Proteomic study of recombinant adenovirus 5 encoding human p53 by matrix-assisted laser desorption/ionization mass spectrometry in combination with database search

Liu, Yan‐Hui; Vellekamp, Gary; Chen, Guodong; Mirza, Urooj A.; Wylie, David; Twarowska, Barbara; Tang, John T.; Porter, Fred; Wang, Shihong; Nagabhushan, T L; Pramanik, Birendra N.

doi:10.1016/s1387-3806(02)00975-2

Cited by 14 publications

(18 citation statements)

References 38 publications

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“…Tryptic digestion of adenovirus proteins is typically preceded by separate denaturation with detergents, reduction and alkylation steps [e.g.,3, 5–7] and takes much longer than the 5 min required for microwave assisted acid cleavage and MALDI-TOF profiling.…”

Section: 0 Results and Discussionmentioning

confidence: 99%

“…Adenoviruses are commonly used as vectors in genetic engineering and gene therapy [1] and, as such, have generated continuing interest in new methods for their rapid characterization [for example, 2–4] The adenovirus particle is a large macromolecular assembly (M r ~ 2×10 8 Da) composed of a double-stranded DNA genome and multiple copies of at least 13 distinct proteins [3, 5–11]. A viral protease is responsible for processing [12–14].…”

Section: 0 Introductionmentioning

confidence: 99%

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Microwave assisted acid cleavage for denaturation and proteolysis of intact human adenovirus

Fenselau

Laine

Swatkoski

2011

International Journal of Mass Spectrometry

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Microwave assisted acid cleavage was applied directly to intact adenovirus type 5 to achieve denaturation and proteolysis in a single reaction. The speed of the digestion, coupled with the simplicity of MALDI analysis, allowed peptide products to be profiled in less than 5 min. Identification of peptides from a range of proteins by MALDI-TOF confirms that both denaturation and proteolysis were achieved using low concentrations of acetic acid (12.5%) and short incubations (1.5 to 2 min) at high temperatures (140° C). These conditions favor production of peptides that carry Asp on their C-termini. When this cleavage reaction is carried out in highly enriched H 2 18 O, a single atom of 18 O is introduced site-specifically into the carboxyl terminal. The labeling reaction is applied to label reporter peptides from human adenovirus type 5 harvested from HeLa cells. Small peptide products of endogenous processing were also observed. Adenoviruses are commonly used as vectors in genetic engineering and gene therapy [1] and, as such, have generated continuing interest in new methods for their rapid characterization [for example, 2-4] The adenovirus particle is a large macromolecular assembly (M r ~ 2×10 8 Da) composed of a double-stranded DNA genome and multiple copies of at least 13 distinct proteins [3,[5][6][7][8][9][10][11]. A viral protease is responsible for processing [12][13][14]. The small proteome of the adenovirus served earlier as a model system for the development in this laboratory of enzyme-catalyzed 18 O-labeling [15]. In the present report adenovirus type 5 is revisited to demonstrate the capability of Asp-selective rapid microwave-assisted acid cleavage [16][17][18][19][20][21][22] Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public Access 1.3Figure 1 illustrates the dominant mechanism by which acid cleavage is thought to occur [28] in a protein, and the pathway by which it is expected that a single atom of 18 O would be incorporated into proteolytic products. 18 O-labeling was first optimized on myoglobin and ovalbumin, and then extended to label peptides from human adenovirus type 5.The effect of acid type and concentration on the extent of incorporation of the isotope label has been evaluated. The site of incorporation is confirmed using laser induced dissociation. Material and methods 2.1Human adenovirus type 5 (ATCC # VR-5) was cultured in HeLa host cells (ATCC # CCL-2) following a published method [15]. HeLa cells were grown in 150 cm 2 culture flasks to about 90% confluence, and exposed to virus stock for 1.5 hr with slow rocking. The viru...

