Adenovirus Composition, Proteolysis, and Disassembly Studied by In-depth Qualitative and Quantitative Proteomics

Marco Benevento,Serena Di Palma,Joost Snijder,Crystal L Moyer,Vijay S Reddy,Glen R Nemerow,Albert J.R Heck

doi:10.1074/jbc.m113.537498

Marco Benevento, Serena Di Palma + Show 5 more

Open Access

https://doi.org/10.1074/jbc.m113.537498

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Abstract

Using high-resolution MS-based proteomics in combination with multiple protease digestion, we profiled, with on average 90% sequence coverage, all 13 viral proteins present in an human adenovirus (HAdV) vector. This in-depth profile provided multiple peptide-based evidence on intrinsic protease activity affecting several HAdV proteins. Next, the generated peptide library was used to develop a targeted proteomics method using selected reaction monitoring (SRM) aimed at quantitative profiling of the stoichiometry of all 13 proteins present in the HAdV. We also used this method to probe the release of specific virus proteins initiated by thermal stimulation, mimicking the early stage of HAdV disassembly during entry into host cells. We confirmed the copy numbers of the most well characterized viral capsid components and established the copy numbers for proteins whose stoichiometry has so far not been accurately defined. We also found that heating HAdV induces the complete release of the penton base and fiber proteins as well as a substantial release of protein VIII and VI. For these latter proteins, maturational proteolysis by the adenoviral protease leads to the differential release of fragments with certain peptides being fully released and others largely retained in the AdV particles. This information is likely to be beneficial for the ongoing interpretation of high resolution cryoEM and x-ray electron density maps.

Highlights

Adenoviruses (AdV) are broadly employed as gene delivery vectors
Despite the moderate complexity and middling dynamic range in protein abundance in the human adenovirus (HAdV) proteome, we observed that full protein coverage could not be reached for each protein of HAdV when employing exclusively the commonly used protease trypsin
In some HAdV proteins, the low frequency of arginine (Arg) and lysine (Lys) results in long and hydrophobic tryptic peptides, while in other proteins, the high frequency of R residues results in small, hydrophilic tryptic peptides that are all missed in LC-MS/MS analysis

Summary

Introduction

Adenoviruses (AdV) are broadly employed as gene delivery vectors. Results: Copy numbers of all AdV proteins were measured, and the release of proteins upon heat stress investigated. The generated peptide library was used to develop a targeted proteomics method using selected reaction monitoring (SRM) aimed at quantitative profiling of the stoichiometry of all 13 proteins present in the HAdV We used this method to probe the release of specific virus proteins initiated by thermal stimulation, mimicking the early stage of HAdV disassembly during entry into host cells. For these latter proteins, maturational proteolysis by the adenoviral protease leads to the differential release of fragments with certain peptides being fully released and others largely retained in the AdV particles. This information is likely to be beneficial for the ongoing interpretation of high resolution cryoEM and x-ray electron density maps

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Conclusion