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Characterization of human adenovirus 35 and derivation of complex vectors.

McVey D, Zuber M, Ettyreddy D, Reiter CD, Brough DE, Nabel GJ, King CR, Gall JG - Virol. J. (2010)

Bottom Line: Sequences essential for E1 and E4 function were identified and removed and the effects of the deletions on viral gene transcription were determined.In addition, the non-essential E3 region was deleted from rAd35 vectors and a sequence was found that did not have an effect on viability but reduced viral fitness.This rAd35 vector with multiple gene deletions has the advantages of multiple blocks to viral replication (i.e., E1 and E4 deletions) and a transgene packaging capacity of 7.6 Kb, comparable to rAd5 vectors.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Research, GenVec, Inc, Gaithersburg, MD 20874, USA.

ABSTRACT

Background: Replication-deficient recombinant adenoviral vectors based on human serotype 35 (Ad35) are desirable due to the relatively low prevalence of neutralizing antibodies in the human population. The structure of the viral genome and life cycle of Ad35 differs from the better characterized Ad5 and these differences require differences in the strategies for the generation of vectors for gene delivery.

Results: Sequences essential for E1 and E4 function were identified and removed and the effects of the deletions on viral gene transcription were determined. In addition, the non-essential E3 region was deleted from rAd35 vectors and a sequence was found that did not have an effect on viability but reduced viral fitness. The packaging capacity of rAd35 was dependent on pIX and vectors were generated with stable genome sizes of up to 104% of the wild type genome size. These data were used to make an E1-, E3-, E4-deleted rAd35 vector. This rAd35 vector with multiple gene deletions has the advantages of multiple blocks to viral replication (i.e., E1 and E4 deletions) and a transgene packaging capacity of 7.6 Kb, comparable to rAd5 vectors.

Conclusions: The results reported here allow the generation of larger capacity rAd35 vectors and will guide the derivation of adenovirus vectors from other serotypes.

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Related in: MedlinePlus

E1B and pIX transcription mapping. (A) Schematic of E1b and pIX sequences, splice junctions shown as ^ with their coordinates that were identified by cDNA analysis, and probe locations (not to scale). Base pair coordinates of the probes are: 1 = 1641-1903, 2 = 2527-3046, 3 = 3283-3359, 4 = 3511-3853. Solid thick lines depict transcripts a, b and c as determined from cDNA and northern blot analysis (panel B) with apparent sizes given to the right in nucleotides (nt). The predicted proteins (boxes) are labeled with the name of their Ad5 homologues pIX, E1b19K, E1b55K [6], and E1b15K [11]. (B) Northern blot analysis of pIX transcripts in cells infected with wild type Ad35. Probes 1, 2, 3, 4 from panel A; a, b, c, = RNAs corresponding to putative mRNAs. (C) Schematic of Ad35 E1 region deletions with Ad35 coordinates corresponding to deletion junctions shown above each line. The viral left ITR, E1A TATAA box, and E1A, E1B, and pIX coding sequences are represented. (D) and (E) Northern blot analysis of transcripts in cells infected with wild type Ad35 (wt) or Ad35 viruses with E1 deletions and hybridized to probe 4. Location of transcripts a, b and c are indicated to the left of each blot.
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Figure 3: E1B and pIX transcription mapping. (A) Schematic of E1b and pIX sequences, splice junctions shown as ^ with their coordinates that were identified by cDNA analysis, and probe locations (not to scale). Base pair coordinates of the probes are: 1 = 1641-1903, 2 = 2527-3046, 3 = 3283-3359, 4 = 3511-3853. Solid thick lines depict transcripts a, b and c as determined from cDNA and northern blot analysis (panel B) with apparent sizes given to the right in nucleotides (nt). The predicted proteins (boxes) are labeled with the name of their Ad5 homologues pIX, E1b19K, E1b55K [6], and E1b15K [11]. (B) Northern blot analysis of pIX transcripts in cells infected with wild type Ad35. Probes 1, 2, 3, 4 from panel A; a, b, c, = RNAs corresponding to putative mRNAs. (C) Schematic of Ad35 E1 region deletions with Ad35 coordinates corresponding to deletion junctions shown above each line. The viral left ITR, E1A TATAA box, and E1A, E1B, and pIX coding sequences are represented. (D) and (E) Northern blot analysis of transcripts in cells infected with wild type Ad35 (wt) or Ad35 viruses with E1 deletions and hybridized to probe 4. Location of transcripts a, b and c are indicated to the left of each blot.

