Viral Proteomics

Summary: Viruses have long been studied not only for their pathology and associated disease but also as model systems for molecular processes and as tools for identifying important cellular regulatory proteins and pathways. Recent advances in mass spectrometry methods coupled with the development of proteomic approaches have greatly facilitated the detection of virion components, protein interactions in infected cells, and virally induced changes in the cellular proteome, resulting in a more comprehensive understanding of viral infection. In addition, a rapidly increasing number of high-resolution structures for viral proteins have provided valuable information on the mechanism of action of these proteins as well as aided in the design and understanding of specific inhibitors that could be used in antiviral therapies. In this paper, we discuss proteomic studies conducted on all eukaryotic viruses and bacteriophages, covering virion composition, viral protein structures, virus-virus and virus-host protein interactions, and changes in the cellular proteome upon 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.     

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: 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.     

Proteomic analysis of cells in the early stages of herpes simplex virus type‐1 infection reveals widespread changes in the host cell proteome

During infection by herpes simplex virus type‐1 (HSV‐1) the host cell undergoes widespread changes in gene expression and morphology in response to viral replication and release. However, relatively little is known about the specific proteome changes that occur during the early stages of HSV‐1 replication prior to the global damaging effects of virion maturation and egress. To investigate pathways that may be activated or utilised during the early stages of HSV‐1 replication, 2‐DE and LC‐MS/MS were used to identify cellular proteome changes at 6 h post infection. Comparative analysis of multiple gels representing whole cell extracts from mock‐ and HSV‐1‐infected HEp‐2 cells revealed a total of 103 protein spot changes. Of these, 63 were up‐regulated and 40 down‐regulated in response to infection. Changes in selected candidate proteins were verified by Western blot analysis and their respective cellular localisations analysed by confocal microscopy. We have identified differential regulation and modification of proteins with key roles in diverse cellular pathways, including DNA replication, chromatin remodelling, mRNA stability and the ER stress response. This work represents the first global comparative analysis of HSV‐1 infected cells and provides an important insight into host cell proteome changes during the early stages of HSV‐1 infection.     

Retroviral proteomics and interactomes: intricate balances of cell survival and viral replication

Overall changes in the host cellular proteome upon retroviral infection intensify from the initial entry of the virus to the incorporation of viral DNA into the host genome, and finally to the consistent latent state of infection. The host cell reacts to both the entry of viral elements and the manipulation of host cellular machinery, resulting in a cascade of signaling events and pathway activation. Cell type- and tissue-specific responses are also characteristic of infection and can be classified based on the differential expression of genes and proteins between normal and disease states. The characterization of differentially expressed proteins upon infection is also critical in identifying potential biomarkers within infected bodily fluids. Biomarkers can be used to monitor the progression of infection, track the effectiveness of specific treatments and characterize the mechanisms of disease pathogenesis. Standard proteomic approaches have been applied to monitor the changes in global protein expression and localization in infected cells, tissues and fluids. Here we report on recent investigations into the characterization of proteomes in response to retroviral infection.     

Retroviral proteomics and interactomes: intricate balances of cell survival and viral replication

Overall changes in the host cellular proteome upon retroviral infection intensify from the initial entry of the virus to the incorporation of viral DNA into the host genome, and finally to the consistent latent state of infection. The host cell reacts to both the entry of viral elements and the manipulation of host cellular machinery, resulting in a cascade of signaling events and pathway activation. Cell type- and tissue-specific responses are also characteristic of infection and can be classified based on the differential expression of genes and proteins between normal and disease states. The characterization of differentially expressed proteins upon infection is also critical in identifying potential biomarkers within infected bodily fluids. Biomarkers can be used to monitor the progression of infection, track the effectiveness of specific treatments and characterize the mechanisms of disease pathogenesis. Standard proteomic approaches have been applied to monitor the changes in global protein expression and localization in infected cells, tissues and fluids. Here we report on recent investigations into the characterization of proteomes in response to retroviral infection.     

