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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

E4 transcriptional analysis. (A) E4 genomic and transcription map with probes used in northern blot analysis (not to scale). Genomic description is as in Figure 5 with the addition of putative splice donor (SD) and splice acceptor (SA) sites. The names of the identified RNAs are given to the right. ^ = splicing; An = polyadenylation.. Base pair coordinates of probes: ORF1 = 34,045-34,412, ORF2 = 33,611-34,015, ORF3 = 33,258-33,611, ORF4 = 32.882-33,214, ORF6 = 32,012-32,887, E4 = 31,855-34,550. (B) Northern blot analysis of RNA from Ad35 wild type-infected 293-ORF6 cells at 8 and 24 hpi. Probes are indicated above each lane and predicted transcripts are labeled on the left of the gel. Numbers on the right denote migration of RNA size standards. (C) Northern analysis at 24 hpi of RNA from wild type Ad35 with the following E4 regions: 1 = wt; 2 = AN; 3 = dORF6. Labeled as in panel B. Identity of transcript h was not determined.
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Figure 6: E4 transcriptional analysis. (A) E4 genomic and transcription map with probes used in northern blot analysis (not to scale). Genomic description is as in Figure 5 with the addition of putative splice donor (SD) and splice acceptor (SA) sites. The names of the identified RNAs are given to the right. ^ = splicing; An = polyadenylation.. Base pair coordinates of probes: ORF1 = 34,045-34,412, ORF2 = 33,611-34,015, ORF3 = 33,258-33,611, ORF4 = 32.882-33,214, ORF6 = 32,012-32,887, E4 = 31,855-34,550. (B) Northern blot analysis of RNA from Ad35 wild type-infected 293-ORF6 cells at 8 and 24 hpi. Probes are indicated above each lane and predicted transcripts are labeled on the left of the gel. Numbers on the right denote migration of RNA size standards. (C) Northern analysis at 24 hpi of RNA from wild type Ad35 with the following E4 regions: 1 = wt; 2 = AN; 3 = dORF6. Labeled as in panel B. Identity of transcript h was not determined.

Mentions: We next determined the transcriptional profile of the wild type Ad35 E4 region at early and late time points prior to deleting portions of the E4 transcription unit. A northern blot using a probe to the entire E4 region (E4 probe; Figure 6A) identified four and five individual transcripts at 8 and 24 hpi, respectively (Figure 6B). Because the five late transcripts appeared to include the four early transcripts and late phase RNA was more abundant, late phase RNA was used for further analysis. Open reading frame-specific probes identified three of the transcripts as having unique 5' open reading frames (Figure 6B). The ORF1 probe hybridized to only one transcript, implying that the ORF1 protein would be expressed from that transcript only. The ORF2 probe hybridized to the same transcript as the ORF1 probe and a second, smaller transcript, thus the ORF1 sequence was likely removed from the ORF2-encoding transcript. Similarly, the ORF3 probe hybridized to an even smaller RNA, suggesting that ORF1 and ORF2 sequences were absent and the ORF3 protein was expressed from this RNA. The ORF4 and ORF6 probes gave patterns indistinguishable from the entire E4 probe. The three predicted alternative spliced transcripts (ORF1, ORF2, and ORF3) could be aligned to the E4 region (annotated in Figure 6A). To determine whether the bands on the blots were E4 transcripts, we conducted northern blot analyses of RNA from cells infected with the E4-deleted rAd35 viruses dORF3-6 and AN. The transcripts identified by the probe for the entire E4 region decreased in size concomitantly with the E4 deletion in the virus (Figure 6C). A single transcript was detected with the ORF1 probe, two transcripts with the ORF2 probe and three transcripts with the ORF3 probe, while the ORF6 probe did not detect any transcripts in the E4-deleted rAd35 infected cells. These results confirmed the identity of the transcripts generated from the E4-deleted viruses as the ORF1, ORF2, and ORF3 transcripts. Interestingly, transcripts for neither ORF4 nor ORF6 were revealed in this analysis. It is possible their abundance was too low for detection by northern blot or the proteins are translated from one of the identified transcripts. The identity of the transcript labeled 'h' could not be determined (Figure 6B &6C). The ORF6 probe, which was not strand-specific, detected the transcript in cells infected with Ad35 with an intact E4 region but not in cells infected with rAd35 with ORF6 deleted (dORF3-6 and AN). However, deletion of these same sequences (dORF3-6 and AN) did not cause any change in the size of the h transcript when tested with the complete E4 probe. Thus, it was likely that the 'h' transcript was not generated from the Ad35 E4 region despite hybridizing to a probe of ORF6 sequences.