Mentions: Based on the exon - intron junctions found by the cDNA library analysis and the conserved structure of human adenovirus E1/pIX regions, a set of probes for detection of RNA transcripts were designed to span the predicted introns and exons (Figure 3A). Northern blot analysis of steady-state RNA from wild type Ad35 infected cells detected three distinct transcripts, 'a', 'b', and 'c' (Figure 3B). A separate northern blot analysis with strand-specific probes demonstrated the three transcripts were generated from the E1/pIX coding strand (data not shown). The largest transcript, transcript 'a,' hybridized to all four probes, consistent with an mRNA encoding E1B 214R and 494R proteins, homologs for Ad5 E1B 19K and 55k proteins, respectively (previously annotated by sequence homology [6]). Transcript 'b' hybridized to probes 1, 3, and 4 but not 2, identifying it as the doubly spliced transcript found by cDNA analysis. Transcript 'b' would be predicted to encode for E1B 19K homolog and a modified form of 55K with a predicted molecular weight of 15K, from removal of an intron as described for Ad5 [11]. The smallest transcript, 'c,' hybridized to only probe 4. In addition, nucleotide 3367 was the 5'-most nucleotide identified with the cDNA library, consistent with the identification of transcript 'c' encoding only pIX. None of the transcripts hybridized to a probe to the second intron (data not shown) suggesting that all three transcripts were spliced in this intergenic region. Thus, two alternative spliced transcripts of E1B were identified and the pIX transcript was identified and shown to have a 5' untranslated region in E1B, as annotated in Figure 3A.


Characterization of human adenovirus 35 and derivation of complex vectors.

McVey D, Zuber M, Ettyreddy D, Reiter CD, Brough DE, Nabel GJ, King CR, Gall JG - Virol. J. (2010)

