scholarly journals Temporal Viral Genome-Protein Interactions Define Distinct Stages of Productive Herpesviral Infection

mBio ◽  
2018 ◽  
Vol 9 (4) ◽  
Author(s):  
Jill A. Dembowski ◽  
Neal A. DeLuca
Keyword(s):  
Life Cycle ◽  
Protein Interactions ◽  
Viral Genome ◽  
Isotope Labeling ◽  
De Novo ◽  
Affinity Purification ◽  
Human Pathogens ◽  
Host Proteins ◽  
Viral Dna ◽  
Hsv 1

ABSTRACTHerpesviruses utilize multiple mechanisms to redirect host proteins for use in viral processes and to avoid recognition and repression by the host. To investigate dynamic interactions between herpes simplex virus type 1 (HSV-1) DNA and viral and host proteins throughout infection, we developed an approach to identify proteins that associate with the infecting viral genome from nuclear entry through packaging. To accomplish this, virus stocks were prepared in the presence of ethynyl-modified nucleotides to enable covalent tagging of viral genomes after infection for analysis of viral genome-protein interactions by imaging or affinity purification. Affinity purification was combined with stable isotope labeling of amino acids in cell culture (SILAC) mass spectrometry to enable the distinction between proteins that were brought into the cell by the virus or expressed within the infected cell before or during infection. We found that input viral DNA progressed within 6 h through four temporal stages where the genomes sequentially (i) interacted with intrinsic antiviral and DNA damage response proteins, (ii) underwent a robust transcriptional switch mediated largely by ICP4, (iii) engaged in replication, repair, and continued transcription, and then (iv) transitioned to a more transcriptionally inert state engagingde novo-synthesized viral structural components while maintaining interactions with replication proteins. Using a combination of genetic, imaging, and proteomic approaches, we provide a new and temporally compressed view of the HSV-1 life cycle based on input genome-proteome dynamics.IMPORTANCEHerpesviruses are highly prevalent and ubiquitous human pathogens. Studies of herpesviruses and other viruses have previously been limited by the ability to directly study events that occur on the viral DNA throughout infection. We present a new powerful approach, which allows for the temporal investigation of viral genome-protein interactions at all phases of infection. This work has integrated many results from previous studies with the discovery of novel factors potentially involved in viral infection that may represent new antiviral targets. In addition, the study provides a new view of the HSV-1 life cycle based on genome-proteome dynamics.

scholarly journals Temporal Viral Genome-Protein Interactions Define Distinct Stages of Productive Herpesviral Infection

2018 ◽  
Author(s):  
Jill A. Dembowski ◽  
Neal A. DeLuca
Keyword(s):  
Protein Interactions ◽  
Viral Genome ◽  
De Novo ◽  
Replication Proteins ◽  
Host Proteins ◽  
Nuclear Entry ◽  
Dynamic Interactions ◽  
Damage Response ◽  
Transcriptional Switch ◽  
Hsv 1

SUMMARYHerpesviruses utilize multiple mechanisms to redirect host proteins for use in viral processes and to avoid recognition and repression by the host. To investigate the dynamic interactions between HSV-1 DNA and viral and host proteins, we developed an approach to identify proteins that associate with the infecting viral genome from nuclear entry through packaging. We found that input viral DNA progressed within six hours through four temporal stages where the genomes: 1. interacted with intrinsic and DNA damage response proteins, 2. underwent a robust transcriptional switch mediated largely by ICP4, 3. engaged in replication, repair, and continued transcription, and then 4. transitioned to a more transcriptionally inert state engaging de novo synthesized viral structural components while maintaining interactions with replication proteins. Using a combination of genetic, imaging, and proteomic approaches, we provide a new and temporally compressed view of the HSV-1 life cycle based on genome-proteome dynamics.

scholarly journals PIPINO: A Software Package to Facilitate the Identification of Protein-Protein Interactions from Affinity Purification Mass Spectrometry Data

2016 ◽  
Vol 2016 ◽  
pp. 1-13
Author(s):  
Stefan Kalkhof ◽  
Stefan Schildbach ◽  
Conny Blumert ◽  
Friedemann Horn ◽  
Martin von Bergen ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Protein Interactions ◽  
Isotope Labeling ◽  
Affinity Purification ◽  
Interaction Network ◽  
Mass Spectrometry Data ◽  
Protein Protein Interactions ◽  
Protein Protein Interaction ◽  
Binding Behavior ◽  
Bona Fide

The functionality of most proteins is regulated by protein-protein interactions. Hence, the comprehensive characterization of the interactome is the next milestone on the path to understand the biochemistry of the cell. A powerful method to detect protein-protein interactions is a combination of coimmunoprecipitation or affinity purification with quantitative mass spectrometry. Nevertheless, both methods tend to precipitate a high number of background proteins due to nonspecific interactions. To address this challenge the software Protein-Protein-Interaction-Optimizer (PIPINO) was developed to perform an automated data analysis, to facilitate the selection of bona fide binding partners, and to compare the dynamic of interaction networks. In this study we investigated the STAT1 interaction network and its activation dependent dynamics. Stable isotope labeling by amino acids in cell culture (SILAC) was applied to analyze the STAT1 interactome after streptavidin pull-down of biotagged STAT1 from human embryonic kidney 293T cells with and without activation. Starting from more than 2,000 captured proteins 30 potential STAT1 interaction partners were extracted. Interestingly, more than 50% of these were already reported or predicted to bind STAT1. Furthermore, 16 proteins were found to affect the binding behavior depending on STAT1 phosphorylation such as STAT3 or the importin subunits alpha 1 and alpha 6.

