Viral proteomics: global evaluation of viruses and their interaction with the host

Viruses constantly adapt to and modulate the host environment during replication and propagation. Both DNA and RNA viruses encode multifunctional proteins that interact with and modify host cell proteins. While viral genomes were the first complete sequences known, the corresponding proteomes are only now elucidated, with some surprising results. Even more daunting is the task to globally monitor the impact of viral infection on the proteome of the host cell and many technical hurdles must still be overcome in order to facilitate robust and reproducible measurements. Further complicating the picture is the dynamic nature of proteins, including post-translational modifications, enzymatic cleavage and activation or destruction by proteolytic events. Nevertheless, several promising studies have been published using high-throughput methods directly measuring protein abundance. Particularly, quantitative or semiquantitative mass spectrometry-based analysis of viral and cellular proteomes are now being used to characterize viruses and their host interaction. In addition, the full set of interactions between viral and host proteins, the interactome, is beginning to emerge, with often unexpected interactions that need to be carefully validated. In this review, we will discuss two major areas of viral proteomics: first, virion proteomics (such as the protein characterization of viral particles) and second, proteoviromics, including the viral protein interactomics and the quantitative analysis of host cell proteome during viral infection.     

Viral proteomics: The emerging cutting-edge of virus research

Viruses replicate and proliferate in host cells while continuously adjusting to and modulating the host environment. They encode a wide spectrum of multifunctional proteins, which interplay with and modify proteins in host cells. Viral genomes were chronologically the first to be sequenced. However, the corresponding viral proteomes, the alterations of host proteomes upon viral infection, and the dynamic nature of proteins, such as post-translational modifications, enzymatic cleavage, and activation or destruction by proteolysis, remain largely unknown. Emerging high-throughput techniques, in particular quantitative or semi-quantitative mass spectrometry-based proteomics analysis of viral and cellular proteomes, have been applied to define viruses and their interactions with their hosts. Here, we review the major areas of viral proteomics, including virion proteomics, structural proteomics, viral protein interactomics, and changes to the host cell proteome upon viral infection.     

Quantitative analysis of cellular proteome alterations in human influenza A virus‐infected mammalian cell lines

Over the last years virus–host cell interactions were investigated in numerous studies. Viral strategies for evasion of innate immune response, inhibition of cellular protein synthesis and permission of viral RNA and protein production were disclosed. With quantitative proteome technology, comprehensive studies concerning the impact of viruses on the cellular machinery of their host cells at protein level are possible. Therefore, 2‐D DIGE and nanoHPLC‐nanoESI‐MS/MS analysis were used to qualitatively and quantitatively determine the dynamic cellular proteome responses of two mammalian cell lines to human influenza A virus infection. A cell line used for vaccine production (MDCK) was compared with a human lung carcinoma cell line (A549) as a reference model. Analyzing 2‐D gels of the proteomes of uninfected and influenza‐infected host cells, 16 quantitatively altered protein spots (at least ±1.7‐fold change in relative abundance, p<0.001) were identified for both cell lines. Most significant changes were found for keratins, major components of the cytoskeleton system, and for Mx proteins, interferon‐induced key components of the host cell defense. Time series analysis of infection processes allowed the identification of further proteins that are described to be involved in protein synthesis, signal transduction and apoptosis events. Most likely, these proteins are required for supporting functions during influenza viral life cycle or host cell stress response. Quantitative proteome‐wide profiling of virus infection can provide insights into complexity and dynamics of virus–host cell interactions and may accelerate antiviral research and support optimization of vaccine manufacturing processes.     

How viruses hijack cell regulation

Viruses, as obligate intracellular parasites, are the pathogens that have the most intimate relationship with their host, and as such, their genomes have been shaped directly by interactions with the host proteome. Every step of the viral life cycle, from entry to budding, is orchestrated through interactions with cellular proteins. Accordingly, viruses will hijack and manipulate these proteins utilising any achievable mechanism. Yet, the extensive interactions of viral proteomes has yielded a conundrum: how do viruses commandeer so many diverse pathways and processes, given the obvious spatial constraints imposed by their compact genomes? One important approach is slowly being revealed, the extensive mimicry of host protein short linear motifs (SLiMs).     