Virion-wide protein interactions of Kaposi's sarcoma-associated herpesvirus

Herpesvirus virions are highly organized structures built through specific protein-protein interactions. Thus, revelation of the protein interactions among virion proteins will shed light on the processes and the mechanisms of virion formation. Recently, we identified 24 virion proteins of Kaposi's sarcoma-associated herpesvirus (KSHV), using a proteomic approach (F. X. Zhu et al., J. Virol. 79:800-811, 2005). In the current study, a comprehensive analysis of protein-protein interaction between KSHV virion proteins was carried out using yeast two-hybrid (Y2H) and coimmunoprecipitation (co-IP) approaches. Every pairwise combination between KSHV tegument and capsid proteins, between tegument and envelope proteins, and among tegument proteins was tested for possible binary interaction. Thirty-seven protein-protein interactions were identified by both Y2H and co-IP analyses. The results revealed interactions between tegument and capsid proteins such as that of open reading frame 64 (ORF64) with ORF25 (major capsid protein [MCP]), ORF62 (triplex-1 [TRI-1]), and ORF26 (TRI-2). Many interactions were detected among the tegument proteins. ORF64 was found to interact with several tegument proteins including ORF11, ORF21, ORF33, ORF45, ORF63, ORF75, and ORF64 itself, suggesting that ORF64 may serve as a hub protein and play a role in recruiting tegument proteins during tegumentation and virion assembly. Our investigation also revealed redundant interactions between tegument proteins and envelope glycoproteins. These interactions are believed to contribute to final envelopment in virion assembly. Overall, this study allows us to establish a virion-wide protein interaction map, which provides insight into the architecture of the KSHV virion and sets up a foundation for exploring the functions of these proteins in viral particle assembly.     

Elucidation of the avian nucleolar proteome by quantitative proteomics using SILAC and changes in cells infected with the coronavirus infectious bronchitis virus

The nucleolus is a dynamic subnuclear compartment involved in ribosome subunit biogenesis, regulation of cell stress and modulation of cellular growth and the cell cycle, among other functions. The nucleolus is composed of complex protein/protein and protein/RNA interactions. It is a target of virus infection with many viral proteins being shown to localize to the nucleolus during infection. Perturbations to the structure of the nucleolus and its proteome have been predicted to play a role in both cellular and infectious disease. Stable isotope labeling with amino acids in cell culture coupled to LC‐MS/MS with bioinformatic analysis using Ingenuity Pathway Analysis was used to investigate whether the nucleolar proteome altered in virus‐infected cells. In this study, the avian nucleolar proteome was defined in the absence and presence of virus, in this case the positive strand RNA virus, avian coronavirus infectious bronchitis virus. Data sets, potential protein changes and the functional consequences of virus infection were validated using independent assays. These demonstrated that specific rather than generic changes occurred in the nucleolar proteome in infectious bronchitis virus‐infected cells.     

Virion proteins of Kaposi's sarcoma-associated herpesvirus

The proteins that compose a herpesvirus virion are thought to contain the functional information required for de novo infection, as well as virion assembly and egress. To investigate functional roles of Kaposi's sarcoma-associated herpesvirus (KSHV) virion proteins in viral productive replication and de novo infection, we attempted to identify virion proteins from purified KSHV by a proteomic approach. Extracellular KSHV virions were purified from phorbol-12-tetradecanoate-13-acetate-induced BCBL-1 cells through double-gradient ultracentrifugation, and their component proteins were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Thirty prominent protein bands were excised and subjected to high-performance liquid chromatography ion trap mass spectrometric analysis. This study led to the identification of 24 virion-associated proteins. These include five capsid proteins, eight envelope glycoproteins, six tegument proteins, and five proteins whose locations in the virions have not yet been defined. Putative tegument proteins encoded by open reading frame 21 (ORF21), ORF33, and ORF45 were characterized and found to be resistant to protease digestion when purified virions were treated with trypsin, confirming that they are located within the virion particles. The ORF64-encoded large tegument protein was found to be associated with capsid but sensitive to protease treatment, suggesting its unique structure and array in KSHV virions. In addition, cellular beta-actin and class II myosin heavy chain type A were found inside KSHV virions and associated with tegument-capsid structure. Identification of KSHV virion proteins makes it possible to study the functional roles of these virion proteins in KSHV replication and pathogenicity.     

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.     

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.     

