Two plasmid mammalian expression system

Abstract

Described is a simple plasma-based mammalian expression system that uses the RNA dependent RNA polymerase (RDRP) enzyme systems' activity for expression of recombinant proteins or RNA from viral minigenomes and rescue of recombinant viruses from cDNAs encoding entire genome(s) of negative stranded RNA viruses for use in synthesizing recombinant viruses and developing vaccines. This system will be used for expression of recombinant proteins, therapeutic RNA molecules including anti-sense and/or selecting interfering RNA and Ribozymes. This system can also be used for gene therapy and producing recombinant viruses for production of new vaccines.

Claims

1. A method of producing non-segmented recombinant negative stranded RNA virus, said method comprising: a. obtaining a two plasmid system comprising: i. one cloning plasmid comprising a manipulatable replicon, wherein the manipulatable replicon is replaced by a cDNA fragment encoding the entire viral genomic RNA, said cDNA fragment is operatively linked to RNA polymerase (RNAp) I promoter; and ii. one helper plasmid comprising N, P, and L genes expressing the N, P, and L proteins respectively, wherein the N, P, and L genes are cloned into a single bi-cistronic DNA cassette containing a gene encoding fusion protein of N and P separated by a 2A peptide sequence, an IRES element, and a gene encoding L protein under the control of RNA polymerase (RNAp) II promoter; b. introducing the cloning plasmid and the helper plasmid into a host cell to produce a recombinant host cell; and c. producing non-segmented recombinant negative stranded RNA virus without the help of replicating helper vaccinia virus or exogenous RNA polymerase.

2. The method as claimed in claim 1, wherein said negative stranded RNA virus is selected from the group consisting of measles virus, Rinderpest virus, peste des petits ruminants virus, canine distemper virus, Newcastle disease virus, and sendai viruses.

3. The method as claimed in claim 2, wherein said negative stranded RNA virus is measles virus.

4. The method as claimed in claim 1, wherein said helper plasmid is selected from the group consisting of SEQ ID NO: 8, and SEQ ID NO: 9.

5. A method of producing recombinant measles virus, said method comprising: a. obtaining a two plasmid system comprising: i. one cloning plasmid comprising a manipulatable replicon, wherein the manipulatable replicon is replaced by a cDNA fragment encoding the entire measles virus genomic RNA, said cDNA fragment is operatively linked to RNA polymerase (RNAp) I promoter; and ii. one helper plasmid comprising N, P, and L genes expressing the N, P, and L proteins respectively, wherein the N, P, and L genes are cloned into a single bi-cistronic DNA cassette containing a gene encoding fusion protein of N and P separated by a 2A peptide sequence, an IRES element, and a gene encoding L protein under the control of RNA polymerase (RNAp) II promoter, said helper plasmid is selected from the group consisting of SEQ ID NO: 8, and SEQ ID NO: 9; b. introducing the cloning plasmid and the helper plasmid into a host cell to produce a recombinant host cell; and c. producing recombinant measles virus without the help of replicating helper vaccinia virus or exogenous RNA polymerase.

Description

BRIEF DESCRIPTION OF THE DRAWING

(1) FIGS. 1A-1C: Schematic diagrams of the cloning plasmids encoding the Measles Minireplicon encoding 2 reporter genes.

(2) FIG. 1A: Basic replicon Design

(3) FIG. 1B: Construct 1: HH-replicon-HDV

(4) FIG. 1C: Construct 2: P1P-replicon-P1T

(5) FIGS. 2A-2C: Schematic representations of the Cloning Plasmids created.

(6) FIG. 2A: Cloning Plasmid 1: Vector pUC57 was used to clone P1P_Replicon_P1T construct.

(7) FIG. 2B: Cloning plasmid 2: Vector pIRES was used to prepare pIRES_P1P_Replicon_P1T construct.

(8) FIG. 2C: Cloning plasmid 3: Vector pIRES was used to prepare pIRES_HH_Replicon_HDV.

(9) FIGS. 3A-3B: Synthesis of cDNA of entire MV-E genome

(10) FIG. 3A: Generation of cDNA encoding the entire antigenome of MV-E: Viral RNA was purified using Genejet RNA purification kit (Fermentas) and reverse transcribed using Superscript II and random hexamer primers. This was used to amplify seven overlapping fragments with Superscript III and specific primers and cloned into pCDNA3.1 in which the multiple cloning site was replaced with a Nhe I_Not I_Pac I_Pme I linker.

