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The Promise of Proteomics in the Study of Oncogenic Viruses

2017; Elsevier BV; Volume: 16; Issue: 4 Linguagem: Inglês

10.1074/mcp.o116.065201

1535-9484

Alison A. McBride,

Virus-based gene therapy research

Oncogenic viruses are responsible for about 15% human cancers. This article explores the promise and challenges of viral proteomics in the study of the oncogenic human DNA viruses, HPV, McPyV, EBV and KSHV. These viruses have coevolved with their hosts and cause persistent infections. Each virus encodes oncoproteins that manipulate key cellular pathways to promote viral replication and evade the host immune response. Viral proteomics can identify cellular pathways perturbed by viral infection, identify cellular proteins that are crucial for viral persistence and oncogenesis, and identify important diagnostic and therapeutic targets. Oncogenic viruses are responsible for about 15% human cancers. This article explores the promise and challenges of viral proteomics in the study of the oncogenic human DNA viruses, HPV, McPyV, EBV and KSHV. These viruses have coevolved with their hosts and cause persistent infections. Each virus encodes oncoproteins that manipulate key cellular pathways to promote viral replication and evade the host immune response. Viral proteomics can identify cellular pathways perturbed by viral infection, identify cellular proteins that are crucial for viral persistence and oncogenesis, and identify important diagnostic and therapeutic targets. Proteomics is the identification and characterization of a collection of proteins found in a specific condition or circumstance. As outlined in Fig. 1, viral proteomics can define widely different collections of proteins related to viral infection. These proteomes can range from the proteins in a virion particle, to the cellular proteins found in complex with a particular viral protein, to global changes in cellular proteins in a diseased tissue following viral infection. This article will describe some of the major advances and challenges in viral proteomics, with particular emphasis on oncogenic DNA viruses. About 15% of human cancers are caused by viral infection (3.Plummer M. de Martel C. Vignat J. Ferlay J. Bray F. Franceschi S. Global burden of cancers attributable to infections in 2012: a synthetic analysis.Lancet Glob. Health. 2016; 4: Se609-SS616Abstract Full Text Full Text PDF Scopus (917) Google Scholar). Seven human tumor viruses have been described to date: Epstein-Barr virus (EBV 1The abbreviations used are: EBV, Epstein-Barr virus; KSHV, Kaposi's sarcoma-associated virus; HBV, hepatitis B virus; HCV, hepatitis C virus; HTLV, human T-lymphotrophic virus; MCPyV, Merkel cell polyoma virus; HPV, human papilloma virus; PTM, post-translational modification. or HHV4); Kaposi's sarcoma associated virus (KSHV or HHV8); Hepatitis B and C viruses (HBV and HCV); Human T-lymphotrophic virus 1 (HTLV-1); Merkel cell polyomavirus (MCPyV); and a group of alpha Human papillomaviruses (HPVs). These are listed in Table I. The DNA viruses MCPyV, EBV, KSHV, and a subset of oncogenic alpha HPVs are direct carcinogens that encode oncogenes which are required for maintenance of the tumor phenotype (1.Law G.L. Korth M.J. Benecke A.G. Katze M.G. Systems virology: host-directed approaches to viral pathogenesis and drug targeting.Nat. Rev. 2013; 11: 455-466Google Scholar); they will be the focus of this article. Fig. 2 shows the vast difference in genome size and coding capacity among these four oncogenic viruses.Table IOncogenic DNA viruses and their oncogenes. For details see (4.Moore P.S. Chang Y. Why do viruses cause cancer? Highlights of the first century of human tumour virology.Nat. Rev. Cancer. 2010; 10: 878-889Crossref PubMed Scopus (460) Google Scholar, 96.Mesri E.A. Feitelson M.A. Munger K. Human viral oncogenesis: a cancer hallmarks analysis.Cell Host Microbe. 2014; 15: 266-282Abstract Full Text Full Text PDF PubMed Scopus (401) Google Scholar, 97.Mesri E.A. Cesarman E. Boshoff C. 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These studies highlight the remarkable progress that has been made in systems virology in the last few decades. Early comparative proteomic studies of oncogenic viruses used 2D gel electrophoresis to identify differential protein expression in virally infected cells or tumors, and later, differentially expressed proteins could often be identified by mass spectrometry techniques. The yeast two hybrid technique, and related mammalian two hybrid techniques, have been extensively used to identify interacting cellular factors of oncogenic viruses (17.Rozenblatt-Rosen O. Deo R.C. Padi M. Adelmant G. Calderwood M.A. Rolland T. Grace M. Dricot A. Askenazi M. Tavares M. Pevzner S.J. Abderazzaq F. Byrdsong D. Carvunis A.R. Chen A.A. Cheng J.W. Correll M. Duarte M. Fan C.Y. Feltkamp M.C. Ficarro S.B. Franchi R. Garg B.K. Gulbahce N. Hao T. Holthaus A.M. James R. Korkhin A. Litovchick L. Mar J.C. Pak T.R. Rabello S. Rubio R. Shen Y. Singh S. Spangle J.M. Tasan M. Wanamaker S. Webber J.T. 