, enabling the genetic engineering of viruses in this superfamily. The DNA transfection system has been established for two rhabdoviruses: rabies virus (Schnell et al 1994) and vesicular stomatitis virus (VSV) (Lawson et al 1995;Whelan et al 1995), with a genome of about 12 kilobases (kb), and for three paramyxoviruses: measles (Radecke et al 1995), Sendai (Garcin et al 1995), and RS (Collins et al 1995) viruses, with an approximate 15 kb genome. These systems, except for that of the measles virus, employed the transfection of cDNA into cells in which the cDNA and the plasmids carrying the viral nucleocapsid protein (NP or N) and RNA polymerase genes are coexpressed by vaccinia virus (VV)-driven bacteriophage T7 RNA polymerase (T7 pol).…”

“…Starting with genomic negative sense (ÿ)RNA was unsuccessful. The reason for this seems to be that unlike ( )RNA, (ÿ)RNA presumably hybridizes to the viral mRNAs supplied in trans (Schnell et al 1994;Lawson et al 1995;Garcin et al 1995), or that T7 pol aberrantly terminates transcription that generates (ÿ)RNA (Whelan et al 1995). In addition, infectious virus recovery is quite rare, since sometimes only one infectious unit is obtained from about 10 7 or even more transfected cells (Schell et al 1994;Lawson et al 1995;Garcin et al 1995).…”