(11) FIG. 3B: Plasmid encoding cDNA of MV-E genome: cDNA encoding entire antigenome of MV-E was synthesized by assembling seven overlapping PCR amplified fragments and cloned in Not I and Pme I sites of pCDNA 3.1()

(12) FIGS. 4A-4B: Schematic representations of the two variants of Helper plasmid created.

(13) FIG. 4A: Helper Plasmid 1: Vector pBiCMV-1 was used to prepare pBiCMV_MV-N_MV-P_IRES_MV-L.

(14) FIG. 4B: Helper plasmid 2: Vector pIRES was used to prepare pIRES_MV-N_p2A_MV-P_MV-L.

(15) FIGS. 5A-5F:

(16) FIG. 5A: Vero cells were co-transfected with Cloning plasmid encoding eGFP and HGH and HPV1 or HPV 2, incubated for 48 hrs at 37 C. and observed for fluorescence and HGH. A: pUC 18 alone;

(17) FIG. 5B: pGFP (positive control);

(18) FIG. 5C: pUC_P1P-replicon-P1T alone;

(19) FIG. 5D: pUC-P1P-replicon-P1T and Helper HPV;

(20) FIG. 5E: pIRES-HH-replicon-HDV alone;

(21) FIG. 5F: pIRES-HH-replicon-HDV and HPV. Note: Helper plasmids 1 and 2 both were able to supply the N, P and L proteins. Representative pictures of HPV1 alone are shown as they were similar.

(22) FIGS. 6A-6B: Rescue of segmented MV: Equal quantities of plasmids pCDNA_MVgenome, Cloning plasmid 1 (pIRES_HH-replicon-HDV) and HPV 1 were cotransfected into Vero cells using Xfect, incubated at 37 C. and observed daily for formation of syncytia. MV-E was harvested from the culture supernatant after syncytia formation covered>80%-90% and titrated using TCID50. Cells were observed simultaneously for expression of EGFP plasmid.

(23) FIG. 6A: Vero cells transfected with pUC-P1P-rep-P1T, pCDNA-MVgenome & Helper plasmid variant 1;

(24) FIG. 6B: Vero cells transfected with pIRES-HH-rep-HDV, pCDNA-MVgenome & Helper plasmid variant 1.

DETAILED DESCRIPTION AND EXAMPLES

(25) The present invention relates to expression system that can be easily used for cellular reconstruction of the RDRP enzyme activity for the expression of recombinant proteins or virus rescue. It comprises of two plasmids1. helper plasmid which expresses N, P and L proteins of MV virus and 2. a Cloning Plasmid which expresses easily manipulatable viral RNA or viral like RNA molecule (minireplicon). The cloning plasmid contains multiple cloning sites (MCS) for easy insertion of DNA encoding target molecule to be expressed. Here MV and RPV are used as model system.

EXAMPLES

(26) The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.

(27) 1. Cells and Viruses

(28) Vero (African green monkey kidney) cells were grown as monolayers in Dulbecco\'s modified Eagle\'s medium (DMEM) supplemented with 5% fetal calf serum (FCS). MRC5 cells were grown as monolayers in DMEM supplemented with 10% FCS. Measles virus (Edmonston) (MV-E) strain was purchased from Serum Institute of India (MVAC, 10.sup.3 TCID50/vial). To prepare a seed stock, Vero or MRC5 cells were seeded in 25 sq. cm flasks at 10.sup.5 cells/flask and incubated for 36 hrs. Cells were then washed with HBSS and seeded with MV-E at a MOI of 0.1 and supplemented with serum free DMEM. Virus was harvested at 24 hr intervals. Virus collected over 72 hrs was pooled together, quantitated and used as seed stock.