Comparative Analysis of T-Cell Spatial Proteomics and the Influence of HIV Expression

As systems biology approaches to virology have become more tractable, highly studied viruses such as HIV can now be analyzed in new unbiased ways, including spatial proteomics. We employed here a differential centrifugation protocol to fractionate Jurkat T cells for proteomic analysis by mass spectrometry; these cells contain inducible HIV-1 genomes, enabling us to look for changes in the spatial proteome induced by viral gene expression. Using these proteomics data, we evaluated the merits of several reported machine learning pipelines for classification of the spatial proteome and identification of protein translocations. From these analyses, we found that classifier performance in this system was organelle dependent, with Bayesian t-augmented Gaussian mixture modeling outperforming support vector machine learning for mitochondrial and endoplasmic reticulum proteins but underperforming on cytosolic, nuclear, and plasma membrane proteins by QSep analysis. We also observed a generally higher performance for protein translocation identification using a Bayesian model, Bayesian analysis of differential localization experiments, on row-normalized data. Comparative Bayesian analysis of differential localization experiment analysis of cells induced to express the WT viral genome versus cells induced to express a genome unable to express the accessory protein Nef identified known Nef-dependent interactors such as T-cell receptor signaling components and coatomer complex. Finally, we found that support vector machine classification showed higher consistency and was less sensitive to HIV-dependent noise. These findings illustrate important considerations for studies of the spatial proteome following viral infection or viral gene expression and provide a reference for future studies of HIV-gene-dropout viruses.     

White spot syndrome virus proteins and differentially expressed host proteins identified in shrimp epithelium by shotgun proteomics and cleavable isotope-coded affinity tag

Shrimp subcuticular epithelial cells are the initial and major targets of white spot syndrome virus (WSSV) infection. Proteomic studies of WSSV-infected subcuticular epithelium of Penaeus monodon were performed through two approaches, namely, subcellular fractionation coupled with shotgun proteomics to identify viral and host proteins and a quantitative time course proteomic analysis using cleavable isotope-coded affinity tags (cICATs) to identify differentially expressed cellular proteins. Peptides were analyzed by offline coupling of two-dimensional liquid chromatography with matrix-assisted laser desorption ionization-tandem time of flight mass spectrometry. We identified 27, 20, and 4 WSSV proteins from cytosolic, nuclear, and membrane fractions, respectively. Twenty-eight unique WSSV proteins with high confidence (total ion confidence interval percentage [CI%], >95%) were observed, 11 of which are reported here for the first time, and 3 of these novel proteins were shown to be viral nonstructural proteins by Western blotting analysis. A first shrimp protein data set containing 1,999 peptides (ion score, > or =20) and 429 proteins (total ion score CI%, >95%) was constructed via shotgun proteomics. We also identified 10 down-regulated proteins and 2 up-regulated proteins from the shrimp epithelial lysate via cICAT analysis. This is the first comprehensive study of WSSV-infected epithelia by proteomics. The 11 novel viral proteins represent the latest addition to our knowledge of the WSSV proteome. Three proteomic data sets consisting of WSSV proteins, epithelial cellular proteins, and differentially expressed cellular proteins generated in the course of WSSV infection provide a new resource for further study of WSSV-shrimp interactions.     

Quantitative proteomic analysis of A549 cells infected with human respiratory syncytial virus subgroup B using SILAC coupled to LC-MS/MS

Human respiratory syncytial virus (HRSV) is a leading cause of serious lower respiratory tract infections in infants. The virus has two subgroups A and B, which differ in prevalence and (nucleotide) sequence. The interaction of subgroup A viruses with the host cell is relatively well characterized, whereas for subgroup B viruses it is not. Therefore quantitative proteomics was used to investigate the interaction of subgroup B viruses with A549 cells, a respiratory cell line. Changes in the cellular proteome and potential canonical pathways were determined using SILAC coupled to LC-MS/MS and Ingenuity Pathway Analysis. To reduce sample complexity and investigate potential trafficking both nuclear and cytoplasmic fractions were analyzed. A total of 904 cellular and six viral proteins were identified and quantified, of which 112 cellular proteins showed a twofold or more change in HRSV-infected cells. Data sets were validated using indirect immunofluorescence confocal microscopy on independent samples. Major changes were observed in constituents of mitochondria including components of the electron transport chain complexes and channels, as well as increases in the abundance of the products of interferon-stimulated genes. This is the first quantitative proteomic analysis of cells infected with HRSV-subgroup B.     