Proteomic Profiling of Purified Rabies Virus Particles

While host proteins incorporated into virions during viral budding from infected cell are known to play essential roles in multiple process of the life cycle of progeny virus, these characteristics have been largely neglected in studies on rabies virus(RABV). Here, we purified the RABV virions with good purity and integrity, and analyzed their proteome by nano LC–MS/MS, followed by the confirmation with immunoblot and immuno-electronic microscopy. In addition to the 5 viral proteins, 49 cellular proteins were reproducibly identified to be incorporated into matured RABV virions. Function annotation suggested that 24 of them were likely involved in virus replication. Furthermore, cryo-EM was employed to observe the purified RABV virions, generating high-resolution pictures of the bullet-shaped virion structure of RABV. This study has provided new insights into the host proteins composition in RABV virion and shed the light for further investigation on molecular mechanisms of RABV infection, as well as the discovery of new anti-RABV therapeutics.     

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.     

The Cellular Proteins Grb2 and DDX3 Are Increased upon Human Cytomegalovirus Infection and Act in a Proviral Fashion

While it is well established that human cytomegalovirus (HCMV) upregulates many cellular proteins and incorporates several of them into its virion, little is known about the functional relevance of such virus-host interactions. Two cellular proteins, Grb2 and DDX3, gained our interest as they appeared enriched in virion particles and this incorporation depended on the viral tegument protein pp65, suggesting a functional relevance. We therefore tested whether the level of these proteins is altered upon HCMV infection and whether they support viral replication. Immunoblotting analyses of cellular fractions showed increased levels of both proteins in infected cells with a maximum at 2 d p.i. and a reduction of the soluble Grb2 fraction. Knockdown of either gene by transfection of siRNAs reduced viral spread not only of the cell culture adapted HCMV strain TB40/E but also of recent clinical isolates. Apparently, Grb2 and DDX3 are proviral cellular factors that are upregulated in infected cells.     

Characterization of early stages in vaccinia virus membrane biogenesis: implications of the 21-kilodalton protein and a newly identified 15-kilodalton envelope protein.

Vaccinia virus (VV) membrane biogenesis is a poorly understood process. It has been proposed that cellular membranes derived from the endoplasmic reticulum-Golgi intermediate compartment (ERGIC) are incorporated in the early stages of virion assembly. We have recently shown that the VV 21-kDa (A17L gene) envelope protein is essential for the formation of viral membranes. In the present work, we identify a 15-kDa VV membrane protein encoded by the A14L gene. This protein is phosphorylated and myristylated during infection and is incorporated into the virion envelope. Both the 21- and 15-kDa proteins are found associated with cellular tubulovesicular elements related to the ERGIC, suggesting that these proteins are transported in these membranes to the nascent viral factories. When synthesis of the 21-kDa protein is repressed, organized membranes are not formed but numerous ERGIC-derived tubulovesicular structures containing the 15-kDa protein accumulate in the boundaries of the precursors of the viral factories. These data suggest that the 21-kDa protein is involved in organizing the recruited viral membranes, while the 15-kDa protein appears to be one of the viral elements participating in the membrane recruitment process from the ERGIC, to initiate virus formation.     

Accumulation of “Old Proteins” and the Critical Need for MS‐based Protein Turnover Measurements in Aging and Longevity

Aging and age‐related diseases are accompanied by proteome remodeling and progressive declines in cellular machinery required to maintain protein homeostasis (proteostasis), such as autophagy, ubiquitin‐mediated degradation, and protein synthesis. While many studies have focused on capturing changes in proteostasis, the identification of proteins that evade these cellular processes has recently emerged as an approach to studying the aging proteome. With advances in proteomic technology, it is possible to monitor protein half‐lives and protein turnover at the level of individual proteins in vivo. For large‐scale studies, these technologies typically include the use of stable isotope labeling coupled with MS and comprehensive assessment of protein turnover rates. Protein turnover studies have revealed groups of highly relevant long‐lived proteins (LLPs), such as the nuclear pore complexes, extracellular matrix proteins, and protein aggregates. Here, the role of LLPs during aging and age‐related diseases and the methods used to identify and quantify their changes are reviewed. The methods available to conduct studies of protein turnover, used in combination with traditional proteomic methods, will enable the field to perform studies in a systems biology context, as changes in proteostasis may not be revealed in studies that solely measure differential protein abundances.