(29) 2. Plasmid Constructions

(30) 2.1 Cloning Plasmid

(31) 2.1.1 Designing the Replicon Construct

(32) The MV leader (ntds 1 to 107), MV trailer (ntds 15786 to 15894) and the intergenic region between the protein coding regions for MV-N and MV-P proteins (ntd. No 1686 to 1806) were selected from the AY 486084.1 sequence from Genbank (Baricevic et al, 2005). Coding regions for the green fluorescence protein (eGFP) and human growth hormone (HGH) to be used as reporter proteins were isolated from U55762.1 and NM-000515.3 respectively. All these sequences were assembled in silico into a MV-E genome like replicon containing 2 gene cassettes. Nucleotide sequences corresponding to recognition sites for Afe I, Age I, Asc I, Mlu I, Nru I, Pci I, Sac II, Xho I, Eco RI, Pac I, Pme I, Pml I, Sbf I and Xba I were arranged into 2 oligonucleotides to synthesize 2 multiple cloning sites (MCS1 and MCS 2) and inserted in the replicon around the EGFP and HGH genes. As a result, the EGFP protein appeared to have been cloned within the MCS1 region at Asc I site and HGH protein within MCS 2 at Pac I site (FIG. 1A). The sequence of the replicon without the reporter genes is given in Seq ID No. 1.

(33) The sequence corresponding to a 5 hammer head ribozyme was reconstructed from Combredet et al, (2003) and attached at the 5 end of the replicon. Similarly, sequence for 3 hepatitis delta virus ribozyme was adopted from Walker et al, (2003) and appended at the 3 end of replicon to generate a HH-replicon-HDV construct (FIG. 1B; Seq ID No. 2).

(34) Sequences encoding the promoter for chinese hamster RNA polymerase I (P1P) was selected from Tower et al, (1989) and a terminator sequence for murine RNA polymerase I terminator (P1T) was selected on basis sequences described by Grummt et al, (1985, 1986). The P1P sequence was added at immediately upstream of the 5 terminus (immediately upstream) and P1T sequence was appended immediately downstream of the 3 terminus of replicon to create P1P-replicon-P1T construct (FIG. 1C; Seq ID No. 3).

(35) 2.1.2 Synthesis of Cloning Plasmids

(36) Sequences corresponding to the HH-replicon-HDV (between Eco RI & Hind III sites) and P1P-replicon-P1T (between Sac I and Hind III sites) were synthesized using the gene synthesis method of Young and Dong (2004) and cloned into pUC57 to create pUC_HH-replicon-HDV and pUC_P1P-replicon-P1T (FIG. 2ACloning plasmid no. 1) respectively. They were then subcloned in between Nhe I and Not I sites of pIRES vector from Clonetech to generate pIRES_HH-replicon-HDV (FIG. 2CCloning plasmid no. 3) and pIRES_P1P-replicon-P1T (FIG. 2BCloning plasmid no. 2) plasmids. These plasmids were used for testing the RNA dependent RNA polymerase (RDRP) mediated expression of GFP and HGH proteins in mammalian cells.

(37) After confirming that these plasmids expressed GFP and HGH under the control of RDRP, the genes for EGFP and HGH were removed by sequential digestion and ligation with Asc I and Pac I to create 3 variants of cloning plasmidsCloning Plasmid variant 1 (HH-replicon-HDV) and Cloning plasmid variant 2 (P1P-replicon-P1T) and Cloning Plasmid Variant 3 (pUC_P1P-replicon-P1T). The different plasmids created are listed in Table 1.

(38) TABLE-US-00001 TABLE 1 Different Minireplicon plasmids created. No Name Description Sequence No 1 Cloning plasmid 1 Replicon under the control of CHO cellular Seq ID No. 6 (pUC-P1P-Rep-P1T) RNA polymerase I promoter and murine RNA polymerase I terminator in pUC57 without reporter genes 2 pUC-HH-Rep-HDV Replicon flanked by Hammerhead and Seq ID No. 7 Hepatitis Delta virus ribozymes at the 5 and 3 termini and cloned in pUC57 vector without reporter genes 3 Cloning plasmid 2 Replicon under the control of CHO cellular Seq ID No. 5 (pIRES-P1P-Rep-P1T) RNA polymerase I promoter and murine RNA polymerase I terminator subcloned into the Nhe I and Not I sites of pIRES vector from Clonetech without reporter genes 4 Cloning plasmid 3 Replicon flanked by Hammerhead and Seq ID No. 4 (pIRES-HH-Rep-HDV) Hepatitis Delta virus ribozymes at the 5 and 3 termini subcloned into the Nhe I and Not I sites of pIRES vector from Clonetech without reporter genes 5 Cloning Plasmid 1 with Replicon containing reporter genes eGFP and reporter genes HGH under the control of CHO cellular RNA Polymerase I promoter and murine RNA polymerase I terminator cloned in pUC57 6 Cloning Plasmid 2 with Replicon containing reporter genes eGFP and Seq No. 32 reporter genes HGH under the control of CHO cellular RNA Polymerase I promoter and murine RNA polymerase I terminator subcloned into Nhe I and Not I sites of pIRES vector from Clonetech. 7 Cloning Plasmid 3 with Replicon containing reporter genes eGFP and Seq No. 33 reporter genes HGH flanked by Hammerhead and Hepatitis Delta virus ribozymes at the 5 and 3 termini subcloned into the Nhe I and Not I sites of pIRES vector from Clonetech
2.1.3 Synthesis of cDNA of Entire MV-E Genome