Enriching the viral–host interactomes with interactions mediated by SH3 domains

Protein–protein interactions play an essential role in the regulation of most cellular processes. The process of viral infection is no exception and many viral pathogenic strategies involve targeting and perturbing host–protein interactions. The characterization of the host protein subnetworks disturbed by invading viruses is a major goal of viral research and may contribute to reveal fundamental biological mechanisms and to identify new therapeutic strategies. To assist in this approach, we have developed a database, VirusMINT, which stores in a structured format most of the published interactions between viral and host proteome. Although SH3 are the most ubiquitous and abundant class of protein binding modules, VirusMINT contains only a few interactions mediated by this domain class. To overcome this limitation, we have applied the whole interactome scanning experiment approach to identify interactions between 15 human SH3 domains and viral proline-rich peptides of two oncogenic viruses, human papillomavirus type 16 and human adenovirus A type 12. This approach identifies 114 new potential interactions between the human SH3 domains and proline-rich regions of the two viral proteomes.     

Proteomic analysis of virus-host interactions in an infectious context using recombinant viruses

RNA viruses exhibit small-sized genomes encoding few proteins, but still establish complex networks of interactions with host cell components to achieve replication and spreading. Ideally, these virus-host protein interactions should be mapped directly in infected cell culture, but such a high standard is often difficult to reach when using conventional approaches. We thus developed a new strategy based on recombinant viruses expressing tagged viral proteins to capture both direct and indirect physical binding partners during infection. As a proof of concept, we engineered a recombinant measles virus (MV) expressing one of its virulence factors, the MV-V protein, with a One-STrEP amino-terminal tag. This allowed virus-host protein complex analysis directly from infected cells by combining modified tandem affinity chromatography and mass spectrometry analysis. Using this approach, we established a prosperous list of 245 cellular proteins interacting either directly or indirectly with MV-V, and including four of the nine already known partners of this viral factor. These interactions were highly specific of MV-V because they were not recovered when the nucleoprotein MV-N, instead of MV-V, was tagged. Besides key components of the antiviral response, cellular proteins from mitochondria, ribosomes, endoplasmic reticulum, protein phosphatase 2A, and histone deacetylase complex were identified for the first time as prominent targets of MV-V and the critical role of the later protein family in MV replication was addressed. Most interestingly, MV-V showed some preferential attachment to essential proteins in the human interactome network, as assessed by centrality and interconnectivity measures. Furthermore, the list of MV-V interactors also showed a massive enrichment for well-known targets of other viruses. Altogether, this clearly supports our approach based on reverse genetics of viruses combined with high-throughput proteomics to probe the interaction network that viruses establish in infected cells.     

病毒感染蛋白质组学研究进展

病毒的侵入会导致宿主细胞蛋白表达模式的改变,这种改变将影响宿主细胞的正常生理功能并决定病毒的致病进程和结果.因此,病毒感染蛋白质组学研究有助于揭示病毒与宿主的相互作用机制和病毒的分子致病机制,以及寻找病毒早期感染的分子标记、建立早期诊断方法、评价治疗效果和预后.本文介绍了病毒感染蛋白质组学研究技术、病毒诱导宿主细胞蛋白质组改变和病毒感染宿主血清差异蛋白质组等方面的研究进展.     

