scholarly journals Rapid Degradation Pathways of Host Proteins During HCMV Infection Revealed by Quantitative Proteomics

2021 ◽  
Vol 10 ◽  
Author(s):  
Kai-Min Lin ◽  
Katie Nightingale ◽  
Lior Soday ◽  
Robin Antrobus ◽  
Michael P. Weekes
Keyword(s):  
Protein Degradation ◽  
Hcmv Infection ◽  
Host Protein ◽  
Restriction Factors ◽  
Host Proteins ◽  
Alternative Pathways ◽  
Viral Immune Evasion ◽  
Proteomic Approach ◽  
Definition Of ◽  
Mechanistic View

Human cytomegalovirus (HCMV) is an important pathogen in immunocompromised individuals and neonates, and a paradigm for viral immune evasion. We previously developed a quantitative proteomic approach that identified 133 proteins degraded during the early phase of HCMV infection, including known and novel antiviral factors. The majority were rescued from degradation by MG132, which is known to inhibit lysosomal cathepsins in addition to the proteasome. Global definition of the precise mechanisms of host protein degradation is important both to improve our understanding of viral biology, and to inform novel antiviral therapeutic strategies. We therefore developed and optimized a multiplexed comparative proteomic analysis using the selective proteasome inhibitor bortezomib in addition to MG132, to provide a global mechanistic view of protein degradation. Of proteins rescued from degradation by MG132, 34–47 proteins were also rescued by bortezomib, suggesting both that the predominant mechanism of protein degradation employed by HCMV is via the proteasome, and that alternative pathways for degradation are nevertheless important. Our approach and data will enable improved mechanistic understanding of HCMV and other viruses, and provide a shortlist of candidate restriction factors for further analysis.

scholarly journals Cellular and Biochemical Mechanisms of the Retroviral Restriction Factor SAMHD1

2013 ◽  
Vol 2013 ◽  
pp. 1-11 ◽  
Author(s):  
Li Wu
Keyword(s):  
Host Protein ◽  
Nucleic Acid Metabolism ◽  
Target Cells ◽  
Restriction Factors ◽  
Host Proteins ◽  
Retroviral Infection ◽  
Deoxynucleoside Triphosphate ◽  
Biochemical Mechanisms ◽  
Hiv 1

Replication of HIV-1 and other retroviruses is dependent on numerous host proteins in the cells. Some of the host proteins, however, function as restriction factors to block retroviral infection of target cells. The host protein SAMHD1 has been identified as the first mammalian deoxynucleoside triphosphate triphosphohydrolase (dNTPase), which blocks the infection of HIV-1 and other retroviruses in non-cycling immune cells. SAMHD1 protein is highly expressed in human myeloid-lineage cells and CD4+ T-lymphocytes, but its retroviral restriction function is only observed in noncycling cells. Recent studies have revealed biochemical mechanisms of SAMHD1-mediated retroviral restriction. In this review, the latest progress on SAMHD1 research is summarized and the mechanisms by which SAMHD1 mediates retroviral restriction are analyzed. Although the physiological function of SAMHD1 is largely unknown, this review provides perspectives about the role of endogenous SAMHD1 protein in maintaining normal cellular function, such as nucleic acid metabolism and the proliferation of cells.

scholarly journals Severe Acute Respiratory Syndrome Coronavirus Nonstructural Protein 2 Interacts with a Host Protein Complex Involved in Mitochondrial Biogenesis and Intracellular Signaling

2009 ◽  
Vol 83 (19) ◽  
pp. 10314-10318 ◽  
Author(s):  
Cromwell T. Cornillez-Ty ◽  
Lujian Liao ◽  
John R. Yates ◽  
Peter Kuhn ◽  
Michael J. Buchmeier
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Intracellular Signaling ◽  
Nonstructural Protein ◽  
Host Protein ◽  
Nonstructural Proteins ◽  
Host Proteins ◽  
Catalytic Activities ◽  
Nonstructural Protein 2 ◽  
Host Signaling ◽  
Prohibitin 1

ABSTRACT The severe acute respiratory syndrome coronavirus (SARS-CoV) generates 16 nonstructural proteins (nsp's) through proteolytic cleavage of a large precursor protein. Although several nsp's exhibit catalytic activities that are important for viral replication and transcription, other nsp's have less clearly defined roles during an infection. In order to gain a better understanding of their functions, we attempted to identify host proteins that interact with nsp's during SARS-CoV infections. For nsp2, we identified an interaction with two host proteins, prohibitin 1 (PHB1) and PHB2. Our results suggest that nsp2 may be involved in the disruption of intracellular host signaling during SARS-CoV infections.

