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. 2000 May 9;97(10):5516-21.
doi: 10.1073/pnas.97.10.5516.

Engineering the Largest RNA Virus Genome as an Infectious Bacterial Artificial Chromosome

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Engineering the Largest RNA Virus Genome as an Infectious Bacterial Artificial Chromosome

F Almazán et al. Proc Natl Acad Sci U S A. .
Free PMC article

Abstract

The construction of cDNA clones encoding large-size RNA molecules of biological interest, like coronavirus genomes, which are among the largest mature RNA molecules known to biology, has been hampered by the instability of those cDNAs in bacteria. Herein, we show that the application of two strategies, cloning of the cDNAs into a bacterial artificial chromosome and nuclear expression of RNAs that are typically produced within the cytoplasm, is useful for the engineering of large RNA molecules. A cDNA encoding an infectious coronavirus RNA genome has been cloned as a bacterial artificial chromosome. The rescued coronavirus conserved all of the genetic markers introduced throughout the sequence and showed a standard mRNA pattern and the antigenic characteristics expected for the synthetic virus. The cDNA was transcribed within the nucleus, and the RNA translocated to the cytoplasm. Interestingly, the recovered virus had essentially the same sequence as the original one, and no splicing was observed. The cDNA was derived from an attenuated isolate that replicates exclusively in the respiratory tract of swine. During the engineering of the infectious cDNA, the spike gene of the virus was replaced by the spike gene of an enteric isolate. The synthetic virus replicated abundantly in the enteric tract and was fully virulent, demonstrating that the tropism and virulence of the recovered coronavirus can be modified. This demonstration opens up the possibility of employing this infectious cDNA as a vector for vaccine development in human, porcine, canine, and feline species susceptible to group 1 coronaviruses.

Figures

Figure 1
Figure 1
Construction of a cDNA encoding an infectious TGEV RNA as a BAC. (a) Strategy for the construction of full-length cDNA clones of TGEV. Full-length TGEV cDNA was assembled from DI-C (16, 17), and subgenomic overlapping cDNA fragments were generated by reverse transcriptase–PCR (RT-PCR). The cDNA fragments were joined at shared restriction sites and assembled as described in Materials and Methods. The genetic map of TGEV (Top) and the defective minigenome DI-C (Middle) is shown. The cDNA clones and relevant restriction sites used to restore the three deletions (Δ1, Δ2, and Δ3) of DI-C are indicated. (Top) Letters indicate the viral genes. L, leader sequence; UTR, untranslated region. Numbers in the genetic map of DI-C indicate the position of the three deletions within the genome. (b) Cloning of the TGEV cDNA in pBeloBAC11. Plasmids pBAC-TGEVΔClaI, pBAC-TGEVClaI, and pBAC-TGEVFL were generated as described in Materials and Methods. Relevant restriction sites are indicated. CMV, CMV immediate-early promoter; poly(A) tail of 24 A residues; HDV, hepatitis delta virus ribozyme; BGH, bovine GH termination and polyadenylation sequences; Sc11, S gene of PUR-C11 strain; SAP, shrimp alkaline phosphatase.
Figure 2
Figure 2
Infectious TGEV recovered from cDNA. (a) Amplification of the rPUR-MAD-Sc11 virus. ST cells were transfected either with plasmid pBAC-TGEVFL encoding an infectious TGEV RNA or with plasmid pBAC-TGEVFL−(ClaI)RS encoding the coronavirus RNA carrying the ClaI fragment inserted in the reverse orientation, or they were mock transfected. The recovered virus was passaged, and the culture supernatants were titrated on ST cells. Error bars represent standard deviations of the mean from six experiments. (b) Cytopathic effect and plaque morphology produced by the indicated virus on ST cells. (c) Analysis of the genetic markers of the recovered rPUR-MAD-Sc11 virus. Nucleotide differences within the positions of the genetic markers between the rPUR-MAD-Sc11 and the parental virus providing the TGEV genome except the S gene (PUR-MAD) or the parental providing the S gene (PUR-C11) are indicated. Only 2 of the 14 nucleotide differences in the S gene between the PUR-MAD and the PUR-C11 strains are indicated. Letters on the top bar indicate the viral genes; L, leader sequence; UTR, untranslated region.
Figure 3
Figure 3
Splicing at TGEV RNA sequence domains with high splicing potential. A databank search of the eight RNA sites with the highest probability to undergo splicing was performed as described (28). The location of the identified sites with highest splicing potential along the TGEV sequence is illustrated. ST cells were transfected with the infectious cDNA, and the potential splice sites were amplified by RT-PCR, by using as template the cytoplasmic RNA at passage zero (p0) or after the first passage (p1). The amplified DNA fragments were analyzed by agarose gel electrophoresis. Letters on the top bar indicate the viral genes; L, leader sequence; UTR, untranslated region; Sc11, S gene of PUR-C11 strain; M, molecular mass markers.
Figure 4
Figure 4
Growth of the TGEV recovered from the cDNA in cell culture and in vivo. (a and b) Growth kinetics of rPUR-MAD-Sc11, PUR-MAD, and PUR-C11 virus on ST cells after infection at high (5 pfu per cell) and low (0.05 pfu per cell) multiplicities of infection, respectively. Mean values of three experiments are indicated. The standard deviation was lower than 30% in all cases (not shown). (c) Surviving newborn National Institutes of Health minipigs infected with rPUR-MAD-Sc11, PUR-MAD, or PUR-C11 virus at 48 h after birth with 2 × 108 pfu per animal. This experiment was performed twice with similar results. (d) Growth of rPUR-MAD-Sc11, PUR-MAD, and PUR-C11 virus in the indicated tissues. Values represent the means of representative tissue samples from three animals killed 3 days after inoculation. Error bars represent standard deviation.

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