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Identification of the Mechanisms Causing Reversion to Virulence in an Attenuated SARS-CoV for the Design of a Genetically Stable Vaccine.

Jimenez-Guardeño JM, Regla-Nava JA, Nieto-Torres JL, DeDiego ML, Castaño-Rodriguez C, Fernandez-Delgado R, Perlman S, Enjuanes L - PLoS Pathog. (2015)

Bottom Line: A SARS-CoV lacking the full-length E gene (SARS-CoV-∆E) was attenuated and an effective vaccine.To increase the genetic stability of the vaccine candidate, we introduced small attenuating deletions in E gene that did not affect the endogenous PBM, preventing the incorporation of novel chimeric proteins in the virus genome.In addition, to increase vaccine biosafety, we introduced additional attenuating mutations into the nsp1 protein.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cell Biology, Centro Nacional de Biotecnología (CNB-CSIC), Campus Universidad Autónoma de Madrid, Madrid, Spain.

ABSTRACT
A SARS-CoV lacking the full-length E gene (SARS-CoV-∆E) was attenuated and an effective vaccine. Here, we show that this mutant virus regained fitness after serial passages in cell culture or in vivo, resulting in the partial duplication of the membrane gene or in the insertion of a new sequence in gene 8a, respectively. The chimeric proteins generated in cell culture increased virus fitness in vitro but remained attenuated in mice. In contrast, during SARS-CoV-∆E passage in mice, the virus incorporated a mutated variant of 8a protein, resulting in reversion to a virulent phenotype. When the full-length E protein was deleted or its PDZ-binding motif (PBM) was mutated, the revertant viruses either incorporated a novel chimeric protein with a PBM or restored the sequence of the PBM on the E protein, respectively. Similarly, after passage in mice, SARS-CoV-∆E protein 8a mutated, to now encode a PBM, and also regained virulence. These data indicated that the virus requires a PBM on a transmembrane protein to compensate for removal of this motif from the E protein. To increase the genetic stability of the vaccine candidate, we introduced small attenuating deletions in E gene that did not affect the endogenous PBM, preventing the incorporation of novel chimeric proteins in the virus genome. In addition, to increase vaccine biosafety, we introduced additional attenuating mutations into the nsp1 protein. Deletions in the carboxy-terminal region of nsp1 protein led to higher host interferon responses and virus attenuation. Recombinant viruses including attenuating mutations in E and nsp1 genes maintained their attenuation after passage in vitro and in vivo. Further, these viruses fully protected mice against challenge with the lethal parental virus, and are therefore safe and stable vaccine candidates for protection against SARS-CoV.

No MeSH data available.


Related in: MedlinePlus

Generation of chimeric membrane genes after 16 serial passages of SARS-CoV lacking E protein in cell culture.(A) Representation of membrane chimeric genes generated after passage of SARS-CoV-∆E (∆E) and SARS-CoV∆[E,6-9b] (∆[E,6-9b] 16 times in Vero E6 cells and ∆E virus in DBT-mACE2 cells. Top, ∆E p1 represents the genomic sequence of viruses lacking E protein at passage 1. Grey boxes indicate E gene with a partial deletion highlighted in light grey (∆). Transcription-regulating sequences (TRSs) of the different genes are shown in blue boxes and the membrane gene (M) is shown in red. Chimeric membrane genes generated after 16 serial passages (p16) are formed by a partial duplication of membrane gene fused to part of SARS-CoV leader sequence (green boxes). TMD1, TMD2 and TMD3 represent the three different transmembrane domains contained within the first part of membrane gene. ATG and STOP represent the start and stop codon of potential proteins, respectively. EITL, RSVL and SLVL represent the last four amino acids of chimeric proteins that form PDZ-binding motifs. (B) Amino acid sequences corresponding to chimeric proteins generated after SARS-CoV-∆E passage in cell culture. New amino acids are shown in red. Presence of native membrane and chimeric membrane proteins was analyzed by Western blot at 24 hpi using two polyclonal antibodies generated to recognize either all membrane proteins, chimeric or not (C) or a unique sequence in the chimeric protein generated after SARS-CoV-∆E passage in DBT-mACE2 cells (D).
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ppat.1005215.g001: Generation of chimeric membrane genes after 16 serial passages of SARS-CoV lacking E protein in cell culture.(A) Representation of membrane chimeric genes generated after passage of SARS-CoV-∆E (∆E) and SARS-CoV∆[E,6-9b] (∆[E,6-9b] 16 times in Vero E6 cells and ∆E virus in DBT-mACE2 cells. Top, ∆E p1 represents the genomic sequence of viruses lacking E protein at passage 1. Grey boxes indicate E gene with a partial deletion highlighted in light grey (∆). Transcription-regulating sequences (TRSs) of the different genes are shown in blue boxes and the membrane gene (M) is shown in red. Chimeric membrane genes generated after 16 serial passages (p16) are formed by a partial duplication of membrane gene fused to part of SARS-CoV leader sequence (green boxes). TMD1, TMD2 and TMD3 represent the three different transmembrane domains contained within the first part of membrane gene. ATG and STOP represent the start and stop codon of potential proteins, respectively. EITL, RSVL and SLVL represent the last four amino acids of chimeric proteins that form PDZ-binding motifs. (B) Amino acid sequences corresponding to chimeric proteins generated after SARS-CoV-∆E passage in cell culture. New amino acids are shown in red. Presence of native membrane and chimeric membrane proteins was analyzed by Western blot at 24 hpi using two polyclonal antibodies generated to recognize either all membrane proteins, chimeric or not (C) or a unique sequence in the chimeric protein generated after SARS-CoV-∆E passage in DBT-mACE2 cells (D).