(39) The MV-E cDNA was cloned from viral particles purified from a batch of MV-E vaccinepurchased from the Serum Institute of India, Pune, India. Viral RNA was extracted from 10.sup.5 lysed virus particles using GeneJet RNA purification kit (Fermentas) according to the manufacturer's RNA purification kit according to the manufacturer's protocol. The viral RNA was reverse transcribed into cDNA using random hexamers and Superscript II DNA polymerase. As Seven overlapping cDNA fragments covering the entire viral genome (as shown in FIG. 3a) were generated by PCR using PfuTurbo DNA polymerase and the following primers

(40) TABLE-US-00002 (1) 5-GCGGCCGCACCAAAC-3; (2) 5-CCTGACCGCGGATGC-3; (3) 5-ACCTCGCATCCGCGG-3; (4) 5-CCTCCAGAGTAATCGATTAAGG-3; (5) 5-AATCGATTACTCTGGAGGAGCAG-3; (6) 5-CTTGCACCCTAAGTTTTAATTAACTAC-3; (7) 5-GAACAATATCGGTAGTTAATTAAAAC-3; (8) 5-TGAGGGACTCGAGCATACTC-3; (9) 5-ATAAGATAGTAGCCATCCTGGAGTAT-3; (10) 5-GTAGGGCCATGTGCTGGG-3; (11) 5-CATAGCCGTAACAAAAAGGGTAC-3; (12) 5-GAGCATCAAGTGAAGGACCATG-3; (13) 5-GCATTGTGGTATTATAGAGCCTATC-3; (14) 5-CGGTTTAAACCAGACAAAGCTG-3

(41) The multiple cloning site from the plasmid pCDNA3.1() was removed by digestion with Nhe I and Pme I and replaced it with a linker containing Nhe I-Not I-Pac I-Pme I sites pCDNA-Not_Pac_Pme. The fragments generated by using different primer pairs 7, 8 (Pac I, Xho I), 9,10 (Xho I, Kpn I), 11,12 (Kpn I, Nco I) and 13, 14 (Nco I, Pme I) and ligated into a Pac I-Pme I digested pCDNA-Not_Pac_Pme to generate a plasmid with the nucleotides from Pac I to the 3 end of the MV-E antigenome called pCDNA_Not_Pac_MVg_Pme. The fragments generated by other three pairs1,2 (Not I, Sac II), 3, 4 (Sac II, Cla II), 5, 6 (Cla I, Pac I) were ligated into the Not I-Pac I digested pCDNA_Not_Pac_MVg_Pme plasmid to create pCDNA_MVgenome (FIG. 3b).

(42) 2.2 Helper Plasmid

(43) RNA was prepared from the purified MV-E virus purchased from Serum Institute of India, Pune, India using the GeneJet RNA purification kit (Fermentas) according to the manufacturer's protocol. 1 g RNA was reverse transcribed using random hexamers and amplified using primers specific for the N (F: 5-GCTAGCATGGCCACACTTTTAAGG-3 and R 5-GCGGCCGCCTAGTCTAGAAGATT-3), P (F 5-GCTAGCATGGCAGAAGAGCAGG-3,R 5-GCGGCCGCCTACTTCATTATTATC-3) and L (F 5-GCTAGCATGGACTCGCTATCTGTCAAC-3, R 5-GCGGCCGCTTAGTCCTTAATCAG-3) protein coding regions using Superscript III (Invitrogen) as described by Martin et al, (2006) and Combredet et al (2003) using standard molecular cloning techniques. Amplified cDNAs were cloned in between the Nhe I and Not I sites of pIRES vector (Clonetech) to generate pIRES_N, pIRES_P and pIRES_L plasmids.

