Proteome analysis of virus-host interactions of cowpox viruses

Kuhpockenviren (CPXV) verursachen die häufigsten zoonotischen Orthopockenvirus (OPV) Infektionen in Europa und Nord- sowie Zentralasien 1. Das Virus besitzt das breiteste Wirtsspektrum der OPV und wird von Nagetieren sowie weiteren Wildtieren aber auch domestizierten Tieren auf den Menschen übertragen 2. CPXV besitzen außerdem die größte genetische Diversität und den größten Satz an OPV Genen, der auch alle open reading frames des Variola Virus (VARV) beinhaltet 2. Das Ansteigen von CPXV Infektionen beim Menschen in einer Gesellschaft mit abnehmender Immunität hat Bedenken wegen des zoonotischen Potentials des Virus hervorgerufen 1. Während das Vaccinia Virus (VACV) in der Literatur als Modell für Untersuchungen der Pathogenität und des Wirtstropismus von OPV dient, sind CPXV-spezifische Studien unterrepräsentiert. Die Charakterisierung der CPXV-Partikel, sowie die Analyse CPXV-stammspezifischer Papthogenitätsunterschiede, sollen als Grundlage dienen das Gefährdungspotential dieser aufkommenden Zoonose zukünftig besser einschätzen zu können. Im Gegensatz zum Vaccinia Viren (VACV) und Affenpockenviren (MPXV) ist die Proteinzusammensetzung des CPXV Mature Virion bisher nicht beschrieben. Die Zusammensetzung des CPXV Mature Virion (MV) Proteoms ist im Rahmen dieser Arbeit erstmals qualitativ und quantitativ mit Nano-Flüssigchromatographie (nLC) und Elektrospray-Ionisation (ESI) Tandem Massenspektrometrie (MS/MS)) basierten Proteomanalysen verschiedener Kuhpockenvirusisolate ebenso untersucht worden, wie die Ubiquitinierung von viralen Proteinen, und die Assoziation von Wirtszellproteinen. Das konservierte MV Proteom von drei verschiedenen CPXV Stämmen unterschiedlicher Ursprungsspezies besteht demnach aus 101 viralen Proteinen, die zu 25 % ubiquitiniert und mit 160 Wirtsproteinen assoziiert sind. Die zehn häufigsten viralen Proteine repräsen¬tieren dabei mehr als 50 mol% des Gesamtpartikels. CPXV und VACV MV unterscheiden sich in mittel und niedrig exprimierten Proteinen, zu denen immunmodulatorische Proteine und ein host-range Faktor zählen, die CPXV Schutz vor der Immunantwort in der frühen Replikationsphase bereits vor der Synthese früher Proteine bieten können. Die Ergebnisse reflektieren dabei die Situation in vivo, die durch einen breiteren Wirtstropismus und ein höheres pathogenes Potential von CPXV gegenüber VACV gekennzeichnet ist. Für Pockenviren ist bisher kein zellulärer Rezeptor beschrieben, es wird aber vermutet, dass zelluläre Faktoren für das attachment und den entry ubiquitär verbreitet sein müssen, da OPV die meisten Säugetierzelllinien penetrieren können. Mit einem Ansatz aus nativer Gelelektrophorese gekoppelt mit nLC-MS/MS und Cluster Analysen des Co-Migrationsverhaltens der Proteine im nativen Gel sind Proteinkomplexe zwischen der Membran humaner Zellen und CPXV während des attachments identifiziert und die Resultate der Cluster Analysen in einem attachment Netzwerk visualisiert worden. Einige der zellulären Membranproteine sind, wie vermutet, ubiquitär in verschiedenen Zelltypen verbreitet und innerhalb der Säugetiere hoch konserviert. Die vorliegenden Analysen der CPXV MV sind damit die bislang umfassendste Charakterisierung von OPV-Partikeln auf Proteomebene. Natürlich auftretende Infektionen und Tierexperimente haben gezeigt, dass die CPXV-Pathogenität von der Wirtsspezies, dem Infektionsweg und dem Virusstamm abhängig ist 3,4. Die Gründe für die Pathogenitätsunterschiede verschiedener CPXV-Stämme sind bisher unbekannt. In dieser Arbeit sind Mechanismen identifiziert worden, die für Pathogenitätsunterschiede verschiedener CPXV Stämme in vivo verantwortlich sein können. Zwei im Rattenmodell verschieden pathogene Stämme sind dafür in den Genomsequenzen, dem Wachstum auf verschiedenen Zelllinien, der viralen Proteinexpression und dem Einfluss auf das Proteom humaner Keratinozyten (HaCaT) miteinander verglichen worden. Die quantitativen shotgun Proteomanalysen der Viruspartikel, des Zellkulturüberstands, der für die Immunmodulation wichtige sekretierte Protein enthält, und des Zytoplasmas, dem Ort der viralen Replikation, sind mittels nLC-MS/MS und stable isotope dimethyl labeling durchgeführt worden. Diese Untersuchungen zeigen, dass sich OPV nicht nur in der Ausstattung und der