Mentions: To determine the stability of SARS-CoV-∆E, or of virus containing deletions of the E protein and several group specific genes including 6, 7a, 7b, 8a, 8b and 9b (SARS-CoV-∆[E,6-9b]), we infected Vero E6 and DBT-mACE2 cells with rSARS-CoV, rSARS-CoV-∆E or rSARS-CoV-∆[E,6-9b]. Supernatants were serially passaged 16 times and the distal third of the genome, from the S gene to the 3´ end (around 8 kb), was sequenced using specific primers (S1 Table). In all cases, an insertion consisting of a partially duplicated M gene fused to the SARS-CoV leader RNA sequence, a 5´ sequence common to coronavirus mRNAs [50–52] was detected upstream of the native M protein (Fig 1A). In contrast, no chimeric proteins were detected after serial passage of the parental virus. All MCH genes encoded the amino terminus and the three transmembrane domains of M and also different PDZ-binding motifs at the carboxy-terminus of the protein (Fig 1B). Genomic evolution occurred rapidly, as the chimeric genes were already detected within 5 passages in both cells lines, Vero E6 and DBT-mACE2.


Identification of the Mechanisms Causing Reversion to Virulence in an Attenuated SARS-CoV for the Design of a Genetically Stable Vaccine.

Jimenez-Guardeño JM, Regla-Nava JA, Nieto-Torres JL, DeDiego ML, Castaño-Rodriguez C, Fernandez-Delgado R, Perlman S, Enjuanes L - PLoS Pathog. (2015)

Generation of chimeric membrane genes after 16 serial passages of SARS-CoV lacking E protein in cell culture.(A) Representation of membrane chimeric genes generated after passage of SARS-CoV-∆E (∆E) and SARS-CoV∆[E,6-9b] (∆[E,6-9b] 16 times in Vero E6 cells and ∆E virus in DBT-mACE2 cells. Top, ∆E p1 represents the genomic sequence of viruses lacking E protein at passage 1. Grey boxes indicate E gene with a partial deletion highlighted in light grey (∆). Transcription-regulating sequences (TRSs) of the different genes are shown in blue boxes and the membrane gene (M) is shown in red. Chimeric membrane genes generated after 16 serial passages (p16) are formed by a partial duplication of membrane gene fused to part of SARS-CoV leader sequence (green boxes). TMD1, TMD2 and TMD3 represent the three different transmembrane domains contained within the first part of membrane gene. ATG and STOP represent the start and stop codon of potential proteins, respectively. EITL, RSVL and SLVL represent the last four amino acids of chimeric proteins that form PDZ-binding motifs. (B) Amino acid sequences corresponding to chimeric proteins generated after SARS-CoV-∆E passage in cell culture. New amino acids are shown in red. Presence of native membrane and chimeric membrane proteins was analyzed by Western blot at 24 hpi using two polyclonal antibodies generated to recognize either all membrane proteins, chimeric or not (C) or a unique sequence in the chimeric protein generated after SARS-CoV-∆E passage in DBT-mACE2 cells (D).
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4626112&req=5

