cDNA corresponding to the antigenome of nonsegmented negative stranded RNA viruses and process for the production of such viruses
The present invention relates, in general, to a methodology for the generation of nonsegmented negative-strand RNA viruses (Pringle, 1991) from cloned deoxyribonucleic acid (cDNA). Such rescued viruses are suitable for use as vaccines, or alternatively, as plasmids in somatic gene therapy applications. The invention also relates to cDNA molecules suitable as tools in this methodology and to helper cell lines allowing the direct rescue of such viruses. Measles virus (MV) is used as a mode for other representatives of the Mononegavirales, in particular the family Paramyxoviridae.
1. A method for producing an infectious non-segmented negative-strand RNA virus of the family Paramyxoviridae comprising:
(a) introducing into a host cell a plasmid comprising a cDNA molecule, said cDNA molecule comprising a (i-)-strand sequence of said negative-strand RNA virus, encoding the entire genome of the virus, operatively linked to an expression control sequence, which allows the synthesis of antigenomic RNA transcripts bearing the authentic 3′-termini, wherein the (+)-strand sequence of said negative-strand RNA virus is operatively linked to a foreign expressible DNA fragment and wherein the length of the genome of the infectious non-segmented negative-strand RNA virus is an integral multiple of six, said host cell comprising (i) stably transfected nucleic acid molecules containing nucleic acid sequences that allow genomic expression of an exogenous RNA polymerase, a viral N protein and a viral P protein, and (ii) a nucleic acid molecule containing nucleic acid sequence encoding a viral L protein; and
(b) recovering the infectious non-segmented negative-strand RNA virus without the help of a helper virus.
2. The method of claim 1 , wherein the host cell expresses a selectable marker.
3. The method of claim 1 , wherein the nucleic acid molecule containing the nucleic acid sequence encoding the viral L protein is stably transfected into the host cell that allows genomic expression of the viral L protein in the host cell.
4. The method of claim 1 , wherein the expression control sequence is an RNA polymerase promoter.
5. The method of claim 1 , wherein the foreign expressible DNA fragment is inserted into a region of said cDNA molecule encoding a viral protein, said insertion being effected in a manner maintaining the reading frame to create a fusion protein, and permitting the expression of said foreign expressible DNA fragment under the control of viral transcriptional control sequences of the viral protein.
6. The method of claim 1 , wherein the foreign expressible DNA fragment is inserted into a region of said cDNA molecule downstream of a viral protein coding region in a manner avoiding formation of a fusion protein, but allowing expression of the downstream expressible DNA fragment either by a stop/restart mechanism where the last A residue of the upstream termination triplet coincides with that of the start codon of the downstream coding region, or by placing an internal ribosome entry site (IRES) between the two coding regions.
7. The method of claim 1 , wherein the foreign expressible DNA fragment is inserted in the 5′ terminal region of the (+) strand sequence of a non-segmented negative-strand RNA virus.
8. The method of claim 1 , wherein said foreign expressible DNA fragment is inserted into a non-coding region of said cDNA molecule and flanked by viral transcription control sequences or heterologous transcription control sequences controlling the expression of the foreign expressible DNA fragment.
9. The method of claim 1 , wherein said plasmid further comprises a genomic ribozyme sequence immediately adjacent to the 3′ terminal nucleotide of said cDNA molecule, and wherein said plasmid optionally further comprises downstream of said genomic ribozyme sequence at least one terminator.
10. The method of claim 9 , wherein the terminator is a T7 terminator.
11. The method of claim 9 , wherein the genomic ribozyme sequence is the hepatitis delta virus genomic ribozyme sequence.
12. The method of claim 1 , wherein said plasmid is capable of replicating in a prokaryotic or a eukaryotic cell.
13. The method of claim 1 , wherein said foreign expressible DNA fragment encodes at least one immunogenic epitope.
14. The method of claim 13 , wherein the foreign expressible DNA fragment encodes an immunogenic epitope of a protein from a pathogen.
15. The method of claim 14 , wherein the foreign expressible DNA fragment encodes an immunogenic epitope of a protein from a virus, a bacterium, or a parasite.
16. The method of claim 13 , wherein the immunogenic epitope is capable of eliciting a protective immune response.
17. The method of claim 1 , wherein said foreign expressible DNA fragment encodes a gene product that a target cell is in need thereof, or a toxin.
18. The method of claim 1 , wherein said negative-strand RNA virus is measles virus or mumps virus.
19. The method of claim 1 , wherein said nucleic acid sequences encoding the viral N protein, viral P protein, and viral L protein are derived from measles virus or mumps virus.
20. The method of claim 1 , wherein said host cell is derived from human embryonic kidney cell line 293.
21. The method of claim 1 , wherein the ratio of the plasmid and the nucleic acid molecule containing the nucleic acid sequence encoding the viral L protein is about 1000:1.
22. The method of claim 1 , wherein said recovery of the infectious non-segmented negative-strand RNA virus is achieved by directly collecting the host cell culture after syncytia formation.
23. The method of claim 1 , wherein step 1(b) further comprising mixing the host cell introduced with the plasmid with other cells competent of being infected and capable of replicating the infectious non-segmented negative-strand RNA virus, before recovering the infectious non-segmented negative-strand RNA virus.
24. A method for producing an infectious non-segmented negative-strand RNA virus of the family Paramyxoviridae comprising:
(a) introducing into a host cell a plasmid comprising a cDNA molecule, said cDNA molecule comprising an entire (+)-strand sequence of said negative-strand RNA virus, encoding the entire genome of the virus, operatively linked to an expression control sequence, which allows the synthesis of antigenomic RNA transcripts bearing the authentic 3′-termini, wherein the (+)-strand sequence of said negative-strand RNA virus is operatively linked to a foreign expressible DNA fragment and wherein the length of the genome of the infectious non-segmented negative-strand RNA virus is an integral multiple of six, said host cell comprising plasmids containing nucleic acid sequences that allow expression of an exogenous RNA polymerase, a viral N protein, a viral P protein, and a viral L protein; and
(b) recovering the infectious non-segmented negative-strand RNA virus without the help of a helper virus.
25. The method of claim 24 , wherein the RNA virus belongs to the subfamily Paramyxovirinae.
26. The method of claim 24 , wherein said negative-strand RNA virus is measles virus or mumps virus.
27. The method of claim 24 , wherein the host cell comprises at least one stably transfected plasmid containing nucleic acid sequence that allows genomic expression of an exogenous RNA polymerase, a viral N protein, a viral P protein, or a viral L protein.
28. The method of claim 15 , wherein the foreign expressible DNA fragment encodes an immunogenic epitope of an envelope protein from a virus.