Proteome analysis of vaccinia virus IHD-W-infected HEK 293 cells with 2-dimensional gel electrophoresis and MALDI-PSD-TOF MS of on solid phase support N-terminally sulfonated peptides

Sebastian Bartel,Daniel Bourquain,Andreas Nitsche,Kai Darsow,Joerg Doellinger,Rainer Buchholz,Harald A Lange

doi:10.1186/1743-422x-8-380

Sebastian Bartel, Daniel Bourquain + Show 5 more

Open Access

https://doi.org/10.1186/1743-422x-8-380

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Abstract

BackgroundDespite the successful eradication of smallpox by the WHO-led vaccination programme, pox virus infections remain a considerable health threat. The possible use of smallpox as a bioterrorism agent as well as the continuous occurrence of zoonotic pox virus infections document the relevance to deepen the understanding for virus host interactions. Since the permissiveness of pox infections is independent of hosts surface receptors, but correlates with the ability of the virus to infiltrate the antiviral host response, it directly depends on the hosts proteome set. In this report the proteome of HEK293 cells infected with Vaccinia Virus strain IHD-W was analyzed by 2-dimensional gel electrophoresis and MALDI-PSD-TOF MS in a bottom-up approach.ResultsThe cellular and viral proteomes of VACV IHD-W infected HEK293 cells, UV-inactivated VACV IHD-W-treated as well as non-infected cells were compared. Derivatization of peptides with 4-sulfophenyl isothiocyanate (SPITC) carried out on ZipTipμ-C18 columns enabled protein identification via the peptides' primary sequence, providing improved s/n ratios as well as signal intensities of the PSD spectra. The expression of more than 24 human proteins was modulated by the viral infection. Effects of UV-inactivated and infectious viruses on the hosts' proteome concerning energy metabolism and proteins associated with gene expression and protein-biosynthesis were quite similar. These effects might therefore be attributed to virus entry and virion proteins. However, the modulation of proteins involved in apoptosis was clearly correlated to infectious viruses.ConclusionsThe proteome analysis of infected cells provides insight into apoptosis modulation, regulation of cellular gene expression and the regulation of energy metabolism. The confidence of protein identifications was clearly improved by the peptides' derivatization with SPITC on a solid phase support. Some of the identified proteins have not been described in the context of poxvirus infections before and need to be further characterised to identify their meaning for apoptosis modulation and pathogenesis.

Highlights

Despite the successful eradication of smallpox by the WHO-led vaccination programme, pox virus infections remain a considerable health threat
ZipTipμ-C18 columns solid phase-supported sulfophenyl isothiocyanate (SPITC) derivatization is preferable compared to an in-solution approach, since s/n ratios as well as signal intensities are increased, and so the sensitivity for recording high quality post source decay (PSD) spectra is enhanced
Proteome analysis of Vaccinia Virus (VACV) IHD-W-infected HEK293 cells in the late replication phase shows the upregulation of the energy metabolism and alteration of gene expression regulation

Summary

Introduction

Despite the successful eradication of smallpox by the WHO-led vaccination programme, pox virus infections remain a considerable health threat. Despite remarkable progress in the control and treatment of infectious diseases, the problem of emerging and re-emerging pathogens is likely to be one of the main issues of medical science and public health in the twenty-first century [1] In this respect viral diseases are of particular concern, because advances in the field of antiviral drugs have lagged behind those regarding bactericidal drugs and antibiotics. It was shown by the emergence of the severe acute respiratory syndrome (SARS) that new members of neglected virus families can cross into humans from unsuspected reservoirs, making rapid advances in our understanding of virushost dynamics necessary [2]. In addition to the threat of zoonoses, the relevance of a deeper understanding of the interactions of poxviruses with their host cells is pointed out by considering the classification of smallpox as a category A bioterrorism agent by the Centers for Disease Control and Prevention (CDC), especially since no acceptable treatment is available and the immunity in the population is declining [5]

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