Sequenz von immunmodulatorischen Proteinen unterscheiden, sondern diese zudem unterschiedlich stark exprimieren können. Die Pathogenitätsunterschiede der Stämme in vivo können demnach die Folge verschieden stark exprimierter viraler Zytokinrezeptoren und damit einer unterschiedlich effektiv inhibierten Immunantwort sein.Cowpox Virus (CPXV) is the cause of most zoonotic orthopoxvirus (OPV) infections in Europe and North- as well as Central Asia 1. The virus has the broadest host range of OPV and is transmitted to humans from rodents and other wild and domestic animals 2. Furthermore, CPXV possess the largest set of OPV genes, which contain all Variola Virus (VARV) open reading frames 2. The rise of human CPXV infections in a society with declining immunity has raised concerns about the virus’ zoonotic potential 1. While Vaccinia Virus (VACV) is usually used as a model for the analysis of pathogenicity and host tropism of OPV, CPXV-specific studies are underrepresented in the literature. The characterization of CPXV-particles, as well as the analysis of strain-specific differences in pathogenicity, can serve as a foundation for the evaluation of the hazard potential of this emerging zoonoses in the future. In contrast to Vaccinia Virus (VACV) and Monkeypox Virus (MPXV), the protein composition of the CPXV Mature Virion (MV) is unknown. In this study, the proteome of CPXV viral particles has been analysed qualitatively and quantitatively using mass spectrometry based proteomic methods (nano-liquid-chromatography (nLC) and electrospray ionisation (ESI) tandem mass spectrometry (MS/MS)) for the first time, as well as the ubiquitination of virus proteins and associated host proteins. The conserved CPXV MV proteome of three various strains from different host species has been determined to consist of 101 viral proteins, 25 % of them are ubiquitinated and 160 host proteins are associated. The ten most abundant viral proteins represent more than 50 mol% of the virus particle. CPXV and VACV MV differ in medium and low abundant proteins, among them are immunomodulators and host range factors, which might protect CPXV from immune response in the early replication phase prior to protein synthesis. The results reflect the in vivo situation, which is characterized by a broader host range and higher pathogenic potential of CPXV compared to VACV. No cellular receptor for OPV has been identified so far, although it is assumed that cellular factors for attachment and entry need to be spread ubiquitously, since OPV can enter most mammalian cell lines. Cellular binding partners of viral membrane proteins during the attachment of CPXV to a human cell membrane have been identified by combining native gel electrophoresis and nLC-MS/MS with cluster analysis of the co-migration behavior of proteins in the gel. The results have been visualized in an attachment network. Some cellular members of the attachment complex are distributed ubiquitously in different cell types and are highly conserved among mammals. To date, the present analysis is the most comprehensive characterization of OPV particles on a proteome level. Naturally occuring infections and animal experiments have shown that pathogenicity of CPXV depends on the host species, the route of infection and the viral strain 3,4. Mechanisms that might be related to strain-specific pathogenicity in vivo have been identified in this thesis. Therefore, the genome sequence, the growth kinetics on different cell lines, the viral protein expression as well as the influence of an infection on the proteome of human keratinocytes (HaCaT) have been compared for two CPXV strains with different pathogenicity in a rat model. The quantitative shotgun proteome analysis of viral particles, the secretome, which contains proteins related with imunmodulation, and the cytoplasm, the place of the virus replication, have been carried using nLC-MS/MS and stable isotope dimethyl labelling. These investigations show for the first time that OPV differ not only in their equipment and sequence of virus-host interactors but also in their degree of expression. The different pathogenicity of the strains in vivo may therefore be the result of an altered expression of viral cytokine receptors, which might modulate the antiviral immune response with different effectivity