Quantitative subcellular proteome and secretome profiling of influenza A virus-infected human primary macrophages

Influenza A viruses are important pathogens that cause acute respiratory diseases and annual epidemics in humans. Macrophages recognize influenza A virus infection with their pattern recognition receptors, and are involved in the activation of proper innate immune response. Here, we have used high-throughput subcellular proteomics combined with bioinformatics to provide a global view of host cellular events that are activated in response to influenza A virus infection in human primary macrophages. We show that viral infection regulates the expression and/or subcellular localization of more than one thousand host proteins at early phases of infection. Our data reveals that there are dramatic changes in mitochondrial and nuclear proteomes in response to infection. We show that a rapid cytoplasmic leakage of lysosomal proteins, including cathepsins, followed by their secretion, contributes to inflammasome activation and apoptosis seen in the infected macrophages. Also, our results demonstrate that P2X₇ receptor and src tyrosine kinase activity are essential for inflammasome activation during influenza A virus infection. Finally, we show that influenza A virus infection is associated with robust secretion of different danger-associated molecular patterns (DAMPs) suggesting an important role for DAMPs in host response to influenza A virus infection. In conclusion, our high-throughput quantitative proteomics study provides important new insight into host-response against influenza A virus infection in human primary macrophages.     

Application of proteomics technology for analyzing the interactions between host cells and intracellular infectious agents

Host-pathogen interactions involve protein expression changes within both the host and the pathogen. An understanding of the nature of these interactions provides insight into metabolic processes and critical regulatory events of the host cell as well as into the mechanisms of pathogenesis by infectious microorganisms. Pathogen exposure induces changes in host proteins at many functional levels including cell signaling pathways, protein degradation, cytokines and growth factor production, phagocytosis, apoptosis, and cytoskeletal rearrangement. Since proteins are responsible for the cell biological functions, pathogens have evolved to manipulate the host cell proteome to achieve optimal replication. Intracellular pathogens can also change their proteome to adapt to the host cell and escape from immune surveillance, or can incorporate cellular proteins to invade other cells. Given that the interactions of intracellular infectious agents with host cells are mainly at the protein level, proteomics is the most suitable tool for investigating these interactions. Proteomics is the systematic analysis of proteins, particularly their interactions, modifications, localization and functions, that permits the study of the association between pathogens with their host cells as well as complex interactions such as the host-vector-pathogen interplay. A review on the most relevant proteomic applications used in the study of host-pathogen interactions is presented.     

Comprehensive Profiling of Lysine Acetylome in Baculovirus Infected Silkworm (Bombyx mori) Cells

Bombyx mori is one of the key lepidopteran model species, and is economically important for silk production and proteinaceous drug expression. Baculovirus and insect host are important natural biological models for studying host–pathogen interactions. The impact of Bombyx mori nucleopolyhedrovirus (BmNPV) infection on the proteome and acetylome of Bombyx mori ovarian (BmN) cells are explored to facilitate a better understanding of infection‐driven interactions between BmNPV and host in vitro. The proteome and acetylome are profiled through six‐plex Tandem mass tag (TMT) labeling‐based quantitative proteomics. A total of 4194 host proteins are quantified, of which 33 are upregulated and 47 are downregulated in BmN cells at 36 h post‐infection. Based on the proteome, quantifiable differential Kac proteins are identified and functionally annotated to gene expression regulation, energy metabolism, substance synthesis, and metabolism after BmNPV infection. Altogether, 644 Kac sites in 431 host proteins and 39 Kac sites in 22 viral proteins are identified and quantified in infected BmN cells. Our study demonstrates that BmNPV infection globally impacts the proteome and acetylome of BmN cells. The viral proteins are also acetylated by the host acetyltransferase. Protein acetylation is essential for cellular self‐regulation and response to virus infection. This study provides new insights for understanding the host–virus interaction mechanisms, and the role of acetylation in BmN cellular response to viral infection.     

