Production of infectious human respiratory syncytial virus from cloned cDNA confirms an essential role for the transcription elongation factor from the 5' proximal open reading frame of the M2 mRNA in gene expression and provides a capability for vaccine development.

Abstract

Infectious human respiratory syncytial virus (RSV) was produced by the intracellular coexpression of five plasmid-borne cDNAs. One cDNA encoded a complete positive-sense version of the RSV genome (corresponding to the replicative intermediate RNA or antigenome), and each of the other four encoded a separate RSV protein, namely, the major nucleocapsid N protein, the nucleocapsid P phosphoprotein, the major polymerase L protein, or the protein from the 5' proximal open reading frame of the M2 mRNA [M2(0RF1)]. RSV was not produced if any of the five plasmids was omitted. The requirement for the M2(0RF1) protein is consistent with its recent identification as a transcription elongation factor and contirms its importance for RSV gene expression. It should thus be possible to introduce defined changes into infectious RSV. This should be useful for basic studies of RSV molecular biology and pathogenesis; in addition, there are immediate applications to the development of live attenuated vaccine strains bearing predetermined defined attenuating mutations.Human respiratory syncytial virus (RSV) is the most important pediatric viral respiratory pathogen worldwide (1-3). This ubiquitous highly infectious agent emerges each year in seasonal epidemics and nearly everyone is infected at least once within the first 2 years of life. RSV disease is responsible for considerable morbidity and mortality and lacks an approved vaccine or highly effective antiviral therapy. Research on RSV is impeded by its poor growth in tissue culture, the instability of the virion, and the lack of a highly permissive experimental animal other than the chimpanzee.Resistance to RSV reinfection induced by natural infection is incomplete but increases incrementally with repeated exposure. Thus, RSV can infect multiple times during childhood and later life, but serious disease usually is limited to the first and sometimes second infections of life. The minimum goal of RSV immunoprophylaxis is to induce sufficient resistance to prevent serious disease associated with the first or second infection.RSV is a member of the pneumovirus genus of the paramyxovirus family (1, 4 The development of methods for introducing designed changes into genomic RNA of nonsegmented negative-strand RNA viruses was impeded by the lack of homologous viral recombination and the lack of infectivity of naked genomic RNA. The supposition that the minimum unit of infectivity for this type of virus is a nucleocapsid competent for RNA synthesis suggested a different strategy to produce infectious virus from viral cDNA. This involved the intracellular coexpression, from separate transfected plasmids, of cDNAencoded genomic or antigenomic RNA and those viral proteins necessary to generate a transcribing and replicating nucleocapsid. cDNA expression would be driven by T7 RNA polymerase supplied by a vaccinia recombinant virus. This approach was developed first by using short internally deleted analogs of genomic or antigenomic RNA ("minigenomes") that were shown to participate in...

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

“…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).…”

Bovine respiratory syncytial virus infection

“…An understanding of the mechanisms by which BRSV is able to establish infection in the bovine respiratory infection, induce an inflammatory response and respiratory disease has been greatly facilitated by advances in reverse genetics. This involves the production of infectious virus from cloned cDNA [32,37]. The recovery of RSV from cDNA requires co-expression in cell culture of a complete copy of the viral RNA genome and the N, P, M2-1 and L proteins engineered to be expressed by bacteriophage T7 RNA polymerase.…”

Crystal structure of the essential transcription antiterminator M2-1 protein of human respiratory syncytial virus and implications of its phosphorylation

“…The virus-encoded components needed for RNA replication are the large protein (L), the nucleocapsid protein (N), and the phosphoprotein (P). However, complete transcription of mRNAs also requires the M2-1 transcription antiterminator protein (3,4).…”