ppat.1005215.g001: Generation of chimeric membrane genes after 16 serial passages of SARS-CoV lacking E protein in cell culture.(A) Representation of membrane chimeric genes generated after passage of SARS-CoV-∆E (∆E) and SARS-CoV∆[E,6-9b] (∆[E,6-9b] 16 times in Vero E6 cells and ∆E virus in DBT-mACE2 cells. Top, ∆E p1 represents the genomic sequence of viruses lacking E protein at passage 1. Grey boxes indicate E gene with a partial deletion highlighted in light grey (∆). Transcription-regulating sequences (TRSs) of the different genes are shown in blue boxes and the membrane gene (M) is shown in red. Chimeric membrane genes generated after 16 serial passages (p16) are formed by a partial duplication of membrane gene fused to part of SARS-CoV leader sequence (green boxes). TMD1, TMD2 and TMD3 represent the three different transmembrane domains contained within the first part of membrane gene. ATG and STOP represent the start and stop codon of potential proteins, respectively. EITL, RSVL and SLVL represent the last four amino acids of chimeric proteins that form PDZ-binding motifs. (B) Amino acid sequences corresponding to chimeric proteins generated after SARS-CoV-∆E passage in cell culture. New amino acids are shown in red. Presence of native membrane and chimeric membrane proteins was analyzed by Western blot at 24 hpi using two polyclonal antibodies generated to recognize either all membrane proteins, chimeric or not (C) or a unique sequence in the chimeric protein generated after SARS-CoV-∆E passage in DBT-mACE2 cells (D).
Mentions: To determine the stability of SARS-CoV-∆E, or of virus containing deletions of the E protein and several group specific genes including 6, 7a, 7b, 8a, 8b and 9b (SARS-CoV-∆[E,6-9b]), we infected Vero E6 and DBT-mACE2 cells with rSARS-CoV, rSARS-CoV-∆E or rSARS-CoV-∆[E,6-9b]. Supernatants were serially passaged 16 times and the distal third of the genome, from the S gene to the 3´ end (around 8 kb), was sequenced using specific primers (S1 Table). In all cases, an insertion consisting of a partially duplicated M gene fused to the SARS-CoV leader RNA sequence, a 5´ sequence common to coronavirus mRNAs [50–52] was detected upstream of the native M protein (Fig 1A). In contrast, no chimeric proteins were detected after serial passage of the parental virus. All MCH genes encoded the amino terminus and the three transmembrane domains of M and also different PDZ-binding motifs at the carboxy-terminus of the protein (Fig 1B). Genomic evolution occurred rapidly, as the chimeric genes were already detected within 5 passages in both cells lines, Vero E6 and DBT-mACE2.

Bottom Line: A SARS-CoV lacking the full-length E gene (SARS-CoV-∆E) was attenuated and an effective vaccine.To increase the genetic stability of the vaccine candidate, we introduced small attenuating deletions in E gene that did not affect the endogenous PBM, preventing the incorporation of novel chimeric proteins in the virus genome.In addition, to increase vaccine biosafety, we introduced additional attenuating mutations into the nsp1 protein.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cell Biology, Centro Nacional de Biotecnología (CNB-CSIC), Campus Universidad Autónoma de Madrid, Madrid, Spain.

ABSTRACT
A SARS-CoV lacking the full-length E gene (SARS-CoV-∆E) was attenuated and an effective vaccine. Here, we show that this mutant virus regained fitness after serial passages in cell culture or in vivo, resulting in the partial duplication of the membrane gene or in the insertion of a new sequence in gene 8a, respectively. The chimeric proteins generated in cell culture increased virus fitness in vitro but remained attenuated in mice. In contrast, during SARS-CoV-∆E passage in mice, the virus incorporated a mutated variant of 8a protein, resulting in reversion to a virulent phenotype. When the full-length E protein was deleted or its PDZ-binding motif (PBM) was mutated, the revertant viruses either incorporated a novel chimeric protein with a PBM or restored the sequence of the PBM on the E protein, respectively. Similarly, after passage in mice, SARS-CoV-∆E protein 8a mutated, to now encode a PBM, and also regained virulence. These data indicated that the virus requires a PBM on a transmembrane protein to compensate for removal of this motif from the E protein. To increase the genetic stability of the vaccine candidate, we introduced small attenuating deletions in E gene that did not affect the endogenous PBM, preventing the incorporation of novel chimeric proteins in the virus genome. In addition, to increase vaccine biosafety, we introduced additional attenuating mutations into the nsp1 protein. Deletions in the carboxy-terminal region of nsp1 protein led to higher host interferon responses and virus attenuation. Recombinant viruses including attenuating mutations in E and nsp1 genes maintained their attenuation after passage in vitro and in vivo. Further, these viruses fully protected mice against challenge with the lethal parental virus, and are therefore safe and stable vaccine candidates for protection against SARS-CoV.

No MeSH data available.


Related in: MedlinePlus