Development of a size-restricted pIX-deleted helper virus for amplification of helper-dependent adenovirus vectors

Sargent, KL; Ng, Philip; Evelegh, Carole; Graham, Felicia L.; Parks, RJ

doi:10.1038/sj.gt.3302107

Cited by 36 publications

(42 citation statements)

References 35 publications

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“…The same could be true of the burst assay, which was harvested after 48 h, but the magnitude of the reduction was so great that other effects of protein IX should be considered. The phenotype may be similar to that of a recently reported protein IX deficient virus with a severe defect in viral burst size that could not be solely explained by a defect in capsid stability (Sargent et al, 2004b). The FMDV 2A sequence used was longer than the PTV-1 sequence and included part of the FMDV 1D capsid protein.…”

Section: Discussionmentioning

confidence: 52%

Expression of heterologous genes in oncolytic adenoviruses using picornaviral 2A sequences that trigger ribosome skipping

Funston

Kallioinen

Felipe

et al. 2008

Journal of General Virology

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Insertion of picornaviral 2A sequences into mRNAs causes ribosomes to skip formation of a peptide bond at the junction of the 2A and downstream sequences, leading to the production of two proteins from a single open reading frame. Adenoviral protein IX is a minor capsid protein that has been used to display foreign peptides on the surface of the capsid. We have used 2A sequences from the foot-and-mouth disease virus (FMDV) and porcine teschovirus 1 (PTV-1) to express protein IX (pIX) and green fluorescent protein (GFP) from pIX-2A-GFP fusion genes in an oncolytic virus derived from human adenovirus 5. GFP was efficiently expressed by constructs containing either 2A sequence. Peptide bond skipping was more efficient with the 58 aa FMDV sequence than with the 22 aa PTV-1 2A sequence, but the virus with the FMDV 2A sequence showed a reduction in plaque size, cytopathic effect, viral burst size and capsid stability. We conclude that ribosome skipping induced by 2A sequences is an effective strategy to express heterologous genes in adenoviruses; however, careful selection or optimization of the 2A sequence may be required if protein IX is used as the fusion partner. INTRODUCTIONReplication-competent oncolytic adenoviruses have been extensively tested in animals, and a virus lacking the E1B 55K gene was recently approved for cancer therapy in China (reviewed by Alemany, 2007). The main factor limiting widespread application of oncolytic adenoviruses is their low efficacy. Many groups have attempted to develop more active viruses, for example by inserting genes for toxic proteins or prodrug-activating enzymes (reviewed by Alemany, 2007). We and others have previously used internal ribosome entry sites (IRESs) to initiate the translation of prodrug-activating enzymes in adenoviruses but the results were disappointing (reviewed by de Felipe, 2002;Fuerer & Iggo, 2004;Lukashev et al., 2005). Ribosome skipping is a recently described mechanism that allows translation of multiple proteins from a single mRNA. It is based on the use of a picornaviral 2A sequence that causes the ribosome to continue translation after skipping the formation of one peptide bond. This process was first characterized in foot-and-mouth disease virus (FMDV) (Ryan & Drew, 1994). The process was originally termed 'cleavage' by analogy with the proteasemediated cleavages occurring at other sites in the FMDV polyprotein but this is misleading because the 'cleavage' results from failure to form a peptide bond during translation. Unlike reinitiation of translation by IRESs, skipping induced by 2A sequences gives approximately equal expression of the proteins upstream and downstream of the 2A site (de Felipe et al., 2006). 2A and 2A-like sequences have previously been used in biotechnology applications, but not in adenoviruses (reviewed by de Felipe et al., 2006). Protein IX is a small cement protein located between the hexons in the capsid of the adenovirus (Furcinitti et al., 1989). It is essential for the packaging of full-length viral genomes (Ghosh-Cho...

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Section: Discussionmentioning

confidence: 52%

Expression of heterologous genes in oncolytic adenoviruses using picornaviral 2A sequences that trigger ribosome skipping

Funston

Kallioinen

Felipe

et al. 2008

Journal of General Virology

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“…Deletion of pIX results in virions that are heat labile, with capsids that reportedly can no longer package full-length Ad DNA (36 kb) but, instead, can accommodate only 35 kb or less (ϳ97% of the wild-type genome). However, we have recently shown that large DNA molecules (up to at least 37.2 kb) can be accommodated in capsids deficient in pIX but that these virions are noninfectious (i.e., do not form plaques) for reasons which are still unclear (16).…”

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confidence: 99%

“…293pIXc4 (16), was grown in complete medium supplemented with 0.4 mg of hygromycin/ml. AdRP2050 helper virus has been described previously (16) and was grown and had its titers determined on 293 cells.…”

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confidence: 99%

Activation of Adenoviral Gene Expression by Protein IX Is Not Required for Efficient Virus Replication

