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.     

Methods and approaches for the comprehensive characterization and quantification of cellular proteomes using mass spectrometry.

Proteomics has been proposed as one of the key technologies in the postgenomic era. So far, however, the comprehensive analysis of cellular proteomes has been a challenge because of the dynamic nature and complexity of the multitude of proteins in cells and tissues. Various approaches have been established for the analyses of proteins in a cell at a given state, and mass spectrometry (MS) has proven to be an efficient and versatile tool. MS-based proteomics approaches have significantly improved beyond the initial identification of proteins to comprehensive characterization and quantification of proteomes and their posttranslational modifications (PTMs). Despite these advances, there is still ongoing development of new technologies to profile and analyze cellular proteomes more completely and efficiently. In this review, we focus on MS-based techniques, describe basic approaches for MS-based profiling of cellular proteomes and analysis methods to identify proteins in complex mixtures, and discuss the different approaches for quantitative proteome analysis. Finally, we briefly discuss novel developments for the analysis of PTMs. Altered levels of PTM, sometimes in the absence of protein expression changes, are often linked to cellular responses and disease states, and the comprehensive analysis of cellular proteome would not be complete without the identification and quantification of the extent of PTMs of proteins.     

Deciphering Spatial Protein–Protein Interactions in Brain Using Proximity Labeling

Cellular biomolecular complexes including protein–protein, protein–RNA, and protein–DNA interactions regulate and execute most biological functions. In particular in brain, protein–protein interactions (PPIs) mediate or regulate virtually all nerve cell functions, such as neurotransmission, cell–cell communication, neurogenesis, synaptogenesis, and synaptic plasticity. Perturbations of PPIs in specific subsets of neurons and glia are thought to underly a majority of neurobiological disorders. Therefore, understanding biological functions at a cellular level requires a reasonably complete catalog of all physical interactions between proteins. An enzyme-catalyzed method to biotinylate proximal interacting proteins within 10 to 300 nm of each other is being increasingly used to characterize the spatiotemporal features of complex PPIs in brain. Thus, proximity labeling has emerged recently as a powerful tool to identify proteomes in distinct cell types in brain as well as proteomes and PPIs in structures difficult to isolate, such as the synaptic cleft, axonal projections, or astrocyte–neuron junctions. In this review, we summarize recent advances in proximity labeling methods and their application to neurobiology.     

Analysis of the interaction of viral RNA replication proteins by using the yeast two-hybrid assay.

The yeast two-hybrid system has been a useful tool in the genetic evaluation of protein-protein interactions. However, the biological relevance of these two-hybrid interactions to viral positive-strand RNA replication has not been demonstrated. The brome mosaic virus (BMV) system has been characterized extensively both genetically and biochemically, providing numerous mutations in the BMV 1a helicase-like and 2a polymerase-like proteins. We have tested wild-type 1a and 18 insertion mutations of 1a and found a perfect correlation between the in planta phenotypes and their ability to interact with 2a in the two-hybrid system. This finding allowed further characterization of the interaction between and among the BMV viral proteins. Using the two-hybrid assay, we have found that the interaction between the helicase-like region of 1a and the N terminus of 2a is stabilized by the presence of the centrally conserved polymerase-like domain of 2a. We have also identified a novel interaction between the 1a helicase-like protein and itself. Additionally, we have found this interaction in two related tripartite RNA viruses, cowpea chlorotic mottle virus and cucumber mosaic virus. We have demonstrated that this protein-protein interaction is specific to homologous pairings of the protein.     

Tools for analyzing and predicting N-terminal protein modifications

The vast majority of the proteins encoded in any genome naturally undergo a large number of different N-terminal modifications, hindering their characterization by routine proteomic approaches. These modifications are often irreversible, usually cotranslational and are crucial, as their occurrence may reflect or affect the status, fate and function of the protein. For example, large signal peptide cleavages and N-blocking mechanisms reflect targeting to various cell compartments, whereas N-ligation events tend to be related to protein half-life. N-terminal positional proteomic strategies hold promise as a new generation of approaches to the fine analysis of such modifications. However, further biological investigation is required to resolve problems associated with particular low-abundance or challenging N-terminal modifications. Recent progress in genomics and bioinformatics has provided us with a means of assessing the impact of these modifications in proteomes. This review focuses on methods for characterizing the occurrence and diversity of N-terminal modifications and for assessing their contribution to function in complete proteomes. Progress is being made towards the annotation of databases containing information for complete proteomes, and should facilitate research into all areas of proteomics.     

