Rescue of a segmented negative-strand RNA virus entirely from cloned complementary DNAs

Abstract

We provide the first report, to our knowledge, of a helper-independent system for rescuing a segmented, negative-strand RNA genome virus entirely from cloned cDNAs. Plasmids were constructed containing full-length cDNA copies of the three Bunyamwera bunyavirus RNA genome segments flanked by bacteriophage T7 promoter and hepatitis delta virus ribozyme sequences. When cells expressing both bacteriophage T7 RNA polymerase and recombinant Bunyamwera bunyavirus proteins were transfected with these plasmids, full-length antigenome RNAs were transcribed intracellularly, and these in turn were replicated and packaged into infectious bunyavirus particles. The resulting progeny virus contained specific genetic tags characteristic of the parental cDNA clones. Reassortant viruses containing two genome segments of Bunyamwera bunyavirus and one segment of Maguari bunyavirus were also produced following transfection of appropriate plasmids. This accomplishment will allow the full application of recombinant DNA technology to manipulate the bunyavirus genome.

Figure 1

Plasmid map of the pT7ribo series. The upper part of the figure shows the sequence around the StuI and SmaI restriction sites that were used to insert blunt-ended DNA fragments. RNA transcripts produced by bacteriophage T7 RNA polymerase would contain two G residues, derived from the cloning site, before the authentic bunyavirus 5′ terminal sequence. The exact 3′ end of the RNA is specified by self-cleavage of the nascent RNA by the hepatitis delta virus (Hep δ) antigenome ribozyme (18). The conserved 11 terminal bases of all three Bunyamwera virus genome segments are shown. T7, T7 promoter; T7 term, T7 transcription termination sequence.

Figure 2

Protein profiles of transfectant and authentic bunyaviruses. BHK cells were infected with transfectant or authentic bunyaviruses and labeled with [³⁵S]methionine, and cell lysates were reacted with anti-Bunyamwera virus and anti-NSm sera. Immunoprecipitated proteins were analyzed by SDS/PAGE. Lanes 1–3, protein profiles of transfectant viruses recovered using the transcription plasmids pT7riboBUNL(+)Xho, pT7riboBUNM(+), and pT7riboBUNS(+); lanes 4–6, protein profiles of transfectant viruses recovered using the transcription plasmids pT7riboBUNL(+)Xho, pT7riboBUNM(+), and pT7riboMAGS(+); lane 7, protein profile of authentic Maguari virus (MAG); lane 8, protein profile of authentic Bunyamwera virus (BUN); and lane 9, immunoprecipitated proteins from mock infected (mi) cells. The positions of the bunyavirus proteins are indicated. The G1, G2, and N proteins of Maguari virus (which are recognized efficiently by the anti-Bunyamwera virus serum) migrate slower than their respective Bunyamwera virus counterparts.

Figure 3

Demonstration of genetic tags in the genome RNAs of transfectant virus by reverse transcription-PCR and restriction enzyme digestion. Virion RNA was extracted from the supernatant fluid of cells infected with transfectant Bunyamwera virus or authentic Bunyamwera virus. Full-length S segment cDNA was reverse-transcribed using primer S3 and amplified by PCR using primers S3 and S4 (19). To amplify a cDNA fragment of the L segment, reverse transcription was performed with primer ab30 (5′-TGCAATTAAATCATCTGGAAG), and PCR was performed with primers ab30 and ab31 (5′-GAGAGATGTAAGCTGAACAC), which amplified a fragment spanning nucleotides 2991–3315. Aliquots of the PCR products were digested with XhoI (lanes 4, 7, 10, and 13) as indicated. Control reactions (lanes 2, 5, 8, and 11) were set up omitting reverse transcriptase in the first step. The S segment PCR product obtained from transfectant virus RNA (lanes 3 and 4) was digested with XhoI to yield fragments of 566 and 395 nt, whereas that derived from authentic virus RNA (lanes 6 and 7) remained resistant to XhoI digestion. The L segment PCR product (324 nt) from transfectant virus (lanes 9 and 10) was cut by XhoI to yield comigrating fragments of 170 and 154 nt, whereas the product derived from authentic virus RNA (lanes 9 and 10) was resistant. Lane 1, BstEII-digested λ DNA markers (sizes in nt indicated).