Rescue of measles viruses from cloned DNA.

Abstract

A system has been established allowing the rescue of replicating measles viruses (MVs) from cloned DNA. On one hand, plasmids were constructed from which MV antigenomic RNAs with the correct termini are transcribed by phage T7 RNA polymerase. On the other hand, helper cells derived from the human embryonic kidney 293 cell line were generated constitutively expressing T7 RNA polymerase together with MV nucleocapsid protein and phosphoprotein. Simultaneous transfection of the helper cells with the MV antigenomic plasmid and with a plasmid encoding the MV polymerase under direction of a T7 promoter led to formation of syncytia from which MVs were easily recovered. A genetic tag comprising three nucleotide changes was present in the progeny virus. As a first application of reverse genetics, a segment of 504 nucleotides from the 5′ non‐coding region of the fusion gene was deleted, leading to an MV variant whose replication behaviour in Vero cells was indistinguishable from that of the laboratory Edmonston B strain. Since no helper virus is involved, this system, in principle, should be applicable to the rescue of any member of the large virus order Mononegavirales, i.e. viruses with a nonsegmented negative‐strand RNA genome.

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).…”

“…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).…”

“…The encapsidation in the normal virus life cycle is thought to initiate at, or very close to, the 5 0 end of the nascent RNA chain, and then to progress strictly coupled with the chain elongation under the continuous supply of NP (Lamb & Kolakofsky 1996). This highly coordinated encapsidation probably does not occur in the initial round of transcription catalysed by T7 pol, because it elongates the chain much faster than mononegavirus polymerases (Chamberlin & Ryan 1982;Iverson & Rose 1981;Radecke et al 1995). The initial transcripts could thus remain naked for some time, or sparsely bound with NP, and hence would be readily attacked by RNases (Radecke et al 1995).…”

“…This highly coordinated encapsidation probably does not occur in the initial round of transcription catalysed by T7 pol, because it elongates the chain much faster than mononegavirus polymerases (Chamberlin & Ryan 1982;Iverson & Rose 1981;Radecke et al 1995). The initial transcripts could thus remain naked for some time, or sparsely bound with NP, and hence would be readily attacked by RNases (Radecke et al 1995). Therefore, it has been presumed that the encapsidation of primary transcripts is the bottleneck in the process leading to virus recovery from cDNA.…”

Morbilliviruses and human disease

“…The development of systems during the last decade which permit the generation of recombinant MVs, 'virus rescue' [187], has opened the door to mechanistic studies, not only of MV persistence and its role in neurological sequelae of MV infection and the role of specific genes in this process, but also dissection of the roles of individual genes in isolation or in combination with others, in attenuation, virulence immunosuppression, and interaction with the innate immune system.…”