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In infected cells, viral and cellular proteins can be defined with a global and unbiased shotgun approach in which all viral and cellular proteins can be identified at a specific time of infection, an intracellular location, or associated with a specific activity (e.g. replication). In a complementary approach, viral interactomes can be defined by determining all interactions among viral and cellular proteins using highly specific protein complex purification techniques. In practice, many of these approaches can be combined to yield important information about viral infection. These approaches are outlined in Fig. 1. Proteomics can define the proteins contained in virion particles. The components of the large, enveloped EBV and KHSV virions have been analyzed by LC-MS/MS, revealing 24–34 viral proteins in the viral capsid, tegument, and envelopes, as well as several host proteins (22.Johannsen E. Luftig M. Chase M.R. Weicksel S. Cahir-McFarland E. Illanes D. Sarracino D. Kieff E. 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Proteins of purified Epstein-Barr virus.Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 16286-16291Crossref PubMed Scopus (323) Google Scholar). Mass spectrometry techniques can also reveal post-translational modifications of virion proteins; Lind et al. identified phosphoproteins in the adenovirus type 2 virion using LC MS/MS techniques but, despite the highly purified nature of virion particles, they had to employ several additional strategies such as TiO2 enrichment and alternative digestion strategies to identify virion associated phosphoproteins (24.Lind S.B. Artemenko K.A. Elfineh L. Zhao Y.H. Bergquist J. Pettersson U. The phosphoproteome of the adenovirus type 2 virion.Virology. 2012; 433: 253-261Crossref PubMed Scopus (15) Google Scholar). These studies provide important insight into the viral life cycle strategy as virion associated proteins are often required to evade the host intrinsic immune system as well as to initiate the immediate-early viral transcriptional process. In comparison, the small nonenveloped polyoma and papilloma virions are very simple with just one major and one minor capsid protein (25.Schowalter R.M. Buck C.B. The Merkel cell polyomavirus minor capsid protein.Plos Pathog. 2013; 9: e1003558Crossref PubMed Scopus (74) Google Scholar, 26.Buck C.B. Cheng N. Thompson C.D. Lowy D.R. Steven A.C. Schiller J.T. Trus B.L. Arrangement of L2 within the papillomavirus capsid.J. Virol. 2008; 82: 5190-5197Crossref PubMed Scopus (250) Google Scholar). One unique, and relatively unstudied, feature of these small DNA viruses is that their genomes are packaged in host nucleosomes (27.Schaffhausen B.S. Benjamin T.L. Deficiency in histone acetylation in nontransforming host range mutants of polyoma virus.Proc. Natl. Acad. Sci. U.S.A. 1976; 73: 1092-1096Crossref PubMed Scopus (72) Google Scholar, 28.Favre M. Breitburd F. Croissant O. Orth G. Chromatin-like structures obtained after alkaline disruption of bovine and human papillomaviruses.J. 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Global profiling of histone modifications in the polyomavirus BK virion minichromosome.Virology. 2015; 483: 1-12Crossref PubMed Scopus (8) Google Scholar). Notably, the authors found that N-terminal acetylation of histone H2A occurred only on the viral genome packaged in virions, and not of those in infected cells (29.Fang C.Y. Shen C.H. Wang M. Chen P.L. Chan M.W. Hsu P.H. Chang D. Global profiling of histone modifications in the polyomavirus BK virion minichromosome.Virology. 2015; 483: 1-12Crossref PubMed Scopus (8) Google Scholar). However, as yet, these studies have not provided functional insight into these modifications. However, it would be surprising if the polyoma and papillomavirus life cycle strategies did not take advantage of epigenetic modulation of virion DNA to facilitate viral infection. Global profiling of chromatin modifications by mass spectrometry is challenging, in part because most modifications are located in the highly basic N-terminal tails of the histones. The Garcia laboratory has developed a very detailed workflow that includes propionic anhydride derivatization of lysine residue side chains before trypsin digestion to circumvent this problem. An additional propionylation step targets the N terminus of the digested peptides to help identify these very short peptides by nano LC-MS/MS (30.Kulej K. Avgousti D.C. Weitzman M.D. Garcia B.A. Characterization of histone post-translational modifications during virus infection using mass spectrometry-based proteomics.Methods. 2015; 90: 8-20Crossref PubMed Scopus (16) Google Scholar). The workflow also incorporates a TiO2 binding step to enrich for phosphopeptides. Garcia and colleagues used this method for dynamic global profiling of adenovirus (30.Kulej K. Avgousti D.C. 