Genetic manipulation of non-segmented negative-strand RNA viruses

Conzelmann, Karl‐Klaus

doi:10.1099/0022-1317-77-3-381

Cited by 78 publications

(39 citation statements)

References 62 publications

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“…For successful rescue of most negativestranded RNA viruses in the Paramyxoviridae and Rhabdoviridae, the N, P, and L proteins have to be provided and expressed from different plasmids to the vector coding for the full-length genome (13,40). We generated three plasmids, pEMC-P, pEMC-N, and pEMC-L, to code for the respective proteins of CDV.…”

Section: Resultsmentioning

confidence: 99%

“…For first-strand cDNA synthesis, 1 g of total RNA was used following the protocol supplied with AMV reverse transcriptase (Promega). The cDNA synthesis was primed with an oligo-dT (12)(13)(14)(15)(16)(17)(18) primer or a primer complementary to base pairs 15,657 through 15,690, for cloning of the full-length cDNA p(ϩ)CDV or CDV-FL-Avr II rev (Table 1), respectively, for amplification of the area carrying the genetic tag. PCR reactions were carried out following protocols supplied with Taq (Life Technologies) or Pwo (Boehringer) DNA polymerases.…”

Section: Cells and Virusesmentioning

confidence: 99%

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Establishment of a Rescue System for Canine Distemper Virus

Gassen¹,

Collins²,

Duprex³

et al. 2000

J Virol

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Canine distemper virus (CDV) has been rescued from a full-length cDNA clone. Besides Measles virus (MV) and Rinderpest virus, a third morbillivirus is now available for genetic analysis using reverse genetics. A plasmid p(؉)CDV was constructed by sequential cloning using the Onderstepoort vaccine strain large-plaque-forming variant. The presence of a T7 promoter allowed transcription of full-length antigenomic RNA by a T7 RNA polymerase, which was provided by a host range mutant of vaccinia virus (MVA-T7). Plasmids expressing the nucleocapsid protein, the phosphoprotein, and the viral RNA-dependent RNA polymerase, also under control of a T7 promoter, have been generated. Infection of HeLa cells with MVA-T7 and subsequent transfection of p(؉)CDV plus the helper plasmids led to syncytium formation and release of infectious recombinant (r) CDV. Comparison of the rescued virus with the parental virus revealed no major differences in the progression of infection or in the shape and size of syncytia. A genetic tag, consisting of two nucleotide changes within the coding region of the L protein, has been identified in the rCDV genome. Expression by rCDV of all the major viral structural proteins has been demonstrated by immunofluorescence.Canine distemper virus (CDV) is an enveloped virus with a monopartite negative-stranded RNA genome. Together with Measles virus (MV) and Rinderpest virus, it belongs to the morbilliviruses which form a serologically closely related genus in the family Paramyxoviridae. CDV primarily affects dogs, but infections of other terrestrial carnivores, in both captivity and the wild, have been reported (1,2,18,21,22,25,26,29,33,36). The mortality rates associated with CDV infection vary among susceptible species and range from 0% in domestic cats to 50% in dogs and 100% in ferrets. One of the currently available vaccines (Onderstepoort strain) efficiently protects dogs, but it is insufficiently attenuated for other species, and high levels of mortality can occur due to its remaining virulence (10). Thus, there is a need to develop more attenuated vaccines for CDV to fully protect susceptible animals.A rescue system for CDV would provide the means to study attenuating effects of defined mutations and might subsequently facilitate generation and examination of new vaccines. Mutations that cause persistence of the virus could also be determined. This system would be useful in gaining a better understanding of CDV infection which can more easily be studied in cell culture and animal models by coexpression of additional reporter genes (e.g., the enhanced green fluorescent protein) from the recombinant viral genome (19,20).The CDV genome is 15,690 nucleotides (nt) in length and consists of a short 3Ј leader region and six genes encoding the N nucleocapsid (N), phospho-(P), matrix (M), fusion (F), hemagglutinin (H) and large (L) proteins (4,5,7,16,32,37,42). They are separated by intergenic regions of 3 nt and are followed by a short 5Ј trailer region. The nonstructural proteins V and C are encoded wit...

