Proteomic approaches to the study of papillomavirus–host interactions

Department of Microbiology and Immunobiology, Harvard Medical School, NRB Room 950, 77 Avenue Louis Pasteur, Boston, MA 02115, USA. Electronic address: .
Virology (Impact Factor: 3.28). 01/2013; 435(1):57-69. DOI: 10.1016/j.virol.2012.09.046
Source: PubMed

ABSTRACT The identification of interactions between viral and host cellular proteins has provided major insights into papillomavirus research, and these interactions are especially relevant to the role of papillomaviruses in the cancers with which they are associated. Recent advances in mass spectrometry technology and data processing now allow the systematic identification of such interactions. This has led to an improved understanding of the different pathologies associated with the many papillomavirus types, and the diverse nature of these viruses is reflected in the spectrum of interactions with host proteins. Here we review a history of proteomic approaches, particularly as applied to the papillomaviruses, and summarize current techniques. Current proteomic studies on the papillomaviruses use yeast-two-hybrid or affinity purification-mass spectrometry approaches. We detail the advantages and disadvantages of each and describe current examples of papillomavirus proteomic studies, with a particular focus on the HPV E6 and E7 oncoproteins.

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    ABSTRACT: Abstract Objective: The aim of our work was to identify by Western Blot technique (WB) the oncoproteins generated by E6 and E7 genes of high risk Human Papillomavirus (HR-HPV), resulting from integration of viral genomes into cervical cell DNA and their interactions with tumor suppressor human proteins p53 and pRb in premalignant or malignant cervical lesions. Methods: The study was performed on 2,500 women from Caserta Local Health Authority who underwent cervical cytology from June 2010 to September 2011. Informed consent of participants was obtained. The cell samples taken by brushing were stored in liquid based cytology (LBC) and in physiological solution for WB analysis. Cytology diagnoses was based on 2001 Bethesda classification. Proteomic research was performed according to size after extraction and SDS (Sodium Dodecyl Sulfate) electrophoresis. Proteins of interest were detected by primary monoclonal antibodies and WB analysis. Results: Cytology results of 2,500 women were positive for abnormalities of epithelial cells in 3.1%. Atypical squamous cells of undetermined significance (ASC-US) were 1.3%, Low Squamous Intraepithelial Lesion (L-SIL) 1.7% and High Squamous Intraepithelial Lesion (H-SIL) or worse 0.2%. The integration of E6 and E7 genes with their oncoproteins expression were found in 3.1% of ASC-US and in all cases of SIL or worse. On the other hand positive interactions of E6/p53 proteins and E7/pRb were found in 4.8% of L-SIL, in 75.0% of H-SIL or worse and in no ASC-US. Conclusions: Results show that the integration of viral genomes (E6 and E7) is present in all H-SIL or worse and in several L-SIL, but only the most advanced lesions, histologically confirmed, have shown the E6/p53 interaction and E7/pRb. Our protocol allows a selection of women with advanced lesions toward cancer to obtain appropriate management.
    12/2014; s4. DOI:10.4172/2157-7099.S4022
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    ABSTRACT: The cDNA libraries are indispensable and critical tools for performing protein-protein interaction studies. In this study, a high quality yeast two-hybrid cDNA library from the LFBK cell line was constructed and characterized. LFBK cell line was originally derived from the swine kidney cells and is highly susceptible to foot-and-mouth disease virus (FMDV) infection. The total RNA was extracted from the LFBK cells and the switching mechanism at the 5' end of RNA template (SMART) technique was employed for the cDNA synthesis. Subsequently, double stranded cDNA was amplified by long-distance PCR, purified and co-transformed with pGADT7-rec vector in yeast strain Y187. The quality parameters of the constructed library were evaluated to qualify the constructed library. Nucleotide sequencing of the randomly selected clones from the library confirmed the swine genotype of LFBK cell line. The LFBK cDNA library was mated with the 2C protein of FMDV in yeast two-hybrid (YTH) system and several putative interaction partners were identified in the preliminary screening. The LFBK library was observed to be of high quality and could potentially be applied to protein interaction studies between FMDV and the host cells using YTH system. Copyright © 2015 The International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.
    Biologicals 01/2015; DOI:10.1016/j.biologicals.2015.01.003 · 1.41 Impact Factor
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    ABSTRACT: The DNA of equine papillomavirus type 2 (EcPV2) is consistently found in equine papillomas and squamous cell carcinomas, indicating a causal association of EcPV2 in the pathogenesis of these tumours; however, little is known about the prevalence of this virus.
    Veterinary Dermatology 06/2014; 25(3). DOI:10.1111/vde.12129 · 1.99 Impact Factor

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