Application of spectral library prediction for parallel reaction monitoring of viral peptides

A major part of the analysis of parallel reaction monitoring (PRM) data is the comparison of observed fragment ion intensities to a library spectrum. Classically, these libraries are generated by data-dependent acquisition (DDA). Here, we test Prosit, a published deep neural network algorithm, for its applicability in predicting spectral libraries for PRM. For this purpose, we targeted 1529 precursors derived from synthetic viral peptides and analyzed the data with Prosit and DDA-derived libraries. Viral peptides were chosen as an example, because virology is an area where in silico library generation could significantly improve PRM assay design. With both libraries a total of 1174 precursors were identified. Notably, compared to the DDA-derived library, we could identify 101 more precursors by using the Prosit-derived library. Additionally, we show that Prosit can be applied to predict tandem mass spectra of synthetic viral peptides with different collision energies. Finally, we used a spectral library predicted by Prosit and a DDA library to identify SARS-CoV-2 peptides from a simulated oropharyngeal swab demonstrating that both libraries are suited for peptide identification by PRM. Summarized, Prosit-derived viral spectral libraries predicted in silico can be used for PRM data analysis, making DDA analysis for library generation partially redundant in the future.Peer Reviewe

Combined Proteomics/Genomics Approach Reveals Proteomic Changes of Mature Virions as a Novel Poxvirus Adaptation Mechanism

DNA viruses, like poxviruses, possess a highly stable genome, suggesting that adaptation of virus particles to specific cell types is not restricted to genomic changes. Cowpox viruses are zoonotic poxviruses with an extraordinarily broad host range, demonstrating their adaptive potential in vivo. To elucidate adaptation mechanisms of poxviruses, we isolated cowpox virus particles from a rat and passaged them five times in a human and a rat cell line. Subsequently, we analyzed the proteome and genome of the non-passaged virions and each passage. While the overall viral genome sequence was stable during passaging, proteomics revealed multiple changes in the virion composition. Interestingly, an increased viral fitness in human cells was observed in the presence of increased immunomodulatory protein amounts. As the only minor variant with increasing frequency during passaging was located in a viral RNA polymerase subunit and, moreover, most minor variants were found in transcription-associated genes, protein amounts were presumably regulated at transcription level. This study is the first comparative proteome analysis of virus particles before and after cell culture propagation, revealing proteomic changes as a novel poxvirus adaptation mechanism

A Next-Generation Sequencing Approach Uncovers Viral Transcripts Incorporated in Poxvirus Virions

Transcripts are known to be incorporated in particles of DNA viruses belonging to the families of Herpesviridae and Mimiviridae, but the presence of transcripts in other DNA viruses, such as poxviruses, has not been analyzed yet. Therefore, we first established a next-generation-sequencing (NGS)-based protocol, enabling the unbiased identification of transcripts in virus particles. Subsequently, we applied our protocol to analyze RNA in an emerging zoonotic member of the Poxviridae family, namely Cowpox virus. Our results revealed the incorporation of 19 viral transcripts, while host identifications were restricted to ribosomal and mitochondrial RNA. Most viral transcripts had an unknown and immunomodulatory function, suggesting that transcript incorporation may be beneficial for poxvirus immune evasion. Notably, the most abundant transcript originated from the D5L/I1R gene that encodes a viral inhibitor of the host cytoplasmic DNA sensing machinery