scholarly journals A Parasite Cysteine Protease Is Key to Host Protein Degradation and Iron Acquisition

2008 ◽  
Vol 283 (43) ◽  
pp. 28934-28943 ◽  
Author(s):  
Theresa C. O'Brien ◽  
Zachary B. Mackey ◽  
Richard D. Fetter ◽  
Youngchool Choe ◽  
Anthony J. O'Donoghue ◽  
...  
Keyword(s):  
Protein Degradation ◽  
Cysteine Protease ◽  
Iron Acquisition ◽  
Host Protein

scholarly journals A Cathepsin B-like Protease Is Required for Host Protein Degradation inTrypanosoma brucei

2004 ◽  
Vol 279 (46) ◽  
pp. 48426-48433 ◽  
Author(s):  
Zachary B. Mackey ◽  
Theresa C. O'Brien ◽  
Doron C. Greenbaum ◽  
Rebecca B. Blank ◽  
James H. McKerrow
Keyword(s):  
Protein Degradation ◽  
Cathepsin B ◽  
Host Protein

scholarly journals Visualization of Host-Polerovirus Interaction Topologies Using Protein Interaction Reporter Technology

2015 ◽  
Vol 90 (4) ◽  
pp. 1973-1987 ◽  
Author(s):  
Stacy L. DeBlasio ◽  
Juan D. Chavez ◽  
Mariko M. Alexander ◽  
John Ramsey ◽  
Jimmy K. Eng ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Protein Interaction ◽  
Protein Interactions ◽  
Interaction Network ◽  
Host Protein ◽  
Host Proteins ◽  
Content Type ◽  
Host Pathogen ◽  
Pathogen Protein ◽  
Binding Interfaces

ABSTRACTDemonstrating direct interactions between host and virus proteins during infection is a major goal and challenge for the field of virology. Most protein interactions are not binary or easily amenable to structural determination. Using infectious preparations of a polerovirus (Potato leafroll virus[PLRV]) and protein interaction reporter (PIR), a revolutionary technology that couples a mass spectrometric-cleavable chemical cross-linker with high-resolution mass spectrometry, we provide the first report of a host-pathogen protein interaction network that includes data-derived, topological features for every cross-linked site that was identified. We show that PLRV virions have hot spots of protein interaction and multifunctional surface topologies, revealing how these plant viruses maximize their use of binding interfaces. Modeling data, guided by cross-linking constraints, suggest asymmetric packing of the major capsid protein in the virion, which supports previous epitope mapping studies. Protein interaction topologies are conserved with other species in theLuteoviridaeand with unrelated viruses in theHerpesviridaeandAdenoviridae. Functional analysis of three PLRV-interacting host proteinsin plantausing a reverse-genetics approach revealed a complex, molecular tug-of-war between host and virus. Structural mimicry and diversifying selection—hallmarks of host-pathogen interactions—were identified within host and viral binding interfaces predicted by our models. These results illuminate the functional diversity of the PLRV-host protein interaction network and demonstrate the usefulness of PIR technology for precision mapping of functional host-pathogen protein interaction topologies.IMPORTANCEThe exterior shape of a plant virus and its interacting host and insect vector proteins determine whether a virus will be transmitted by an insect or infect a specific host. Gaining this information is difficult and requires years of experimentation. We used protein interaction reporter (PIR) technology to illustrate how viruses exploit host proteins during plant infection. PIR technology enabled our team to precisely describe the sites of functional virus-virus, virus-host, and host-host protein interactions using a mass spectrometry analysis that takes just a few hours. Applications of PIR technology in host-pathogen interactions will enable researchers studying recalcitrant pathogens, such as animal pathogens where host proteins are incorporated directly into the infectious agents, to investigate how proteins interact during infection and transmission as well as develop new tools for interdiction and therapy.

scholarly journals Molecular Basis of the Ternary Interaction between NS1 of the 1918 Influenza A Virus, PI3K, and CRK

Viruses ◽  
2020 ◽  
Vol 12 (3) ◽  
pp. 338
Author(s):  
Alyssa Dubrow ◽  
Sirong Lin ◽  
Nowlan Savage ◽  
Qingliang Shen ◽  
Jae-Hyun Cho
Keyword(s):  
Influenza A Virus ◽  
Molecular Basis ◽  
Influenza A ◽  
Host Protein ◽  
Structural Protein ◽  
Host Proteins ◽  
Ternary Interaction ◽  
Important Virulence Factor ◽  
1918 Influenza ◽  
Phosphoinositide 3 Kinase