Towards functional repertoire of the earliest proteins

The conserved protein sequence motifs present in all prokaryotic proteomes, “omnipresent motifs,” presumably, correspond to the earliest proteins of the Last Universal Cellular Ancestor, from which all the proteomes have descended. Fifteen proteomes, each representing one of the total 15 diverse phyla of 131 Eubacteria and Archea, from which the omnipresent elements have been originally derived, are exhaustively screened. All those proteins which harbor the omnipresent motifs are identified. Six “omnipresent” protein types are revealed which are located in all 15 proteomes: ABC cassettes, FtsH proteases, translation initiation factors, translation elongation factors, isoleucyl-tRNA synthases, and RNA polymerases β’. In addition to the omnipresent motifs, these proteins also contain other highly conserved motifs, standing for additional modules of the proteins. Remarkably, the identified tentative earliest proteins are responsible for only three basic functions: supply of monomers (ABC transporters and proteases), protein synthesis (initiation and elongation factors, aminoacyl-tRNA synthases), and RNA synthesis (polymerases). No enzymes involved in metabolic activities are present in the list of the earliest proteins derived by this approach. Some of the omnipresent sequence motifs are found, indeed, in the metabolic enzymes (e.g. NTP binding motifs), but these enzymes do not make a sequence matching collection of 15 sequences, i.e. they are not omnipresent. Future analysis of less conserved sequence motifs may reveal at what degree of conservation (stage of evolution) the metabolic enzymes could have entered the scene.     

Adenosine‐to‐Inosine RNA Editing in Mouse and Human Brain Proteomes

Proteogenomics is based on the use of customized genome or RNA sequencing databases for interrogation of shotgun proteomics data in search for proteome‐level evidence of genome variations or RNA editing. In this work, the products of adenosine‐to‐inosine RNA editing in human and murine brain proteomes are identified using publicly available brain proteome LC‐MS/MS datasets and an RNA editome database compiled from several sources. After filtering of false‐positive results, 20 and 37 sites of editing in proteins belonging to 14 and 32 genes are identified for murine and human brain proteomes, respectively. Eight sites of editing identified with high spectral counts overlapped between human and mouse brain samples. Some of these sites have been previously reported using orthogonal methods, such as α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA) glutamate receptors, CYFIP2, coatomer alpha. Also, differential editing between neurons and microglia is demonstrated in this work for some of the proteins from primary murine brain cell cultures. Because many edited sites are still not characterized functionally at the protein level, the results provide a necessary background for their further analysis in normal and diseased cells and tissues using targeted proteomic approaches.     

The application of yeast hybrid systems in protein interaction analysis

Yeast hybrid systems have been widely used due to their convenience and low cost. Based on these systems, many methods have been developed to analyze protein–protein, protein–DNA and protein–RNA interactions. In this paper, we are reviewing these different yeast hybrid systems. According to the number of hybrid proteins, yeast hybrid systems can be divided into three categories, yeast one-hybrid, yeast two-hybrid and yeast three-hybrid systems. Alternatively, yeast hybrid systems can be categorized according to the subcellular localization of the protein interaction process in the cell into nuclear protein–protein interactions, cytosol protein–protein interactions and membrane protein–protein interactions. Throughout the review, we focus on the progress and limitations of each yeast hybrid system over the recent years.     

Organic phase separation opens up new opportunities to interrogate the RNA-binding proteome

Protein–RNA interactions regulate all aspects of RNA metabolism and are crucial to the function of catalytic ribonucleoproteins. Until recently, the available technologies to capture RNA-bound proteins have been biased toward poly(A) RNA-binding proteins (RBPs) or involve molecular labeling, limiting their application. With the advent of organic–aqueous phase separation–based methods, we now have technologies that efficiently enrich the complete suite of RBPs and enable quantification of RBP dynamics. These flexible approaches to study RBPs and their bound RNA open up new research avenues for systems-level interrogation of protein–RNA interactions.     

Cell physiology and protein secretion of Bacillus licheniformis compared to Bacillus subtilis

The genome sequence of Bacillus subtilis was published in 1997 and since then many other bacterial genomes have been sequenced, among them Bacillus licheniformis in 2004. B. subtilis and B. licheniformis are closely related and feature similar saprophytic lifestyles in the soil. Both species can secrete numerous proteins into the surrounding medium enabling them to use high-molecular-weight substances, which are abundant in soils, as nutrient sources. The availability of complete genome sequences allows for the prediction of the proteins containing signals for secretion into the extracellular milieu and also of the proteins which form the secretion machinery needed for protein translocation through the cytoplasmic membrane. To confirm the predicted subcellular localization of proteins, proteomics is the best choice. The extracellular proteomes of B. subtilis and B. licheniformis have been analyzed under different growth conditions allowing comparisons of the extracellular proteomes and conclusions regarding similarities and differences of the protein secretion mechanisms between the two species.     

