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Proteomic analysis of purified turkey adenovirus 3 virions.

ABSTRACT: Turkey adenovirus 3 (TAdV-3) causes high mortality and significant economic losses to the turkey industry. However, little is known about the molecular determinants required for viral replication and pathogenesis. Moreover, TAdV-3 does not grow well in cell culture, thus detailed structural studies of the infectious particle is particularly challenging. To develop a better understanding of virus-host interactions, we performed a comprehensive proteomic analysis of proteinase K treated purified TAdV-3 virions isolated from spleens of infected turkeys, by utilizing one-dimensional liquid chromatography mass spectrometry. Our analysis resulted in the identification of 13 viral proteins associated with TAdV-3 virions including a novel uncharacterized TaV3gp04 protein. Further, we detected 18 host proteins in purified virions, many of which are involved in cell-to cell spread, cytoskeleton dynamics and virus replication. Notably, seven of these host proteins have not yet been reported to be present in any other purified virus. In addition, five of these proteins are known antiviral host restriction factors. The availability of reagents allowed us to identify two cellular proteins (collagen alpha-1 (VI) chain and haemoglobin) in the purified TAdV-3 preparations. These results represent the first comprehensive proteomic profile of TAdV-3 and may provide information for illustrating TAdV-3 replication and pathogenesis.

SUBMITTER: Kumar P

PROVIDER: S-EPMC4497381 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Proteomic analysis of purified turkey adenovirus 3 virions.

Kumar Pankaj P van den Hurk Jan J Ayalew Lisanework E LE Gaba Amit A Tikoo Suresh K SK

Veterinary research 20150709

Turkey adenovirus 3 (TAdV-3) causes high mortality and significant economic losses to the turkey industry. However, little is known about the molecular determinants required for viral replication and pathogenesis. Moreover, TAdV-3 does not grow well in cell culture, thus detailed structural studies of the infectious particle is particularly challenging. To develop a better understanding of virus-host interactions, we performed a comprehensive proteomic analysis of proteinase K treated purified T ...[more]

PMID: 26159706

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Project description:BACKGROUND:Purified protein derivative (PPD) has been used for more than half a century as an antigen for the diagnosis of tuberculosis infection based on delayed type hypersensitivity. Although designated as "purified," in reality, the composition of PPD is highly complex and remains ill-defined. In this report, high resolution mass spectrometry was applied to understand the complexity of its constituent components. A comparative proteomic analysis of various PPD preparations and their functional characterization is likely to help in short-listing the relevant antigens required to prepare a less complex and more potent reagent for diagnostic purposes. RESULTS:Proteomic analysis of Connaught Tuberculin 68 (PPD-CT68), a tuberculin preparation generated from M. tuberculosis, was carried out in this study. PPD-CT68 is the protein component of a commercially available tuberculin preparation, Tubersol, which is used for tuberculin skin testing. Using a high resolution LTQ-Orbitrap Velos mass spectrometer, we identified 265 different proteins. The identified proteins were compared with those identified from PPD M. bovis, PPD M. avium and PPD-S2 from previous mass spectrometry-based studies. In all, 142 proteins were found to be shared between PPD-CT68 and PPD-S2 preparations. Out of the 354 proteins from M. tuberculosis-derived PPDs (i.e. proteins in either PPD-CT68 or PPD-S2), 37 proteins were found to be shared with M. avium PPD and 80 were shared with M. bovis PPD. Alignment of PPD-CT68 proteins with proteins encoded by 24 lung infecting bacteria revealed a number of similar proteins (206 bacterial proteins shared epitopes with 47 PPD-CT68 proteins), which could potentially be involved in causing cross-reactivity. The data have been deposited to the ProteomeXchange with identifier PXD000377. CONCLUSIONS:Proteomic and bioinformatics analysis of different PPD preparations revealed commonly and differentially represented proteins. This information could help in delineating the relevant antigens represented in various PPDs, which could further lead to development of a lesser complex and better defined skin test antigen with a higher specificity and sensitivity.

