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Link to original content: https://pubmed.ncbi.nlm.nih.gov/16891412
Rewiring the severe acute respiratory syndrome coronavirus (SARS-CoV) transcription circuit: engineering a recombination-resistant genome - PubMed Skip to main page content
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. 2006 Aug 15;103(33):12546-51.
doi: 10.1073/pnas.0605438103. Epub 2006 Aug 4.

Rewiring the severe acute respiratory syndrome coronavirus (SARS-CoV) transcription circuit: engineering a recombination-resistant genome

Boyd Yount  1 , Rhonda S Roberts, Lisa Lindesmith, Ralph S Baric
Affiliations

Rewiring the severe acute respiratory syndrome coronavirus (SARS-CoV) transcription circuit: engineering a recombination-resistant genome

Boyd Yount et al. Proc Natl Acad Sci U S A. .

Abstract

Live virus vaccines provide significant protection against many detrimental human and animal diseases, but reversion to virulence by mutation and recombination has reduced appeal. Using severe acute respiratory syndrome coronavirus as a model, we engineered a different transcription regulatory circuit and isolated recombinant viruses. The transcription network allowed for efficient expression of the viral transcripts and proteins, and the recombinant viruses replicated to WT levels. Recombinant genomes were then constructed that contained mixtures of the WT and mutant regulatory circuits, reflecting recombinant viruses that might occur in nature. Although viable viruses could readily be isolated from WT and recombinant genomes containing homogeneous transcription circuits, chimeras that contained mixed regulatory networks were invariantly lethal, because viable chimeric viruses were not isolated. Mechanistically, mixed regulatory circuits promoted inefficient subgenomic transcription from inappropriate start sites, resulting in truncated ORFs and effectively minimize viral structural protein expression. Engineering regulatory transcription circuits of intercommunicating alleles successfully introduces genetic traps into a viral genome that are lethal in RNA recombinant progeny viruses.

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Conflict of interest statement

Conflict of interest statement: No conflicts declared.