Comparison of the Cowpox Virus and Vaccinia Virus Mature Virion Proteome: Analysis of the Species- and Strain-Specific Proteome

Cowpox virus (CPXV) causes most zoonotic orthopoxvirus (OPV) infections in Europe and Northern as well as Central Asia. The virus has the broadest host range of OPV and is transmitted to humans from rodents and other wild or domestic animals. Increasing numbers of human CPXV infections in a population with declining immunity have raised concerns about the virus’ zoonotic potential. While there have been reports on the proteome of other human-pathogenic OPV, namely vaccinia virus (VACV) and monkeypox virus (MPXV), the protein composition of the CPXV mature virion (MV) is unknown. This study focused on the comparative analysis of the VACV and CPXV MV proteome by label-free single-run proteomics using nano liquid chromatography and high-resolution tandem mass spectrometry (nLC-MS/MS). The presented data reveal that the common VACV and CPXV MV proteome contains most of the known conserved and essential OPV proteins and is associated with cellular proteins known to be essential for viral replication. While the species-specific proteome could be linked mainly to less genetically-conserved gene products, the strain-specific protein abundance was found to be of high variance in proteins associated with entry, host-virus interaction and protein processing

Pipasic: similarity and expression correction for strain-level identification and quantification in metaproteomics

Motivation: Metaproteomic analysis allows studying the interplay of organisms or functional groups and has become increasingly popular also for diagnostic purposes. However, difficulties arise owing to the high sequence similarity between related organisms. Further, the state of conservation of proteins between species can be correlated with their expression level, which can lead to significant bias in results and interpretation. These challenges are similar but not identical to the challenges arising in the analysis of metagenomic samples and require specific solutions. Results: We introduce Pipasic (peptide intensity-weighted proteome abundance similarity correction) as a tool that corrects identification and spectral counting-based quantification results using peptide similarity estimation and expression level weighting within a non-negative lasso framework. Pipasic has distinct advantages over approaches only regarding unique peptides or aggregating results to the lowest common ancestor, as demonstrated on examples of viral diagnostics and an acid mine drainage dataset

The Pitch Rise Paradigm: A New Task for Real-Time Endoscopy of Non-Stationary Phonation

As standard stroboscopy is restricted to the recording of periodic vocal fold vibrations, observations of non-stationary laryngeal mechanisms demand real-time recording systems, the most advanced being the high-speed video technique. It allows the registration of laryngeal parameters during a variation of the fundamental frequency. The aim of this study was to compare amplitude and frequency parameters of vocal fold vibration during stationary and non-stationary phonation, i.e. a monotonous pitch rise. Twenty-nine young female adults with no incidence of voice disorders were examined while performing two diff erent phonation tasks: sustained phonation with a constant frequency and a monotonous pitch rise. Endoscopic recordings and the acoustic signals were acquired simultaneously. Both acoustic and laryngeal parameters were derived for short time intervals of 17.8 ms for the constant pitch and pitch rise conditions. Instantaneous frequency, sound pressure level, vibratory amplitudes of the vocal folds and the type of glottal closure were compared. At the beginning of the pitch rise, the acoustic and laryngeal parameters were similar to the parameters that occurred within the sustained phonation conditions. In contrast, the laryngeal parameters at the middle and at the end of the pitch rise diff ered substantially from those during sustained phonation. For the fi rst time, quantitative measures of the growing glottal chink and the vibration amplitude decrease during pitch increase could be taken. In general, the image evaluation of the pitch rise paradigm can be subdivided into the starting, the raising and the final phase. As each phase can be considered as quasi-stationary, existing software modules are capable of analysing the process by treating each phase separately. Hence, the pitch rise condition may be suitable for clinical examination to detect information of voice disturbances that cannot be visualized during sustained phonation