Project description:BackgroundTuberculin skin test based on in vivo intradermal inoculation of purified protein derivative from Mycobacterium bovis (bPPD) is the diagnostic test for the control and surveillance of bovine tuberculosis (bTB).MethodsProteomic analysis was performed on different bPPD preparations from M. bovis, strain AN5. Proteins were precipitated from bPPD solutions by TCA precipitation. The proteome of bPPD preparations was investigated by bottom-up proteomics, which consisted in protein digestion and nano-LC-MS/MS analysis. Mass spectrometry analysis was performed on a Q-exactive hybrid quadrupole-Orbitrap mass spectrometer coupled online to an Easy nano-LC1000 system.ResultsThree hundred and fifty-six proteins were identified and quantified by at least 2 peptides (99% confidence per peptide). One hundred and ninety-eight proteins, which had not been previously described, were detected; furthermore, the proteomic profile shared 80 proteins with previous proteomes from bPPDs from the United Kingdom and Brazil and 139 protein components from bPPD from Korea. Locus name of M. bovis (Mb) with orthologs from M. tuberculosis H37Rv, comparative gene and protein length, molecular mass, functional categories, gene name and function of each protein were reported. Ninety-two T cell mycobacterial antigens responsible for delayed-type hypersensitivity were detected, fifty-two of which were not previously reported in any bPPD proteome. Data are available via ProteomeXchange with identifier PXD005920.ConclusionsThis study represents the highest proteome coverage of bPPD preparations to date. Since proteins perform cellular functions essential to health and/or disease, obtaining knowledge of their presence and variance is of great importance in understanding disease states and for advancing translational studies. Therefore, to better understand Mycobacterium tuberculosis complex biology during infection, survival, and persistence, the reproducible evaluation of the proteins that catalyze and control these processes is critically important. More active and more specific tuberculins would be desirable. Indeed, many antigens contained within bPPD are currently responsible for the cross-reactivity resulting in false-positive results as they are shared between non-tuberculous and tuberculous mycobacteria.

Project description:Pseudorabies virus (PRV), a member of the Alphaherpesvirinae, has a complex multilayered extracellular virion that is structurally conserved among other herpesviruses. PRV virions contain a double-stranded DNA genome within a proteinaceous capsid surrounded by the tegument, a layer of viral and cellular proteins. The envelope layer, which encloses the capsid and tegument, contains viral transmembrane proteins anchored in a phospholipid bilayer. The viral and host proteins contained within virions execute important functions during viral spread and pathogenesis, but a detailed understanding of the composition of PRV virions has been lacking. In this report, we present the first comprehensive proteomic characterization of purified PRV virions by mass spectrometry using two complementary approaches. To exclude proteins present in the extracellular medium that may nonspecifically associate with virions, we also analyzed virions treated with proteinase K and samples prepared from mock-infected cells. Overall, we identified 47 viral proteins associated with PRV virions, 40 of which were previously localized to the capsid, tegument, and envelope layers using traditional biochemical approaches. Additionally, we identified seven viral proteins that were previously undetected in virions, including pUL8, pUL20, pUL32, pUL40 (RR2), pUL42, pUL50 (dUTPase), and Rsp40/ICP22. Furthermore, although we did not enrich for posttranslational modifications, we detected phosphorylation of four virion proteins: pUL26, pUL36, pUL46, and pUL48. Finally, we identified 48 host proteins associated with PRV virions, many of which have known functions in important cellular pathways such as intracellular signaling, mRNA translation and processing, cytoskeletal dynamics, and membrane organization. This analysis extends previous work aimed at determining the composition of herpesvirus virions and provides novel insights critical for understanding the mechanisms underlying PRV entry, assembly, egress, spread, and pathogenesis.

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Proteomic analysis of purified turkey adenovirus 3 virions.

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Proteomic analysis of purified turkey adenovirus 3 virions.

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