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Oncogenic viruses establish persistent infections and the late stages of infection must be induced by manipulation of the host cell (e.g. differentiation). Quasivirus particles (recircularized viral genomes packaged in a cell line overexpressing the capsid proteins) can be used to generate papillomavirus and polyomavirus particles to study the early stages of infection, and in theory epitope tagged versions of viral proteins could be packaged in these recombinant particles to facilitate their detection and localization (33.McKinney C. Kim M.J. Chen D. McBride A.A. Brd4 activates early viral transcription upon HPV18 infection of primary keratinocytes.mBio. 2016; (in press)Crossref PubMed Scopus (22) Google Scholar, 34.van Doorslaer K. Porter S. McKinney C. Stepp W.H. Mcbride A.A. Novel recombinant papillomavirus genomes expressing selectable genes.Sci Rep-Uk. 2016; (in press)Crossref Scopus (10) Google Scholar). More efficient methods are also being established to induce the late stages of infection. Throughout the course of infection, there can be dramatic changes in cellular organelles as well as the formation of viral replication and assembly factories. Subcellular fractionation and analysis of the protein components in these intracellular structures can provide great insight into the infectious process and reveal ways in which viruses manipulate cellular organization. Baquero-Pérez and Whitehouse took advantage of the fact that KSHV replication and transcription centers are associated with the nuclear envelope and this allowed them to purify, and then identify cellular factors enriched here using LC-MS/MS (35.Baquero-Perez B. Whitehouse A. Hsp70 Isoforms Are Essential for the Formation of Kaposi's Sarcoma-Associated Herpesvirus Replication and Transcription Compartments.Plos Pathog. 2015; 11: e1005274Crossref PubMed Scopus (46) Google Scholar). These studies revealed that the molecular chaperone hsp70 was crucial for the formation of these compartments (35.Baquero-Perez B. Whitehouse A. Hsp70 Isoforms Are Essential for the Formation of Kaposi's Sarcoma-Associated Herpesvirus Replication and Transcription Compartments.Plos Pathog. 2015; 11: e1005274Crossref PubMed Scopus (46) Google Scholar). Bartee et al. used SILAC (stable isotope labeling with amino acids in cell culture) and 2D-LC-MS/MS to compare the proteomics of plasma, golgi, and endoplasmic reticulum membranes of cells in the presence or absence of the KSHV K5 protein, which was known to downregulate MHC class I molecules on the surface of cells (36.Bartee E. McCormack A. Fruh K. Quantitative membrane proteomics reveals new cellular targets of viral immune modulators.Plos Pathog. 2006; 2: e107Crossref PubMed Scopus (186) Google Scholar). Using this approach, they identified three additional immunomodulatory proteins that were underrepresented in the presence of K5 (36.Bartee E. McCormack A. Fruh K. Quantitative membrane proteomics reveals new cellular targets of viral immune modulators.Plos Pathog. 2006; 2: e107Crossref PubMed Scopus (186) Google Scholar). Similar approaches with other oncogenic viruses should reveal a wealth of additional information about how these viruses manipulate cellular function and organization. Exosomes are small membrane bound vesicles that are secreted from cells into bodily fluids, and are thought to regulate the cellular microenvironment, particularly when secreted from tumor cells. Exosomes by their very nature contain an easily purified source of proteins. LC-MS/MS analyses of exosomes secreted from EBV and KSHV infected cells have shown that they contain complex mixtures of proteins that are dramatically modulated by viral infection (37.Meckes Jr, D.G. Gunawardena H.P. Dekroon R.M. Heaton P.R. Edwards R.H. Ozgur S. Griffith J.D. Damania B. Raab-Traub N. Modulation of B-cell exosome proteins by gamma herpesvirus infection.Proc. Natl. Acad. Sci. U.S.A. 2013; 110: SE2925-SE2933Crossref Scopus (176) Google Scholar). Another study examined the protein content of exosomes secreted from MCpyV positive and negative Merkel cell carcinoma cell lines; using LC-MS/MS, proteins involved in cellular motility and oncogenesis were identified (38.Konstantinell A. Bruun J.A. Olsen R. Aspar A. Skalko-Basnet N. Sveinbjornsson B. Moens U. Secretomic analysis of extracellular vesicles originating from polyomavirus-negative and polyomavirus-positive Merkel cell carcinoma cell lines.Proteomics. 2016; 16: 2587-2591Crossref PubMed Scopus (19) Google Scholar). There is strong interest in defining the contents of the exosomes in the quest for tumor biomarkers because they can be isolated noninvasively from body fluids such as saliva. Activity based protein profiling (ABBP) uses highly specific probes that consist of a reactive warhead (that creates an irreversible bond between probe and enzyme), a tag that specifically binds to the catalytic sites of the targeted enzymes, and a reporter that allows their detection or purification (39.Blais D.R. Nasheri N. McKay C.S. Legault M.C. Pezacki J.P. Activity-based protein profiling of host-virus interactions.Trends Biotechnol. 2012; 30: 89-99Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar, 40.Strmiskova M. Desrochers G.F. Shaw T.A. Powdrill M.H. Lafreniere M.A. Pezacki J.P. Chemical methods for probing virus–host proteomic interactions.ACS Infectious Diseases. 2016; (in press)Crossref PubMed Scopus (7) Google Scholar). Comparative ABPP can compare the activity of a class of enzymes in the presence or absence of viral infection. 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