Targeted Proteomics for Studying Pathogenic Bacteria

Mass spectrometry‐based proteomics has been extensively used to map bacterial proteomes, which has led to a better understanding of the molecular mechanisms underlying bacterial infection and bacteria–host interactions. Quantitative proteomics using selected or parallel reaction monitoring is considered one of the most sensitive and specific quantitative MS‐based approaches and has significantly advanced proteome studies of pathogenic bacteria. Here, recent applications of targeted proteomics for bacteria identification, biomarker discovery, and the characterization of bacterial virulence and antimicrobial resistance are reviewed among others. Results of such studies are expected to further contribute to improve the fight against the most common human pathogenic bacteria.     

Magnetic Fractionation and Proteomic Dissection of Cellular Organelles Occupied by the Late Replication Complexes of Semliki Forest Virus

Alphavirus replicase complexes are initially formed at the plasma membrane and are subsequently internalized by endocytosis. During the late stages of infection, viral replication organelles are represented by large cytopathic vacuoles, where replicase complexes bind to membranes of endolysosomal origin. In addition to viral components, these organelles harbor an unknown number of host proteins. In this study, a fraction of modified lysosomes carrying functionally intact replicase complexes was obtained by feeding Semliki Forest virus (SFV)-infected HeLa cells with dextran-covered magnetic nanoparticles and later magnetically isolating the nanoparticle-containing lysosomes. Stable isotope labeling with amino acids in cell culture combined with quantitative proteomics was used to reveal 78 distinct cellular proteins that were at least 2.5-fold more abundant in replicase complex-carrying vesicles than in vesicles obtained from noninfected cells. These host components included the RNA-binding proteins PCBP1, hnRNP M, hnRNP C, and hnRNP K, which were shown to colocalize with the viral replicase. Silencing of hnRNP M and hnRNP C expression enhanced the replication of SFV, Chikungunya virus (CHIKV), and Sindbis virus (SINV). PCBP1 silencing decreased SFV-mediated protein synthesis, whereas hnRNP K silencing increased this synthesis. Notably, the effect of hnRNP K silencing on CHIKV- and SINV-mediated protein synthesis was opposite to that observed for SFV. This study provides a new approach for analyzing the proteome of the virus replication organelle of positive-strand RNA viruses and helps to elucidate how host RNA-binding proteins exert important but diverse functions during positive-strand RNA viral infection.     

种子蛋白质组的研究进展

蛋白质组学是通过对全套蛋白质动态的研究,来阐明生物体、组织、细胞和亚细胞全部蛋白质的表达模式及功能模式。大量可用的核苷酸序列信息和灵敏高速的质谱鉴定技术,使得蛋白质组学方法为分析模式植物和农作物的复杂功能开辟了新的途径。目前,种子蛋白质组研究主要集中在两个方面：一方面是鉴定尽可能多的蛋白,以创建种子特定生命时期的蛋白质组参照图谱;另一方面主要集中在差异蛋白质组,通过比较分析不同蛋白质组,以探明关键功能蛋白。该文综述了近年来种子蛋白质组的研究进展,内容包括种子发育过程中蛋白质组的变化,与种子休眠/萌发相关的蛋白质组、翻译后修饰蛋白质组、细胞与亚细胞差异蛋白质组以及环境因子对种子蛋白质组的影响;并对种子蛋白质组研究的热点问题进行了展望。     

种子蛋白质组的研究进展

蛋白质组学是通过对全套蛋白质动态的研究, 来阐明生物体、组织、细胞和亚细胞全部蛋白质的表达模式及功能模式。大量可用的核苷酸序列信息和灵敏高速的质谱鉴定技术, 使得蛋白质组学方法为分析模式植物和农作物的复杂功能开辟了新的途径。目前, 种子蛋白质组研究主要集中在两个方面: 一方面是鉴定尽可能多的蛋白, 以创建种子特定生命时期的蛋白质组参照图谱; 另一方面主要集中在差异蛋白质组, 通过比较分析不同蛋白质组, 以探明关键功能蛋白。该文综述了近年来种子蛋白质组的研究进展, 内容包括种子发育过程中蛋白质组的变化, 与种子休眠/萌发相关的蛋白质组、翻译后修饰蛋白质组、细胞与亚细胞差异蛋白质组以及环境因子对种子蛋白质组的影响; 并对种子蛋白质组研究的热点问题进行了展望。