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Section: 0 Results and Discussionmentioning

confidence: 99%

Section: 0 Introductionmentioning

confidence: 99%

Microwave assisted acid cleavage for denaturation and proteolysis of intact human adenovirus

Fenselau

Laine

Swatkoski

2011

International Journal of Mass Spectrometry

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“…These findings indicate that either the immature core of Ad2ts1 is more ordered than the mature core of Ad35f or there is significantly more molecular mass within the Ad2ts1 core. The proteome of the mature Ad5 virion has been analyzed in detail by mass spectrometry (7,20,21). Numerous biochemical and polyacrylamide gel electrophoresis analyses (17,18,29,30) have indicated that the protein composition of Ad2ts1 is similar to that of the mature virion but with precursor forms of multiple viral proteins and a ϳ5-fold reduction in the encapsidation of protease.…”

Section: Resultsmentioning

confidence: 99%

Cryo-Electron Microscopy Structure of Adenovirus Type 2 Temperature-Sensitive Mutant 1 Reveals Insight into the Cell Entry Defect

Silvestry

Lindert

Smith

et al. 2009

J Virol

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The structure of the adenovirus type 2 temperature-sensitive mutant 1 (Ad2ts1) was determined to a resolution of 10 Å by cryo-electron microscopy single-particle reconstruction. Ad2ts1 was prepared at a nonpermissive temperature and contains the precursor forms of the capsid proteins IIIa, VI, and VIII; the core proteins VII, X (mu), and terminal protein (TP); and the L1-52K protein. Cell entry studies have shown that although Ad2ts1 can bind the coxsackievirus and Ad receptor and undergo internalization via ␣v integrins, this mutant does not escape from the early endosome and is targeted for degradation. Comparison of the Ad2ts1 structure to that of mature Ad indicates that Ad2ts1 has a different core architecture. The Ad2ts1 core is closely associated with the icosahedral capsid, a connection which may be mediated by preproteins IIIa and VI. Density within hexon cavities is assigned to preprotein VI, and membrane disruption assays show that hexon shields the lytic activity of both the mature and precursor forms of protein VI. The internal surface of the penton base in Ad2ts1 appears to be anchored to the core by interactions with preprotein IIIa. Our structural analyses suggest that these connections to the core inhibit the release of the vertex proteins and lead to the cell entry defect of Ad2ts1.Cryo-electron microscopy (cryo-EM) studies of adenovirus (Ad) combined with atomic resolution structures of component proteins (hexon, penton base, fiber, and protease) have led to a detailed structural model for the mature Ad virion (31). While the Ad protein capsid is icosahedral, the core does not follow the overall symmetry of the particle, and thus the core is not well represented in cryo-EM structures (43). The core is composed of the 36-kb double-stranded DNA (dsDNA) genome complexed with four viral proteins (V, VII, mu, and terminal protein [TP]) and the virally encoded cysteine protease. The core of the mature virion may also contain a few copies of the L1-52K protein (7), a possible scaffolding protein that is present in higher copy numbers in assembling virions (18).The capsid contains the major capsid proteins, hexon, penton base, and fiber, together with four minor capsid proteins (IIIa, VI, VIII, and IX). Cryo-EM difference mapping analyses have led to revised assignments for the locations of the minor capsid proteins, with protein IX on the exterior and the other three proteins on the inner capsid surface (9, 38). A scanning transmission EM study indicated that four trimers of protein IX stabilize the group of nine hexons in the center of each facet (11). However, more recent cryo-EM studies indicated that only the N-terminal domain of protein IX forms these trimeric assemblies (37, 38), while the C-terminal domain, which has a long predicted ␣-helix with strong propensity for coiled coil formation, associates in helical bundles at the facet edges (38). Two cryo-EM studies support the assignment of the tetrameric helical bundle on the capsid exterior to the C-terminal domain of protein IX (10, 23). Cur...