Correlation between Psychometric Tests and Mismatch Negativity in Preschool Children

The objective was to determine whether mismatch negativity (MMN) is suitable to supplement subjective psychometric subtests of central hearing. We assessed 13 healthy children and 32 children with central auditory processing disorder (CAPD). Three different types of sound deviants were presented in a multi-deviant MMN design. At group level, the incidence of MMN was always higher in clinically diagnosed controls. Children with better results in the subtest Auditory Memory Span had a higher incidence of MMN. The controls also had peak latencies that occurred significantly earlier in frontal, central and temporal electrode sites. The area under the curve (AUC) displayed an asymmetric distribution in CAPD children, who tended to have a left-hemispheric dominance. AUC, peak latency, and the incidence of MMN reflected the discriminative ability of CAPD children. Hence, these characteristics could be used for investigating children with deficits in central hearing and can supplement psychometric tests

Direct translation of incoming retroviral genomes

Viruses that carry a positive-sense, single-stranded (+ssRNA) RNA translate their genomes soon after entering the host cell to produce viral proteins, with the exception of retroviruses. A distinguishing feature of retroviruses is reverse transcription, where the +ssRNA genome serves as a template to synthesize a double-stranded DNA copy that subsequently integrates into the host genome. As retroviral RNAs are produced by the host cell transcriptional machinery and are largely indistinguishable from cellular mRNAs, we investigated the potential of incoming retroviral genomes to directly express proteins. Here we show through multiple, complementary methods that retroviral genomes are translated after entry. Our findings challenge the notion that retroviruses require reverse transcription to produce viral proteins. Synthesis of retroviral proteins in the absence of productive infection has significant implications for basic retrovirology, immune responses and gene therapy applications

Leishmania Encodes a Bacterium-like 2,4-Dienoyl-Coenzyme A Reductase That Is Required for Fatty Acid β-Oxidation and Intracellular Parasite Survival

Leishmaniaspp. are protozoan parasites that cause a spectrum of im-portant diseases in humans. These parasites develop as extracellular promastigotesin the digestive tract of their insect vectors and as obligate intracellular amastigotesthat infect macrophages and other phagocytic cells in their vertebrate hosts.Promastigote-to-amastigote differentiation is associated with marked changes in me-tabolism, including the upregulation of enzymes involved in fatty acid -oxidation,which may reflect adaptation to the intracellular niche. Here, we have investigatedthe function of one of these enzymes, a putative 2,4-dienoyl-coenzyme A (CoA) re-ductase (DECR), which is specifically required for the -oxidation of polyunsaturatedfatty acids. TheLeishmaniaDECR shows close homology to bacterial DECR proteins,suggesting that it was acquired by lateral gene transfer. It is present in othertrypanosomatids that have obligate intracellular stages (i.e.,Trypanosoma cruziandAngomonas) but is absent from dixenous parasites with an exclusively extracellularlifestyle (i.e.,Trypanosoma brucei). A DECR-green fluorescent protein (GFP) fusionprotein was localized to the mitochondrion in both promastigote and amastigotestages, and the levels of expression increased in the latter stages. ALeishmania ma-jorΔdecrnull mutant was unable to catabolize unsaturated fatty acids and accumu-lated the intermediate 2,4-decadienoyl-CoA, confirming DECR’s role in -oxidation.Strikingly, theL. majorΔdecrmutant was unable to survive in macrophages and wasavirulent in BALB/c mice. These findings suggest that -oxidation of polyunsaturatedfatty acids is essential for intracellular parasite survival and that the bacterial originof key enzymes in this pathway could be exploited in developing new therapies.Peer Reviewe