(44) 2.2.1 Synthesis of Helper Plasmid Variant 1

(45) N protein gene was amplified from pIRES_N and subcloned into Eco RI and Pst I sites of pBiCMV1 to generate pBiCMV_N plasmid. The P protein sequence was then amplified and cloned in Nhe I and Eag I sites to create the pBiCMV_NP construct. The L protein sequence was then subcloned in the Eag I and Sal I sites of pBiCMV_NP plasmid to generate pBiCMV_NPL plasmid. This plasmid contains a bidirectional CMV promoter and can express the N and P proteins. However, the L sequence will be transcribed as a bicistronic RNA with P and will not be translated. Therefore, a mammalian beta globin IRES element (ires) described first by Chappell et al, (2000) and later on confirmed by Touzlet et al, (2008) to promote efficient translation was inserted immediately upstream of L coding region. An oligonucleotide encoding a pentameric IRES element flanked by a site for Eag I at 5 end and the first 10 nucleotides of L protein at 3 end (5 GGCCGTTCTG ACATCCGGCG GGTTTCTGAC ATCCGGCGGG TTTCTGACAT CCGGCGGGTT TCTGACATCC GGCGGGTTTC TGACATCCGG CGGGTGACTC ACAACGGATC CAACAGACAT ATGGACTCGC 3) was synthesized and inserted by site directed mutagenesis into pBiCMV_NPL to generate create pBiCMV_NPiresL plasmid which will also be called Helper Plasmid Variant 1 (HPV1) (Seq ID No. 8). This plasmid is shown in FIG. 4A as Helper Plasmid 1

(46) 2.2.2 Synthesis of Helper Plasmid Variant 2

(47) N protein sequence was amplified and subcloned in between the Nhe I and Xho I sites to obtain pIRES_N. P protein sequence was then amplified from pIRES_P and cloned into the Eco RI and Mlu I sites to create pIRES_NP. Finally, the L sequence was amplified from pIRES_L and cloned into pIRES_NP between the Sal I and Not I sites to obtain pIRES_NPL. In this form, this plasmid will express N and L proteins but not P. Therefore, a strategy based on the recently described 2A peptide vectors was used to promote the expression of P protein (szymczak and Vignali (2005)). The N and P open reading frames from pIRES_NPL were fused by inserting the oligonucleotide (5 ATCTTCTAGA CGGCTCCGGA GCCACGAACT TCTCTCTGTT AAAGCAAGCA GGAGACGTGG AAGAAAACCC CGGTCCCATG GCAGAAGAGC A 3) which encodes the porcine teschovirus 2Apeptide described by Szymczak et al (2007) flanked on the 5 end by the codons immediately before stop codon of MV N protein and on the 3 end by the first few codons of MV P protein by site directed mutagenesis to fuse the N and P protein regions into a single N2AP fusion protein and obtain pIRES_N2aPL plasmid which will also be called Helper Plasmid Variant 2 (HPV2) (Seq ID No. 9). This plasmid is shown in FIG. 4B as Helper Plasmid 2.

(48) The plasmids HPV1 (pBiCMV_NPiresL) and HPV2(pIRES_N2aPL) are represented schematically in FIGS. 4A-4B.

(49) 2.2.3 Synthesis of Equivalent Helper Plasmids Encoding N, P and L Proteins of Other Negative Stranded RNA Viruses

(50) The cloning strategy used for generation of these helper plasmids was then tested for its applicability to other negative stranded RNA virusesmainly MV, Rinderpest (RPV), peste des petits ruminants (PPRV) canine distemper (CDV), newcastle disease (NDV) and sendai viruses (SeV). Coding regions of the nucleocapsid, phosphoprotein and large proteins were analysed for the presence of restriction enzymes Eco RI, Pst I, Nhe I, Eag I, Sal I, Xho I, Mlu I and Not I. Sites for Eco RI and Pst I were absent in the nucleocapsid proteins of MV and CDV. Similarly, sites for Nhe I and Xho I were absent in the nucleocapsid proteins of MV and SeV. However, variable number of sites for enzymes Eco RI, Pst I, Nhe I and Xho I were detected in the nucleocapsid of other viruses (Table 2).