Sargent¹,

Meulenbroek²,

Parks

2004

J Virol

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The adenovirus (Ad) protein IX (pIX) is a minor component of the Ad capsid and is in part responsible for virion stability; virions lacking pIX are heat labile and lose their infectivity if the DNA content is greater than ϳ35 kb. More recently, pIX has been identified as a transcriptional activator and, in transient-transfection assays, was shown to enhance expression from the E1A, E4, and major late Ad promoters by as much as 70-fold. In this study, we examined the role of pIX's ability to activate transcription during Ad replication. In transient-transfection assays, pIX had a minimal effect on expression from the E1A promoter, increasing expression by only 1.4-fold. We used helper-dependent Ad vectors, which had all Ad protein coding sequences deleted with the exception of E1A and which had capsids that either contained or lacked pIX, to show that pIX derived from decapsidation of the infecting virion does not influence expression of E1A. Similarly, expression of pIX from the Ad genome did not alter the expression levels of E1A. Viruses that had pIX deleted showed a threefold reduction in virus yield and expression of late genes compared to those of a similar virus which encoded pIX. This phenotype could not be rescued by growing the virus in cells which constitutively express pIX. Our results indicate that, although pIX can affect transcription from a variety of viral promoters, it does not appear to play a significant role in activation of Ad promoters during normal Ad replication.The adenovirus (Ad) protein IX (pIX) is a minor component of the Ad virion that associates with the hexons that make up the facets of the icosahedron (1). There are 240 molecules of pIX per Ad virion, and within the virion, pIX is found as a trimer. pIX is expressed at an intermediate time during infection (after initiation of early gene expression but before expression of late genes); however, the levels of pIX increase dramatically after DNA replication has initiated (1). Although originally thought to be dispensable for virion formation (4), pIX was shown to be essential for the packaging of full-length viral DNA molecules (6). Deletion of pIX results in virions that are heat labile, with capsids that reportedly can no longer package full-length Ad DNA (36 kb) but, instead, can accommodate only 35 kb or less (ϳ97% of the wild-type genome). However, we have recently shown that large DNA molecules (up to at least 37.2 kb) can be accommodated in capsids deficient in pIX but that these virions are noninfectious (i.e., do not form plaques) for reasons which are still unclear (16).In addition to its role as a structural protein, pIX has been implicated as a transcriptional activator and appears to be involved in virus-induced nuclear reorganization (10,14). Whereas the N-terminal region of pIX is required for incorporation of the protein into virions, the C terminus, specifically a leucine repeat, is important for its transcriptional activity (14). In transient-transfection assays, pIX was shown to enhance expression from the Ad earl...

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“…23 Alternatively, viral genomes can attach to the capsids and become internalized in cells without being really encapsidated. 24 This suggests that further reduction of HV contamination, if necessary, would require additional mechanisms different from the selective cleavage of C. Other methods based on delayed HV encapsidation 11 or genome size restrictions 25 can be complemented with the strategy described here. An advantage of AdTetCre over standard HVs is the fact that the residual contaminants are highly attenuated, in terms of virus replication and expression of the transgene.…”

Section: Discussionmentioning

confidence: 99%

Self-inactivating helper virus for the production of high-capacity adenoviral vectors

et al. 2011

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Standard methods for producing high-capacity adenoviral vectors (HC-Ads) are based on co-infection with a helper adenovirus (HV). To avoid HV encapsidation, its packaging signal (C) is flanked by recognition sequences for recombinases expressed in the producing cells. However, accumulation of HV and low yield of HC-Ad are frequently observed, due in part to insufficient recombinase expression. We describe here a novel HV (AdTetCre) in which C is flanked by loxP sites that can be excised by a chimeric MerCreMer recombinase encoded in the same viral genome. Efficient modulation of cleavage was obtained by simultaneous control of MerCreMer expression using a tet-on inducible system, and translocation to the nucleus by 4-hydroxytamoxifen (TAM). Encapsidation of AdTetCre was strongly inhibited by TAM plus doxycicline. Using AdTetCre and 293Cre4 cells for the production of HC-Ads, we found that cellular and virus-encoded recombinases cooperate to minimize HV contamination. The method was highly reproducible and allowed the routine production of different HC-Ads in a medium-scale laboratory setting in adherent cells, with titers 410 10 infectious units and o0.1% HV contamination. The residual HVs lacked C and were highly attenuated. We conclude that self-inactivating HVs based on virally encoded recombinases are promising tools for the production of

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Development of a size-restricted pIX-deleted helper virus for amplification of helper-dependent adenovirus vectors

Cited by 36 publications

References 35 publications

Expression of heterologous genes in oncolytic adenoviruses using picornaviral 2A sequences that trigger ribosome skipping

Expression of heterologous genes in oncolytic adenoviruses using picornaviral 2A sequences that trigger ribosome skipping

Activation of Adenoviral Gene Expression by Protein IX Is Not Required for Efficient Virus Replication

Self-inactivating helper virus for the production of high-capacity adenoviral vectors

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