The 1918 influenza A virus (IAV) caused the worst flu pandemic in human history. Non-structural protein 1 (NS1) is an important virulence factor of the 1918 IAV and antagonizes host antiviral immune responses. NS1 increases virulence by activating phosphoinositide 3-kinase (PI3K) via binding to the p85β subunit of PI3K. Intriguingly, unlike the NS1 of other human IAV strains, 1918 NS1 hijacks another host protein, CRK, to form a ternary complex with p85β, resulting in hyperactivation of PI3K. However, the molecular basis of the ternary interaction between 1918 NS1, CRK, and PI3K remains elusive. Here, we report the structural and thermodynamic bases of the ternary interaction. We find that the C-terminal tail (CTT) of 1918 NS1 remains highly flexible in the complex with p85β. Thus, the CTT of 1918 NS1 in the complex with PI3K can efficiently hijack CRK. Notably, our study indicates that 1918 NS1 enhances its affinity to p85β in the presence of CRK, which might result in enhanced activation of PI3K. Our results provide structural insight into how 1918 NS1 hijacks two host proteins simultaneously.

scholarly journals Genomic Approach for Analysis of Surface Proteins in Chlamydia pneumoniae

2002 ◽  
Vol 70 (1) ◽  
pp. 368-379 ◽  
Author(s):  
Silvia Montigiani ◽  
Fabiana Falugi ◽  
Maria Scarselli ◽  
Oretta Finco ◽  
Roberto Petracca ◽  
...  
Keyword(s):  
Chlamydia Pneumoniae ◽  
Genetic Manipulation ◽  
Respiratory Infections ◽  
Surface Protein ◽  
Surface Proteins ◽  
Mass Spectrometry Analysis ◽  
Western Blots ◽  
Spectrometry Analysis ◽  
Proteomic Approach ◽  
Definition Of

ABSTRACT Chlamydia pneumoniae, a human pathogen causing respiratory infections and probably contributing to the development of atherosclerosis and heart disease, is an obligate intracellular parasite which for replication needs to productively interact with and enter human cells. Because of the intrinsic difficulty in working with C. pneumoniae and in the absence of reliable tools for its genetic manipulation, the molecular definition of the chlamydial cell surface is still limited, thus leaving the mechanisms of chlamydial entry largely unknown. In an effort to define the surface protein organization of C. pneumoniae, we have adopted a combined genomic-proteomic approach based on (i) in silico prediction from the available genome sequences of peripherally located proteins, (ii) heterologous expression and purification of selected proteins, (iii) production of mouse immune sera against the recombinant proteins to be used in Western blotting and fluorescence-activated cell sorter (FACS) analyses for the identification of surface antigens, and (iv) mass spectrometry analysis of two-dimensional electrophoresis (2DE) maps of chlamydial protein extracts to confirm the presence of the FACS-positive antigens in the chlamydial cell. Of the 53 FACS-positive sera, 41 recognized a protein species with the expected size on Western blots, and 28 of the 53 antigens shown to be surface-exposed by FACS were identified on 2DE maps of elementary-body extracts. This work represents the first systematic attempt to define surface protein organization in C. pneumoniae.

scholarly journals Insights Into the Polerovirus–Plant Interactome Revealed by Coimmunoprecipitation and Mass Spectrometry

2015 ◽  
Vol 28 (4) ◽  
pp. 467-481 ◽  
Author(s):  
Stacy L. DeBlasio ◽  
Richard Johnson ◽  
Jaclyn Mahoney ◽  
Alexander Karasev ◽  
Stewart M. Gray ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Carbon Fixation ◽  
Dynamic Range ◽  
High Resolution Mass Spectrometry ◽  
Protein Complexes ◽  
Interaction Network ◽  
Host Protein ◽  
Host Proteins ◽  
Leafroll Virus ◽  
Readthrough Protein

Identification of host proteins interacting with the aphidborne Potato leafroll virus (PLRV) from the genus Polerovirus, family Luteoviridae, is a critical step toward understanding how PLRV and related viruses infect plants. However, the tight spatial distribution of PLRV to phloem tissues poses challenges. A polyclonal antibody raised against purified PLRV virions was used to coimmunoprecipitate virus-host protein complexes from Nicotiana benthamiana tissue inoculated with an infectious PLRV cDNA clone using Agrobacterium tumefaciens. A. tumefaciens-mediated delivery of PLRV enabled infection and production of assembled, insect-transmissible virus in most leaf cells, overcoming the dynamic range constraint posed by a systemically infected host. Isolated protein complexes were characterized using high-resolution mass spectrometry and consisted of host proteins interacting directly or indirectly with virions, as well as the nonincorporated readthrough protein (RTP) and three phosphorylated positional isomers of the RTP. A bioinformatics analysis using ClueGO and STRING showed that plant proteins in the PLRV protein interaction network regulate key biochemical processes, including carbon fixation, amino acid biosynthesis, ion transport, protein folding, and trafficking.