(51) TABLE-US-00003 TABLE 2 Presence of sites for Eco RI, Pst I, Nhe I and Xho I in the N protein of various negative stranded RNA viruses Virus Gen bank No Eco RI Pst I Nhe I Xho I MV AY 486084.1 0 0 0 0 RPV AB 547190.1 1 3 1 0 PPRV HQ197753.1 0 2 0 1 CDV AB 687721.2 0 1 2 0 NDV HQ008337.1 0 0 1 2 Sendai NC_001552.1 0 1 0 0

(52) Sites for enzymes Eag I, Sal I and Not I were absent from the L proteins of MV, PPRV, CDV and NDV. RPV and SeV contained 1 site for Sal I in their L proteins (Table 3).

(53) TABLE-US-00004 TABLE 3 Presence of sites for Eag I, Sal I and Not I in the L proteins of various negative stranded RNA viruses Virus Gen bank No Eag I Sal I Not I MV AY 486084.1 0 0 0 RPV AB 547190.1 0 1 1 PPRV HQ197753.1 0 0 0 CDV AB 687721.2 0 0 0 NDV HQ008337.1 0 0 0 Sendai NC_001552.1 0 1 1

(54) However, the genes for both these proteins encode a single protein each. Therefore, it would be easily possible to make synonymous mutations in their protein coding regions and eliminate the sites for these restriction enzymes. Therefore, the same cloning strategy can be easily used to clone the nucleocapsid and Large protein coding regions in a helper plasmid construct similar to either helper plasmid variant 1 or helper plasmid variant 2.

(55) Similar to the above results, analysis of the phosphoprotein coding regions of these viruses revealed the presence of a variable number of sites for enzymes Nhe I, Eag I, Eco RI and Mlu I (Table 4). Sites for these enzymes were absent from the phosphoprotein coding regions of MV, CDV and NDV.

(56) TABLE-US-00005 TABLE 3 Presence of sites for Nhe I, Eag I, Eco RI and Mlu I in the P of various negative stranded RNA viruses Virus Gen bank No Nhe I Eag I Eco RI Mlu I MV AY 486084.1 0 0 0 0 RPV AB 547190.1 1 0 3 0 RPV Z30697.2 0 0 0 0 PPRV HQ197753.1 0 0 1 0 CDV AB 687721.2 0 0 0 0 NDV HQ008337.1 0 0 0 0 Sendai NC_001552.1 0 0 1 0

(57) Although the P protein of RPV (AB547190) sequence is digested by Nhe I and Eco RI the regions corresponding to the recognition sites of these enzymes varies across different strains of RPV (e.g. Z30697.2 in Genbank). On the other hand, the Eco RI site in the P protein of PPRV appears to be highly conserved across most PPRV strains. However, this region of the P protein coding sequence does not overlap with the coding regions of C and V proteins which are also coded by the P gene transcript. Thus, it would be possible to introduce synonymous mutations in the P proteins of RPV and PPRV to enable the use of our proposed strategy for preparing the helper plasmids for MV, CDV, RPV, PPRV and NDV.

(58) Therefore, the same restriction enzymes may be used to synthesize helper plasmid constructs equivalent to those described as Helper Plasmid Variant 1 and Helper Plasmid Variant 2 from the nucleocapsid (N or NP), phosphoprotein (P) and large (L) proteins of other negative stranded RNA viruses. Such variants will be useful as helper plasmids for reconstitution of corresponding viral RNA dependent RNA polymerase enzyme and its exploitation for protein or RNA expression and also generation of recombinant viruses as novel vaccines and/or therapeutic agents.

(59) 3. Expression of Recombinant Proteins by Plasmid Encoded RDRP

(60) First the capacity of cloning plasmids to express RNA molecules which can serve as substrate for MV RNA dependent RNA polymerase (RDRP) was evaluated using a system similar to the one described by Martin et al, (2006). Briefly, Vero cells were transfected with Cloning plasmid, and individual plasmids expressing the N, P and L proteins of MV-E at a ratio of 1:1:1:0.5 in lipofectamine (Invitrogen) according to the manufacturer's protocol. Cells were incubated at 37 C. in 5% CO.sub.2 for 48 hrs and evaluated for expression of green fluorescent protein (eGFP) by microscopy and fluorescence measurement using microplate reader.