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)

E4 transcriptional analysis. (A) E4 genomic and transcription map with probes used in northern blot analysis (not to scale). Genomic description is as in Figure 5 with the addition of putative splice donor (SD) and splice acceptor (SA) sites. The names of the identified RNAs are given to the right. ^ = splicing; An = polyadenylation.. Base pair coordinates of probes: ORF1 = 34,045-34,412, ORF2 = 33,611-34,015, ORF3 = 33,258-33,611, ORF4 = 32.882-33,214, ORF6 = 32,012-32,887, E4 = 31,855-34,550. (B) Northern blot analysis of RNA from Ad35 wild type-infected 293-ORF6 cells at 8 and 24 hpi. Probes are indicated above each lane and predicted transcripts are labeled on the left of the gel. Numbers on the right denote migration of RNA size standards. (C) Northern analysis at 24 hpi of RNA from wild type Ad35 with the following E4 regions: 1 = wt; 2 = AN; 3 = dORF6. Labeled as in panel B. Identity of transcript h was not determined.
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Figure 6: E4 transcriptional analysis. (A) E4 genomic and transcription map with probes used in northern blot analysis (not to scale). Genomic description is as in Figure 5 with the addition of putative splice donor (SD) and splice acceptor (SA) sites. The names of the identified RNAs are given to the right. ^ = splicing; An = polyadenylation.. Base pair coordinates of probes: ORF1 = 34,045-34,412, ORF2 = 33,611-34,015, ORF3 = 33,258-33,611, ORF4 = 32.882-33,214, ORF6 = 32,012-32,887, E4 = 31,855-34,550. (B) Northern blot analysis of RNA from Ad35 wild type-infected 293-ORF6 cells at 8 and 24 hpi. Probes are indicated above each lane and predicted transcripts are labeled on the left of the gel. Numbers on the right denote migration of RNA size standards. (C) Northern analysis at 24 hpi of RNA from wild type Ad35 with the following E4 regions: 1 = wt; 2 = AN; 3 = dORF6. Labeled as in panel B. Identity of transcript h was not determined.
Mentions: We next determined the transcriptional profile of the wild type Ad35 E4 region at early and late time points prior to deleting portions of the E4 transcription unit. A northern blot using a probe to the entire E4 region (E4 probe; Figure 6A) identified four and five individual transcripts at 8 and 24 hpi, respectively (Figure 6B). Because the five late transcripts appeared to include the four early transcripts and late phase RNA was more abundant, late phase RNA was used for further analysis. Open reading frame-specific probes identified three of the transcripts as having unique 5' open reading frames (Figure 6B). The ORF1 probe hybridized to only one transcript, implying that the ORF1 protein would be expressed from that transcript only. The ORF2 probe hybridized to the same transcript as the ORF1 probe and a second, smaller transcript, thus the ORF1 sequence was likely removed from the ORF2-encoding transcript. Similarly, the ORF3 probe hybridized to an even smaller RNA, suggesting that ORF1 and ORF2 sequences were absent and the ORF3 protein was expressed from this RNA. The ORF4 and ORF6 probes gave patterns indistinguishable from the entire E4 probe. The three predicted alternative spliced transcripts (ORF1, ORF2, and ORF3) could be aligned to the E4 region (annotated in Figure 6A). To determine whether the bands on the blots were E4 transcripts, we conducted northern blot analyses of RNA from cells infected with the E4-deleted rAd35 viruses dORF3-6 and AN. The transcripts identified by the probe for the entire E4 region decreased in size concomitantly with the E4 deletion in the virus (Figure 6C). A single transcript was detected with the ORF1 probe, two transcripts with the ORF2 probe and three transcripts with the ORF3 probe, while the ORF6 probe did not detect any transcripts in the E4-deleted rAd35 infected cells. These results confirmed the identity of the transcripts generated from the E4-deleted viruses as the ORF1, ORF2, and ORF3 transcripts. Interestingly, transcripts for neither ORF4 nor ORF6 were revealed in this analysis. It is possible their abundance was too low for detection by northern blot or the proteins are translated from one of the identified transcripts. The identity of the transcript labeled 'h' could not be determined (Figure 6B &6C). The ORF6 probe, which was not strand-specific, detected the transcript in cells infected with Ad35 with an intact E4 region but not in cells infected with rAd35 with ORF6 deleted (dORF3-6 and AN). However, deletion of these same sequences (dORF3-6 and AN) did not cause any change in the size of the h transcript when tested with the complete E4 probe. Thus, it was likely that the 'h' transcript was not generated from the Ad35 E4 region despite hybridizing to a probe of ORF6 sequences.

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