scholarly journals Strategies for understanding dynamic, personalized profiles of host-derived proteins and microbes from human stool

2019 ◽  
Author(s):  
Ellen Casavant ◽  
Les Dethlefsen ◽  
Kris Sankaran ◽  
Daniel Sprockett ◽  
Susan Holmes ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Protein Profile ◽  
Host Protein ◽  
Host Proteins ◽  
Protein Marker ◽  
Gastrointestinal Health ◽  
Wide Range ◽  
Subject Variability ◽  
Human Stool ◽  
Over Time

AbstractMeasuring host proteins through noninvasive stool-based assays opens new avenues for characterizing states of gastrointestinal health. However, the extent to which these proteins vary over time and between healthy subjects is poorly characterized. Here, we characterize technical and biological sources of variability in mass spectrometry-based measurements of host proteins in stool. We identify the proteins that most vary over time within an individual, and among different individuals. Finally, we examine and compare temporal and inter-individual variation in host protein and bacterial taxonomic profiles of the same fecal specimens. To address these issues, five self-reported healthy individuals were each sampled eight times over four weeks. First, we demonstrate that mass spectrometry-based identification and label-free quantification of stool proteins exhibit non-significant variability (p>0.05) between both technical and preparative replicates for a subset of 78 proteins, supporting the utility of this method for biomarker measurement. Second, although 13 human stool proteins varied significantly in relative abundance over time within individuals, 58 proteins varied significantly (at least four-fold) between subjects. The average pair-wise difference between individuals was greater than the average within-subject difference for both the proteome and microbiome datasets (p<0.0001). Fecal host proteins, like the traditional fecal protein marker, calprotectin, unambiguously pointed to innate and adaptive immune responses. For example, one subject’s fecal protein profile suggested a sub-clinical inflammatory state. From these data, we conclude that host-centric protein measurements in stool reveal a wide range of variation during states of apparent health, and add a valuable complementary insight into host-microbiota relationships.IMPORTANCEHuman proteins in stool hold untapped potential for characterizing gastrointestinal health. To fully harness this potential and create a baseline of healthy stool protein abundances and identifications, it will be important to establish the extent to which these proteins might vary in the absence of disease. This study quantifies the major sources of variation in stool protein abundance data. We assessed technical, preparative, temporal, and inter-subject variability of human protein abundances in stool and found that among these sources, differences between subjects accounted for the greatest amount of variation, followed by temporal differences, and then technical factors. Our paired microbiome analysis found matching patterns of temporal and inter-subject variability. By characterizing multiple variance parameters in host stool protein abundances, our analysis helps to contextualize a wide range of future disease-focused stool studies as well as elucidate host-microbe interactions.

scholarly journals A map of direct SARS-CoV-2 protein interactions implicates specific human host processes

2021 ◽  
Author(s):  
Dae-Kyum Kim ◽  
Benjamin Weller ◽  
Chung-Wen Lin ◽  
Dayag Sheykhkarimli ◽  
Jennifer J Knapp ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Membrane Trafficking ◽  
Host Protein ◽  
Host Proteins ◽  
Systematic Map ◽  
High Data ◽  
Protein Ubiquitination ◽  
Human Proteins ◽  
Physical Interactions ◽  
Key Steps

Key steps in viral propagation, immune suppression and pathology are mediated by direct, binary physical interactions between viral and host proteins. To understand the biology of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, we generated an unbiased systematic map of binary physical interactions between viral and host interactions, complementing previous co-complex association maps by conveying more direct mechanistic understanding and enabling targeted disruption of direct interactions. To this end, we deployed two parallel strategies, identifying 205 virus-host and 27 intraviral binary interactions amongst 171 host and 19 viral proteins, with orthogonal validation by an internally benchmarked NanoLuc two-hybrid system to ensure high data quality. Host proteins interacting with SARS-CoV-2 proteins were enriched in various cellular processes, including immune signaling and inflammation, protein ubiquitination, and membrane trafficking. Specific subnetworks provide new hypotheses related to viral modulation of host protein homeostasis and T-cell regulation. The direct virus-host protein interactions we identified can now be prioritized as targets for therapeutic intervention. More generally, we provide a resource of systematic maps describing which SARS-CoV-2 and human proteins interact directly.

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