gms | German Medical Science

International Conference on SARS - one year after the (first) outbreak

08. - 11.05.2004, Lübeck

Coronavirus reverse genetics and vaccine development

Talk

  • corresponding author presenting/speaker Luis Enjuanes - Department of Molecular and Cell Biology. Centro Nacional de Biotecnología, CSIC. Campus Universidad Autónoma. Madrid. Spain
  • Fernando Almazán - Department of Molecular and Cell Biology. Centro Nacional de Biotecnología, CSIC. Campus Universidad Autónoma. Madrid. Spain
  • Isabel Sola - Department of Molecular and Cell Biology. Centro Nacional de Biotecnología, CSIC. Campus Universidad Autónoma. Madrid. Spain
  • Javier Ortego - Department of Molecular and Cell Biology. Centro Nacional de Biotecnología, CSIC. Campus Universidad Autónoma. Madrid. Spain
  • Sonia Zúñiga - Department of Molecular and Cell Biology. Centro Nacional de Biotecnología, CSIC. Campus Universidad Autónoma. Madrid. Spain
  • Sara Alonso - Department of Molecular and Cell Biology. Centro Nacional de Biotecnología, CSIC. Campus Universidad Autónoma. Madrid. Spain
  • David Escors - Department of Molecular and Cell Biology. Centro Nacional de Biotecnología, CSIC. Campus Universidad Autónoma. Madrid. Spain
  • Carmen Galán - Department of Molecular and Cell Biology. Centro Nacional de Biotecnología, CSIC. Campus Universidad Autónoma. Madrid. Spain
  • Jose L. Moreno - Department of Molecular and Cell Biology. Centro Nacional de Biotecnología, CSIC. Campus Universidad Autónoma. Madrid. Spain
  • M. Carmen Capiscol - Department of Molecular and Cell Biology. Centro Nacional de Biotecnología, CSIC. Campus Universidad Autónoma. Madrid. Spain
  • Marta L. Dediego - Department of Molecular and Cell Biology. Centro Nacional de Biotecnología, CSIC. Campus Universidad Autónoma. Madrid. Spain

International Conference on SARS - one year after the (first) outbreak. Lübeck, 08.-11.05.2004. Düsseldorf, Köln: German Medical Science; 2004. Doc04sars3.01

Die elektronische Version dieses Artikels ist vollständig und ist verfügbar unter: http://www.egms.de/de/meetings/sars2004/04sars014.shtml

Veröffentlicht: 26. Mai 2004

© 2004 Enjuanes et al.
Dieser Artikel ist ein Open Access-Artikel und steht unter den Creative Commons Lizenzbedingungen (http://creativecommons.org/licenses/by-nc-nd/3.0/deed.de). Er darf vervielf&aauml;ltigt, verbreitet und &oauml;ffentlich zug&aauml;nglich gemacht werden, vorausgesetzt dass Autor und Quelle genannt werden.


Gliederung

Text

The development of an infectious coronavirus cDNA clone has facilitated coronavirus reverse genetics to study gene function and use of coronaviruses as expression vectors. We have engineered a transmissible gastroenteritis coronavirus (TGEV) cDNA clone in E. coli as a bacterial artificial chromosome (BAC). The stability of the TGEV BAC clone in E. coli has been considerably improved by insertion of a 133-nucleotide synthetic intron to disrupt toxic sequences, allowing stable plasmid amplification for at least 200 generations, and efficient recovery of infectious TGEV. Using TGEV as a coronavirus model, we have previously described the nature and length of the transcription-regulating sequences (TRS). It was shown that the TRS core sequence (CS, 5'-CUAAAC-3') alone was sufficient for transcription when located in an appropriate context. These results and the conservation of the CS preceding all TGEV genes led us to define the TRS as a module composed by the CS plus 5' and 3' flanking sequences (5'TRS and 3'TRS, respectively). A sequence partially identical to the TRS preceding each gene (body TRS, TRS-B) is located at the 3' end of the leader (TRS-L). In the transcription process, during the synthesis of the negative RNA strand, a sequence complementary to the TRS-B (cTRS-B) hybridises with the TRS-L guiding the discontinuous synthesis. The influence of the leader-body TRS base-pairing was studied generating four groups of TGEV mutants allowing standard base-pairing and also non-Watson-Crick base-pairing with the body CS. Analysis of RNA from cells infected with these recombinant TGEVs has shown that non-canonical leader-body junction sites were used, leading to the synthesis of new subgenomic mRNAs (sgmRNAs). Sequences adjacent to the CS had an important role in transcription. The relative amount of sgmRNAs was closely related to the ΔG of the TRS-L and cTRS-B base-pairing. A minimum ΔG threshold was required for sgmRNA synthesis. Our data demonstrate the relevance of leader and body TRS identity in the selection of the fusion site, that could be predicted in silico. According to these data a model for coronavirus transcription involving three main steps has been proposed: (i) complex formation between the TRS-L and the 3' end of the virus genome; (ii) continuous complementarity scanning between the TRS-L and the nascent minus strand; and (iii) template switch to complete a minus copy of each mRNA. Using the TGEV infectious cDNA clone, replication-competent propagation-deficient vectors have been developed by deleting essential genes. These vectors were grown in packaging cell lines to high titers. In addition, virus replicons producing low or no cytopathic effect in human cells have been constructed. Replicon amplification was dramatically increased by providing the nucleoprotein (N) either in cis or in trans. Therefore, N protein probably is a replication/transcription enhancer. Vector biosafety has been improved by identifying and relocating the genome-packaging signal. Following the same strategy, similar replicons have been developed for SARS-CoV and are being used to develop vaccines and in antivirals selection to protect against SARS.