Differential amplification of adenovirus vectors by flanking the packaging signal with attB/attP-ΦC31 sequences: Implications for helper-dependent adenovirus production

Alba, Raúl; Hearing, Patrick; Bosch, Assumpció; Chillón, Miguel

doi:10.1016/j.virol.2007.05.014

Cited by 36 publications

(43 citation statements)

References 27 publications

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“…Adenovirus 5/attB (Adv) was provided by Carmen San Martín (CNB, CSIC). This recombinant virus carries a packaging modification that lengthens the viral cycle and GFP to facilitate detection (28,29). Vero cells were infected with Adv at an MOI of 1 PFU/cell for 16 hpi.…”

Section: Methodsmentioning

confidence: 99%

Cholesterol Flux Is Required for Endosomal Progression of African Swine Fever Virions during the Initial Establishment of Infection

Cuesta-Geijo

Chiappi

Galindo

et al. 2016

J Virol

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African swine fever virus (ASFV) is a major threat for porcine production that has been slowly spreading in Eastern Europe since its first appearance in the Caucasus in 2007. ASFV enters the cell by endocytosis and gains access to the cytosol to start replication from late endosomes and multivesicular bodies. Cholesterol associated with low-density lipoproteins entering the cell by endocytosis also follows a trafficking pathway similar to that of ASFV. Here we show that cholesterol plays an essential role in the establishment of infection as the virus traffics through the endocytic pathway. In contrast to the case for other DNA viruses, such as vaccinia virus or adenovirus 5, cholesterol efflux from endosomes is required for ASFV release/entry to the cytosol. Accumulation of cholesterol in endosomes impairs fusion, resulting in retention of virions inside endosomes. ASFV also remodels intracellular cholesterol by increasing its cellular uptake and redistributes free cholesterol to viral replication sites. Our analysis reveals that ASFV manipulates cholesterol dynamics to ensure an appropriate lipid flux to establish productive infection. IMPORTANCESince its appearance in the Caucasus in 2007, African swine fever (ASF) has been spreading westwards to neighboring European countries, threatening porcine production. Due to the lack of an effective vaccine, ASF control relies on early diagnosis and widespread culling of infected animals. We investigated early stages of ASFV infection to identify potential cellular targets for therapeutic intervention against ASF. The virus enters the cell by endocytosis, and soon thereafter, viral decapsidation occurs in the acid pH of late endosomes. We found that ASFV infection requires and reorganizes the cellular lipid cholesterol. ASFV requires cholesterol to exit the endosome to gain access to the cytoplasm to establish productive replication. Our results indicate that there is a differential requirement for cholesterol efflux for vaccinia virus or adenovirus 5 compared to ASFV.

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Section: Methodsmentioning

confidence: 99%

Cholesterol Flux Is Required for Endosomal Progression of African Swine Fever Virions during the Initial Establishment of Infection

Cuesta-Geijo

Chiappi

Galindo

et al. 2016

J Virol

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“…Ad5/FC31 carries two exogenous sequences flanking ⌿ ( Fig. 2A) (48). This mutant produces a negligible amount of mature virions compared to the control virus amount at 36 h postinfection (hpi) but reaches the same virus production yield as the control at 56 hpi.…”

Section: Importancementioning

confidence: 99%

“…Replication studies showed that genome production levels are similar in Ad5/FC31 and control virus. However, only 1 to 5% of the Ad5/FC31 genome was encapsidated at 36 hpi, indicating that low virus production levels at this time of infection were due to packaging problems (48). Electrophoretic mobility shift assays (EMSA) suggested that one or several nuclear proteins were binding to the exogenous sequences and interfering with correct interaction of packaging proteins and ⌿ (49).…”

Section: Importancementioning

confidence: 99%

Structures of Adenovirus Incomplete Particles Clarify Capsid Architecture and Show Maturation Changes of Packaging Protein L1 52/55k

Condezo

Marabini

Ayora

et al. 2015

J Virol

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Adenovirus is one of the most complex icosahedral, nonenveloped viruses. Even after its structure was solved at near-atomic resolution by both cryo-electron microscopy and X-ray crystallography, the location of minor coat proteins is still a subject of debate. The elaborated capsid architecture is the product of a correspondingly complex assembly process, about which many aspects remain unknown. Genome encapsidation involves the concerted action of five virus proteins, and proteolytic processing by the virus protease is needed to prime the virion for sequential uncoating. Protein L1 52/55k is required for packaging, and multiple cleavages by the maturation protease facilitate its release from the nascent virion. Light-density particles are routinely produced in adenovirus infections and are thought to represent assembly intermediates. Here, we present the molecular and structural characterization of two different types of human adenovirus light particles produced by a mutant with delayed packaging. We show that these particles lack core polypeptide V but do not lack the density corresponding to this protein in the X-ray structure, thereby adding support to the adenovirus cryo-electron microscopy model. The two types of light particles present different degrees of proteolytic processing. Their structures provide the first glimpse of the organization of L1 52/55k protein inside the capsid shell and of how this organization changes upon partial maturation. Immature, full-length L1 52/55k is poised beneath the vertices to engage the virus genome. Upon proteolytic processing, L1 52/55k disengages from the capsid shell, facilitating genome release during uncoating. IMPORTANCEAdenoviruses have been extensively characterized as experimental systems in molecular biology, as human pathogens, and as therapeutic vectors. However, a clear picture of many aspects of their basic biology is still lacking. Two of these aspects are the location of minor coat proteins in the capsid and the molecular details of capsid assembly. Here, we provide evidence supporting one of the two current models for capsid architecture. We also show for the first time the location of the packaging protein L1 52/55k in particles lacking the virus genome and how this location changes during maturation. Our results contribute to clarifying standing questions in adenovirus capsid architecture and provide new details on the role of L1 52/55k protein in assembly.A denoviruses (AdVs) (1) are among the most complex nonenveloped, icosahedral viruses. The AdV capsid is an icosahedron with a ϳ950-Å maximum diameter and triangulation number pseudo-Tϭ25. Each capsid facet has 12 trimers of the major coat protein, hexon. A pentamer of penton base protein sits at each vertex, in complex with a trimer of the projecting fiber. In addition, correct assembly requires four different minor coat proteins: IIIa, VI, VIII, and IX (2). The icosahedral shell encloses a nonicosahedral core with a linear, double-stranded DNA (dsDNA) genome (35 kbp in human AdV type 5 [H...

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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%

“…9,10 However, some HV genomes escape this cleavage, are encapsidated and contaminate the final preparations of HC-Ad. The extent of contamination varies between different methods and laboratories, 7,11 ranging between 0.01 and 1%, but these data are difficult to compare because of the lack of a standardized method for quantification of HC-Ad. In addition, high levels of HV accumulation can interfere with the packaging of HC-Ad during the amplification process, resulting in very low yields of vectors.…”

Section: Introductionmentioning

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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Differential amplification of adenovirus vectors by flanking the packaging signal with attB/attP-ΦC31 sequences: Implications for helper-dependent adenovirus production

Cited by 36 publications

References 27 publications

Cholesterol Flux Is Required for Endosomal Progression of African Swine Fever Virions during the Initial Establishment of Infection

Cholesterol Flux Is Required for Endosomal Progression of African Swine Fever Virions during the Initial Establishment of Infection

Structures of Adenovirus Incomplete Particles Clarify Capsid Architecture and Show Maturation Changes of Packaging Protein L1 52/55k

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

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