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“…Our results suggest that HAdV-5 protease can cleave pVIII at all three potential protease cleavage sites. Earlier studies have suggested the usage of only two potential cleavage sites, at amino acid 111 and amino acid 157, of HAdV-5 pVIII (12,(26)(27)(28). Although maturation of progeny adenovirus particles requires cleavage of adenovirus proteins, including pVIII, to occur in the nucleus of virus-infected cells (8,18), the cleavage of adenovirus proteins (9) including pVIII (this study) appears to occur in the cytoplasm of the transfected cells.…”

Section: Figmentioning

confidence: 64%

Proteolytic Cleavage of Bovine Adenovirus 3-Encoded pVIII

Gaba

Ayalew

Makadiya

et al. 2017

J Virol

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Proteolytic maturation involving cleavage of one nonstructural and six structural precursor proteins including pVIII by adenovirus protease is an important aspect of the adenovirus life cycle. The pVIII encoded by bovine adenovirus 3 (BAdV-3) is a protein of 216 amino acids and contains two potential protease cleavage sites. Here, we report that BAdV-3 pVIII is cleaved by adenovirus protease at both potential consensus protease cleavage sites. Usage of at least one cleavage site appears essential for the production of progeny BAdV-3 virions as glycine-to-alanine mutation of both protease cleavage sites appears lethal for the production of progeny virions. However, mutation of a single protease cleavage site of BAdV-3 pVIII significantly affects the efficient production of infectious progeny virions. Further analysis revealed no significant defect in endosome escape, genome replication, capsid formation, and virus assembly. Interestingly, cleavage of pVIII at both potential cleavage sites appears essential for the production of stable BAdV-3 virions as BAdV-3 expressing pVIII containing a glycine-to-alanine mutation of either of the potential cleavage sites is thermolabile, and this mutation leads to the production of noninfectious virions.IMPORTANCE Here, we demonstrated that the BAdV-3 adenovirus protease cleaves BAdV-3 pVIII at both potential protease cleavage sites. Although cleavage of pVIII at one of the two adenoviral protease cleavage sites is required for the production of progeny virions, the mutation of a single cleavage site of pVIII affects the efficient production of infectious progeny virions. Further analysis indicated that the mutation of a single protease cleavage site (glycine to alanine) of pVIII produces thermolabile virions, which leads to the production of noninfectious virions with disrupted capsids. We thus provide evidence about the requirement of proteolytic cleavage of pVIII for production of infectious progeny virions. We feel that our study has significantly advanced the understanding of the requirement of adenovirus protease cleavage of pVIII.KEYWORDS adenovirus protease, BAdV-3, proteolytic cleavage, thermolabile, pVIII A denoviruses are complex, large, nonenveloped viruses that have a linear doublestranded DNA genome (1). The in-depth molecular characterization of human adenoviruses and their properties, such as ease of genome manipulation, broad tropism, rapid growth to high titers in tissue culture, and transgene carrying capacity of up to 30 kb, have made adenoviruses a popular tool for gene transfer into mammalian cells (2, 3). However, although adenoviruses have been extensively studied for more than 5 decades, some aspects of adenovirus biology, such as virus assembly and final virus maturation, are not fully understood. Moreover, localization of some of the minor capsid proteins and their role(s) in the adenovirus life cycle are not clear yet.As with many other viruses and bacteriophages, proteolytic maturation is a key step in the life cycle of adenoviruses. The k...

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Proteomic study of recombinant adenovirus 5 encoding human p53 by matrix-assisted laser desorption/ionization mass spectrometry in combination with database search

Cited by 14 publications

References 38 publications

Microwave assisted acid cleavage for denaturation and proteolysis of intact human adenovirus

Microwave assisted acid cleavage for denaturation and proteolysis of intact human adenovirus

Cryo-Electron Microscopy Structure of Adenovirus Type 2 Temperature-Sensitive Mutant 1 Reveals Insight into the Cell Entry Defect

Proteolytic Cleavage of Bovine Adenovirus 3-Encoded pVIII

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