The International Protein Index: an integrated database for proteomics experiments

Despite the complete determination of the genome sequence of several higher eukaryotes, their proteomes remain relatively poorly defined. Information about proteins identified by different experimental and computational methods is stored in different databases, meaning that no single resource offers full coverage of known and predicted proteins. IPI (the International Protein Index) has been developed to address these issues and offers complete nonredundant data sets representing the human, mouse and rat proteomes, built from the Swiss-Prot, TrEMBL, Ensembl and RefSeq databases.     

Protein microarrays and their applications

In recent years, the importance of proteomic works, such as protein expression, detection and identification, has grown in the fields of proteomic and diagnostic research. This is because complete genome sequences of humans, and other organisms, progress as cellular processing and controlling are performed by proteins as well as DNA or RNA. However, conventional protein analyses are time-consuming; therefore, high throughput protein analysis methods, which allow fast, direct and quantitative detection, are needed. These are so-called protein microarrays or protein chips, which have been developed to fulfill the need for high-throughput protein analyses. Although protein arrays are still in their infancy, technical development in immobilizing proteins in their native conformation on arrays, and the development of more sensitive detection methods, will facilitate the rapid deployment of protein arrays as high-throughput protein assay tools in proteomics and diagnostics. This review summarizes the basic technologies that are needed in the fabrication of protein arrays and their recent applications.     

Sample preparation for the analysis of membrane proteomes by mass spectrometry

The low abundance and highly hydrophobic nature of most membrane proteins make their analysis more difficult than that for common soluble proteins. Successful membrane protein identification is largely dependent on the sample preparation including the enrichment and dissolution of the membrane proteins. A series of conventional and newly developed methods has been applied to the enrichment of low-abundance membrane proteins at membrane and/or protein levels and to the dissolution of hydrophobic membrane proteins. However, all the existing methods have inherent advantages and limitations. Up to now, there has been no unique method that can universally be employed to solve all the problems and more efforts are needed in improving sample preparation for the analysis of membrane proteomes.     

The Interactomes of Influenza Virus NS1 and NS2 Proteins Identify New Host Factors and Provide Insights for ADAR1 Playing a Supportive Role in Virus Replication

Influenza A NS1 and NS2 proteins are encoded by the RNA segment 8 of the viral genome. NS1 is a multifunctional protein and a virulence factor while NS2 is involved in nuclear export of viral ribonucleoprotein complexes. A yeast two-hybrid screening strategy was used to identify host factors supporting NS1 and NS2 functions. More than 560 interactions between 79 cellular proteins and NS1 and NS2 proteins from 9 different influenza virus strains have been identified. These interacting proteins are potentially involved in each step of the infectious process and their contribution to viral replication was tested by RNA interference. Validation of the relevance of these host cell proteins for the viral replication cycle revealed that 7 of the 79 NS1 and/or NS2-interacting proteins positively or negatively controlled virus replication. One of the main factors targeted by NS1 of all virus strains was double-stranded RNA binding domain protein family. In particular, adenosine deaminase acting on RNA 1 (ADAR1) appeared as a pro-viral host factor whose expression is necessary for optimal viral protein synthesis and replication. Surprisingly, ADAR1 also appeared as a pro-viral host factor for dengue virus replication and directly interacted with the viral NS3 protein. ADAR1 editing activity was enhanced by both viruses through dengue virus NS3 and influenza virus NS1 proteins, suggesting a similar virus-host co-evolution.     

Practical lessons from protein structure prediction

Despite recent efforts to develop automated protein structure determination protocols, structural genomics projects are slow in generating fold assignments for complete proteomes, and spatial structures remain unknown for many protein families. Alternative cheap and fast methods to assign folds using prediction algorithms continue to provide valuable structural information for many proteins. The development of high-quality prediction methods has been boosted in the last years by objective community-wide assessment experiments. This paper gives an overview of the currently available practical approaches to protein structure prediction capable of generating accurate fold assignment. Recent advances in assessment of the prediction quality are also discussed.