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Section: Resultsmentioning

confidence: 99%

Section: Cells and Virusesmentioning

confidence: 99%

Establishment of a Rescue System for Canine Distemper Virus

Gassen¹,

Collins²,

Duprex³

et al. 2000

J Virol

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“…In many aspects, the organization of the calicivirus genome with regard to the sequences coding for the nonstructural proteins is very similar to that of picornaviruses (7,8,14,18,31). However, the respective sequences are found in the 5Ј region of the caliciviral genome, whereas the picornavirus RNA is organized the other way around, with the structural genes orientated to the 5Ј end of the genome and the sequences coding for the nonstructural proteins located at the 3Ј end.…”

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confidence: 99%

Feline Calicivirus: Recovery of Wild-Type and Recombinant Viruses after Transfection of cRNA or cDNA Constructs

Thumfart¹,

Meyers²

2002

J Virol

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The RNA genome of the vaccine strain 2024 of feline calicivirus was cloned as cDNA and analyzed by nucleotide sequencing. A full-length DNA copy of the viral genome was established and proved to be a source of infectious cRNA after in vitro transcription and RNA transfection. Virus could also be recovered when the DNA construct was introduced into cells containing phage T7 RNA polymerase that was provided by vaccinia virus MVA-T7. After insertion of the sequence encoding the green fluorescent protein into the structural protein-encoding region of the infectious cDNA clone, a defective replicon was recovered that was able to replicate autonomously and was packaged into virus particles when the structural proteins were provided in trans.

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“…Enzymatic activities have been predicted for several conserved blocks and\or motifs on the basis of L protein sequence comparisons (Poch et al, 1989(Poch et al, , 1990. However, their functional evaluation was only recently possible using the newly developed reverse genetic tools (Schnell et al, 1994 ;Conzelmann, 1996). Block III of the central ' polymerase ' domain, particularly the core motif AQGDNQ (motif C ; see Fig.…”

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confidence: 99%

The complete Mokola virus genome sequence: structure of the RNA-dependent RNA polymerase.

Mercier

Jacob

Tordo

1997

Journal of General Virology

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The genome sequence of the rabies-related virus Mokola virus (genus Lyssavirus) has been completed by sequencing the L gene, which consists of 6384 nucleotides encoding a 2127 amino acid polymerase. Alignment of the Mokola virus L protein with other polymerases from the virus order Mononegavirales defined three domains : a divergent NH 2 -terminal domain, a highly conserved central domain carrying most of the functional motifs and a COOHterminal domain with alternating conserved and divergent regions. A statistical study outlined the stringency of conservation of glycine, acidic (D, E) and basic (K, R, H) amino acids in polymerases, particularly as key residues of the conserved motifs.Mokola virus is a rabies-related virus that forms genotype 3 of the genus Lyssavirus (family Rhabdoviridae) (Bourhy et al., 1993). It is among the most genetically distant from rabies virus (genotype 1), and modern anti-rabies vaccines fail to protect against Mokola virus infection. Mokola virus has been responsible for a few cases of human and animal encephalomyelitis, largely dispersed throughout sub-saharian Africa (Foggin, 1982). The induced pathology is similar to that of rabies, although no typical ' furious ' form has been associated with Mokola virus infection (King et al., 1994). In mice, Mokola virus is less virulent than rabies virus since it does not kill when injected by a peripheral route (Perrin et al., 1996). In vitro, Mokola virus can multiply in mosquito (Aedes albopictus) cells whereas rabies virus cannot (Aitken et al., 1984). This characteristic is shared only by very distant lyssaviruses such as Obodhiang and Kotokan, which are true arboviruses (Buckley, 1975). Indeed, Mokola virus has been isolated from insectivorous mammals such as shrews (Shope et al., 1970).The Mokola virus genome is a single negative-stranded RNA molecule (order Mononegavirales) which encodes five proteins. Two interact with the viral membrane : the matrix Author for correspondence : Noe$ l Tordo.Fax j33 1 40 61 32 56. e-mail ntordo!pasteur.frThe nucleotide sequence reported in this paper will appear in the EMBL nucleotide sequence database under accession number Y09762.protein (M or M2), which is involved in virion morphology, and the transmembrane glycoprotein (G), which provides cell receptor recognition, and against which neutralizing antibodies are directed. The other three proteins in the ribonucleocapsid structure participate in transcription and replication : the nucleoprotein (N) tightly encapsidates the genome, making it a template for the polymerase complex which comprises the cofactor phosphoprotein (P or M1) and the RNA-dependent RNA polymerase (L). The latter is a nucleotidyl transferase (RNA synthesis), a guanylyl transferase (capping) and a poly(A) synthetase (polyadenylation). In several mononegavirales, L protein has also been shown to be a specific kinase for the phosphoprotein, although the functional role of this phosphorylation remains to be demonstrated (Gao & Lenard, 1995).We have completed the sequence of the Moko...

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Genetic manipulation of non-segmented negative-strand RNA viruses

Cited by 78 publications

References 62 publications

Establishment of a Rescue System for Canine Distemper Virus

Establishment of a Rescue System for Canine Distemper Virus

Feline Calicivirus: Recovery of Wild-Type and Recombinant Viruses after Transfection of cRNA or cDNA Constructs

The complete Mokola virus genome sequence: structure of the RNA-dependent RNA polymerase.

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