(61) In a subsequent experiment, Vero cells were transfected with equal proportions of one cloning plasmid (pUC-P1P-replicon-P1T or pIRES-HH-replicon-HDV or pIRES-P1P-replicon-P1T) and one helper plasmid (Helper variant 1 or Helper variant 2) in lipofectamine (Invitrogen) or xfect (Clonetech) and incubated at 37 C. in 5% CO.sub.2 for 48 hrs and evaluated for the expression of green fluorescent protein (eGFP) by microscopy and fluorescence.

(62) The results are shown in FIGS. 5A-5F. Briefly, Vero cells were co-transfected with Cloning plasmid encoding eGFP and HGH and Helper Plasmid Variant 1 (HPV 1) or Helper Plasmid Variant 2 (HPV 2), incubated for 48 hrs at 37 C. and observed for fluorescence and expression of HGH. FIG. 5A shows that transfection of control plasmidpUC 18 alonedoes not result in eGFP expression (Negative Control). FIG. 5B shows that transfection of a standard plasmid expressing eGFP leads to GFP expression (Positive control). FIG. 5C shows that transfection of Vero cells with Cloning Plasmid (pUC_P1P-replicon-P1T) alone does not result in the expression of eGFP. Similarly, transfection of Heper Plasmid (HPV) alone does not result in the expression of eGFP. In contrast, co-transfection of Cloning Plasmid along with the Helper Plasmid leads to the expression of eGFP (FIGS. 5D, 5E and 5F) indicating that GFP is expressed as a result of RdRP mediated expression.

(63) 4. Rescue of MV-E

(64) The capacity of the helper plasmids to rescue MV-E from cDNA was tested. Plasmid pCDNA_MVgenome was cotransfected with Helper plasmid variant 1 or Helper plasmid variant 2 in Vero cells using Xfect and incubated overnight at 37 C. Transfection medium was replaced by fresh medium and cells were incubated further for two days. When syncytia involved 80% to 90% of cell layer, virus was harvested by scraping infected cells, freeze-thawing of cells and medium and centrifugation to remove cellular debris. Collected virus was titrated using the TCID50 titration method. Briefly, Vero cells were seeded into 96 well plate (7500 cells/well) and infected by serial 1:10 dilutions of virus sample in DMEM containing 5% DCS. After incubation at 37 C. for 7 days, cells were stained with crystal violet and virus dilution that resulted in infection of 50% of test unit was determined. The 50% end point described as tissue culture infectious dose (TCID50) was calculated by the Kaber method. Virus rescued from the pCDNA_MVgenome+Helper plasmid had titers of 10.sup.6 to 10.sup.7 TCID50/mL.

(65) 5. Rescue of Segmented MV-E using Plasmid Encoded RDRP

(66) The capacity of the helper plasmids to rescue recombinant segmented MV-E from cDNA was tested. Vero cells were cotransfected with pCDNA_MVgenome, Cloning plasmid encoding eGFP and either HPV 1 or HPV 2 in equal proportions using Xfect and incubated overnight at 37 C. Transfection medium was replaced by fresh medium and continued to incubate with daily observation for syncytia formation. When syncytia involved 80% to 90% of cell layer, virus was harvested by scraping infected cells, freeze-thawing of cells and medium and centrifugation to remove cellular debris. Collected virus containing was titrated using the TCID50 titration method. Briefly, Vero cells were seeded into 96 well plate (7500 cells/well) and infected by serial 1:10 dilutions of virus sample in DMEM containing 5% DCS. After incubation at 37 C for 7 days, cells were stained with crystal violet and virus dilution that resulted in infection of 50% of test unit was determined. The 50% end point described as tissue culture infectious dose (TCID50) was calculated by the Kaber method. Virus rescued from the originally transfected cells had titers of 10.sup.6 to 10.sup.7 TCID50/mL.

(67) As shown in FIGS. 6A-6B, Cells infected with the virus harvested from the originally transfected vero cells also expressed eGFP indicating successful packaging eGFP encoding minireplicon along with MV-E genome into virions and its transfer to fresh cells.