Figures

Fig. 1.
Fig. 1.
Genome organization of SARS-CoV recombinant viruses. ORF7a/b of SARS-CoV was replaced with the Renilla luciferase gene, resulting in icSARS-CoV Luc. Second- and third-generation constructs were engineered, which contained two or three mutations in the ORF7a/b TRS, altering the ACGAAC to the double (TRS-1, ACGGAT) or triple (TRS-2, CCGGAT) mutant in icSARS-CoV Luc1 and icSARS-CoV Luc2, respectively. The TRS-2 circuit was placed throughout the icSARS-CoV CRG genome or at select sites within the icSARS-CoV PRG genome to allow for structural gene expression. A series of chimeric viruses was assembled by using the icSARS-CoV and icSARS-CoV CRG molecular clones. (A) Genetic organization of the icSARS-CoV luciferase replacement viruses. Red boxes represent WT virus TRS sites. The TRS-B sites are marked by arrows. (B) Organization of the icSARS-CoV CRG and icSARS-CoV PRG recombinant viruses. TRS sites are indicated by small red (icSARS-CoV) or blue (icSARS-CoV CRG) squares, respectively. (C) Genetic organization of chimeric viruses.
Fig. 2.
Fig. 2.
Phenotype of icSARS-CoV Luc recombinant viruses. Infected cells were lysed at various times after infection. (A) Renilla luciferase light units were quantified in triplicate. ■, icSARS-CoV Luc; □, icSARS-CoV Luc1; ▵, icSARS-CoV Luc2. (B and C) At 12 h after infection, cell lysates were separated on polyacrylamide gels and probed with antisera directed against the Renilla luciferase protein (B) or the SARS N protein (C). Lane 1, icSARS-CoV; lane 2, icSARS-CoV Luc; lane 3, icSARS-CoV Luc1; lane 4, icSARS-CoV Luc2. (D) Overall mRNA 7 levels were reduced in icSARS-CoV Luc2-infected cultures. Lane 1, icSARS-CoV; lane 2, icSARS-CoV Luc; lane 3, icSARS-CoV Luc2.
Fig. 3.
Fig. 3.
Phenotypic characterization of SARS-CoV recombinants encoding redesigned TRS networks. The icSARS-CoV CRG and icSARS-CoV PRG were inoculated onto Vero cells at a multiplicity of infection of 0.1. In addition, RNA and protein were harvested at 12 h after infection. (A) Recombinant virus growth in Vero E6 cells. ■, WT Urbani; □, icSARS-CoV; ▴, icSARS-CoV PRG; ▵, icSARS-CoV CRG no. 2. (B) Northern blot analysis of Urbani. Lane 1, icSARS-CoV; lane 2, icSARS-CoV PRG; lane 3, two different plaque-purified icSARS-CoV CRG virus-infected cells; lane 4, CRG no. 2; lane 5, CRG no. 3. Asterisks mark some transcripts noted in icSARS-CoV PRG- and CRG-infected cells. (C) Western blot analysis evaluating S (Top), N (Middle), or ORF3a (Bottom) expression.
Fig. 4.
Fig. 4.
Structure of icSARS-CoV recombinant virus ORF3a transcripts. Cultures of cells were infected with recombinant viruses. The leader-containing amplicons were purified by gel electrophoresis, subcloned, and then sequenced. (A) Organization of icSARS-CoV PRG ORF3a leader-containing transcripts. (B) ORF3a transcripts expressed in cultures transfected with chimeric recombinant genome no. 2. Cross-over sites are shown in gray boxes, and arrows represent the direction of template switching based on the transcription attenuation model for CoV RNA synthesis. Underlined ATG represents the start codon.
Fig. 5.
Fig. 5.
Infectivity of WT and chimeric recombinant RNAs. Cultures were transfected with RNA transcripts encoding the icSARS-CoV, icSARS-CoV CRG, icSARS-CoV Rec1, icSARS-CoV Rec2, and icSARS-CoV Rec3 genomes. Cells were plated onto monolayers and overlaid with medium containing agarose for determination of infectious centers or placed into 25-cm2 flasks. (A) Infectious center assay of recombinant transcripts. (B) Virus titers at different times after transfection. ■, icSARS-CoV; □, icSARS-CoV CRG.
Fig. 6.
Fig. 6.
Leader–body TRS junctions in WT and chimeric recombinant viruses. Intracellular RNA was isolated at 24 and 51 h after transfection. Using leader- and ORF7-specific primer pairs, leader-containing amplicons were separated on agarose gels, excised, subcloned, and sequenced. (A) Leader-containing amplicons in transfected cultures. (B) Leader–body junctions in mRNA 5 transcripts encoding the SARS M glycoprotein. Although the expected leader–body junctions were noted for icSARS-CoV and icSARS-CoV CRG, noncanonical mRNA 5 TRS sites were identified in chimeric viruses. Cross-over site locations are shown by gray boxes, and arrows represent the direction of template switching. Underlining marks the ATG start codon.
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References

    1. Kew O. M., Sutter R. W., de Gourville E. M., Dowdle W. R., Pallansch M. A. Annu. Rev. Microbiol. 2005;59:587–635. - PubMed
    1. Seligman S. J., Gould E. A. Lancet. 2004;363:2073–2075. - PubMed
    1. Poon L. L., Guan Y., Nicholls J. M., Yuen K. Y., Peiris J. S. M. Lancet Infect. Dis. 2004;4:663–671. - PMC - PubMed
    1. Guan Y., Zheng B. J., He Y. Q., Liu X. L., Xhuang Z. X., Cheung C. L., Luo S. W., Li P. H., Zhang L. J., Guan Y. J., et al. Science. 2003;302:276–278. - PubMed
    1. Li W., Shi Z., Yu M., Ren W., Smith C., Epstein J. H., Wang H., Crameri G., Hu Z., Zhang H., et al. Science. 2005;310:676–679. - PubMed

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