E1B and pIX transcription mapping. (A) Schematic of E1b and pIX sequences, splice junctions shown as ^ with their coordinates that were identified by cDNA analysis, and probe locations (not to scale). Base pair coordinates of the probes are: 1 = 1641-1903, 2 = 2527-3046, 3 = 3283-3359, 4 = 3511-3853. Solid thick lines depict transcripts a, b and c as determined from cDNA and northern blot analysis (panel B) with apparent sizes given to the right in nucleotides (nt). The predicted proteins (boxes) are labeled with the name of their Ad5 homologues pIX, E1b19K, E1b55K [6], and E1b15K [11]. (B) Northern blot analysis of pIX transcripts in cells infected with wild type Ad35. Probes 1, 2, 3, 4 from panel A; a, b, c, = RNAs corresponding to putative mRNAs. (C) Schematic of Ad35 E1 region deletions with Ad35 coordinates corresponding to deletion junctions shown above each line. The viral left ITR, E1A TATAA box, and E1A, E1B, and pIX coding sequences are represented. (D) and (E) Northern blot analysis of transcripts in cells infected with wild type Ad35 (wt) or Ad35 viruses with E1 deletions and hybridized to probe 4. Location of transcripts a, b and c are indicated to the left of each blot.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 3: E1B and pIX transcription mapping. (A) Schematic of E1b and pIX sequences, splice junctions shown as ^ with their coordinates that were identified by cDNA analysis, and probe locations (not to scale). Base pair coordinates of the probes are: 1 = 1641-1903, 2 = 2527-3046, 3 = 3283-3359, 4 = 3511-3853. Solid thick lines depict transcripts a, b and c as determined from cDNA and northern blot analysis (panel B) with apparent sizes given to the right in nucleotides (nt). The predicted proteins (boxes) are labeled with the name of their Ad5 homologues pIX, E1b19K, E1b55K [6], and E1b15K [11]. (B) Northern blot analysis of pIX transcripts in cells infected with wild type Ad35. Probes 1, 2, 3, 4 from panel A; a, b, c, = RNAs corresponding to putative mRNAs. (C) Schematic of Ad35 E1 region deletions with Ad35 coordinates corresponding to deletion junctions shown above each line. The viral left ITR, E1A TATAA box, and E1A, E1B, and pIX coding sequences are represented. (D) and (E) Northern blot analysis of transcripts in cells infected with wild type Ad35 (wt) or Ad35 viruses with E1 deletions and hybridized to probe 4. Location of transcripts a, b and c are indicated to the left of each blot.
Mentions: Based on the exon - intron junctions found by the cDNA library analysis and the conserved structure of human adenovirus E1/pIX regions, a set of probes for detection of RNA transcripts were designed to span the predicted introns and exons (Figure 3A). Northern blot analysis of steady-state RNA from wild type Ad35 infected cells detected three distinct transcripts, 'a', 'b', and 'c' (Figure 3B). A separate northern blot analysis with strand-specific probes demonstrated the three transcripts were generated from the E1/pIX coding strand (data not shown). The largest transcript, transcript 'a,' hybridized to all four probes, consistent with an mRNA encoding E1B 214R and 494R proteins, homologs for Ad5 E1B 19K and 55k proteins, respectively (previously annotated by sequence homology [6]). Transcript 'b' hybridized to probes 1, 3, and 4 but not 2, identifying it as the doubly spliced transcript found by cDNA analysis. Transcript 'b' would be predicted to encode for E1B 19K homolog and a modified form of 55K with a predicted molecular weight of 15K, from removal of an intron as described for Ad5 [11]. The smallest transcript, 'c,' hybridized to only probe 4. In addition, nucleotide 3367 was the 5'-most nucleotide identified with the cDNA library, consistent with the identification of transcript 'c' encoding only pIX. None of the transcripts hybridized to a probe to the second intron (data not shown) suggesting that all three transcripts were spliced in this intergenic region. Thus, two alternative spliced transcripts of E1B were identified and the pIX transcript was identified and shown to have a 5' untranslated region in E1B, as annotated in Figure 3A.

Bottom Line: Sequences essential for E1 and E4 function were identified and removed and the effects of the deletions on viral gene transcription were determined.In addition, the non-essential E3 region was deleted from rAd35 vectors and a sequence was found that did not have an effect on viability but reduced viral fitness.This rAd35 vector with multiple gene deletions has the advantages of multiple blocks to viral replication (i.e., E1 and E4 deletions) and a transgene packaging capacity of 7.6 Kb, comparable to rAd5 vectors.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Research, GenVec, Inc, Gaithersburg, MD 20874, USA.

ABSTRACT

Background: Replication-deficient recombinant adenoviral vectors based on human serotype 35 (Ad35) are desirable due to the relatively low prevalence of neutralizing antibodies in the human population. The structure of the viral genome and life cycle of Ad35 differs from the better characterized Ad5 and these differences require differences in the strategies for the generation of vectors for gene delivery.

Results: Sequences essential for E1 and E4 function were identified and removed and the effects of the deletions on viral gene transcription were determined. In addition, the non-essential E3 region was deleted from rAd35 vectors and a sequence was found that did not have an effect on viability but reduced viral fitness. The packaging capacity of rAd35 was dependent on pIX and vectors were generated with stable genome sizes of up to 104% of the wild type genome size. These data were used to make an E1-, E3-, E4-deleted rAd35 vector. This rAd35 vector with multiple gene deletions has the advantages of multiple blocks to viral replication (i.e., E1 and E4 deletions) and a transgene packaging capacity of 7.6 Kb, comparable to rAd5 vectors.

Conclusions: The results reported here allow the generation of larger capacity rAd35 vectors and will guide the derivation of adenovirus vectors from other serotypes.

Show MeSH
Related in: MedlinePlus