scholarly journals RNA Polymerase II Promoter-Proximal Pausing and Release to Elongation Are Key Steps Regulating Herpes Simplex Virus 1 Transcription

2019 ◽  
Vol 94 (5) ◽  
Author(s):  
Claire H. Birkenheuer ◽  
Joel D. Baines
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Rna Polymerase Ii ◽  
Viral Genome ◽  
Viral Dna ◽  
Herpes Simplex Virus 1 ◽  
Pol Ii ◽  
Late Genes ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACT Herpes simplex virus 1 (HSV-1) genes are transcribed by cellular RNA polymerase II (Pol II). Expression of viral immediate early (α) genes is followed sequentially by early (β), late (γ1), and true late (γ2) genes. We used precision nuclear run-on with deep sequencing to map and to quantify Pol II on the HSV-1(F) genome with single-nucleotide resolution. Approximately 30% of total Pol II relocated to viral genomes within 3 h postinfection (hpi), when it occupied genes of all temporal classes. At that time, Pol II on α genes accumulated most heavily at promoter-proximal pause (PPP) sites located ∼60 nucleotides downstream of the transcriptional start site, while β genes bore Pol II more evenly across gene bodies. At 6 hpi, Pol II increased on γ1 and γ2 genes while Pol II pausing remained prominent on α genes. At that time, average cytoplasmic mRNA expression from α and β genes decreased, relative to levels at 3 hpi, while γ1 relative expression increased slightly and γ2 expression increased more substantially. Cycloheximide treatment during the first 3 h reduced the amount of Pol II associated with the viral genome and confined most of the remaining Pol II to α gene PPP sites. Inhibition of both cyclin-dependent kinase 9 activity and viral DNA replication reduced Pol II on the viral genome and restricted much of the remaining Pol II to PPP sites. IMPORTANCE These data suggest that viral transcription is regulated not only by Pol II recruitment to viral genes but also by control of elongation into viral gene bodies. We provide a detailed map of Pol II occupancy on the HSV-1 genome that clarifies features of the viral transcriptome, including the first identification of Pol II PPP sites. The data indicate that Pol II is recruited to late genes early in infection. Comparing α and β gene occupancy at PPP sites and gene bodies suggests that Pol II is released more efficiently into the bodies of β genes than α genes at 3 hpi and that repression of α gene expression late in infection is mediated by prolonged promoter-proximal pausing. In addition, DNA replication is required to maintain full Pol II occupancy on viral DNA and to promote elongation on late genes later in infection.

scholarly journals Purification of viral genome-linked protein VPg from potato virus A-infected plants reveals several post-translationally modified forms of the protein

2008 ◽  
Vol 89 (6) ◽  
pp. 1509-1518 ◽  
Author(s):  
Anders Hafrén ◽  
Kristiina Mäkinen
Keyword(s):  
Potato Virus ◽  
Protein Interactions ◽  
Viral Genome ◽  
Affinity Purification ◽  
Infection Process ◽  
Dependent Manner ◽  
Affinity Tag ◽  
Post Translational Modifications ◽  
Potato Virus A ◽  
Protein Sample

In order to be able to analyse post-translational modifications and protein interactions of viral genome-linked protein VPg taking place during potato virus A (PVA) infection, an affinity tag-based purification system was developed by inserting a sequence encoding a six-histidine and haemagglutinin (HisHA) tag to the 3′ end of the VPg coding sequence within the infectious cDNA clone of PVA. The engineered virus was fully functional and the HisHA tag-encoding sequence remained stable in the PVA genome throughout the infection process. Purification under denaturing conditions resulted in a protein sample that contained multiple VPg and NIa forms carrying post-translational modifications that altered their isoelectric points. Non-modified tagged VPg (pI 8) was a minor product in the protein sample derived from total leaf proteins, but when the replication-associated membranes were used as starting material, its relative amount increased. Further characterization demonstrated that some of the PVA VPg isoforms were modified by multiple phosphorylation events. Purity of the proteins derived from the native purifications with either of the tags was evaluated. A clearly purer VPg sample was obtained by performing tandem affinity purification utilizing both tags sequentially. NIb, CI and HC-Pro co-purified in an affinity-tagged VPg-dependent manner, indicating that the system was able to isolate protein complexes operating during PVA infection.

scholarly journals Purification of Components of the Translation Elongation Factor Complex of Plasmodium falciparum by Tandem Affinity Purification

2007 ◽  
Vol 6 (4) ◽  
pp. 584-591 ◽  
Author(s):  
Sachiko Takebe ◽  
William Harold Witola ◽  
Bernd Schimanski ◽  
Arthur Günzl ◽  
Choukri Ben Mamoun
Keyword(s):  
Plasmodium Falciparum ◽  
Life Cycle ◽  
Protein Interactions ◽  
Affinity Purification ◽  
Elongation Factor ◽  
Tandem Affinity Purification ◽  
Complex Life Cycle ◽  
Spectrometric Analysis ◽  
Translation Elongation Factor ◽  
Translation Elongation

ABSTRACT Plasmodium falciparum is the causative agent of severe human malaria, responsible for over 2 million deaths annually. Of the 5,300 polypeptides predicted to control the parasite life cycle in mosquitoes and humans, 60% are of unknown function. A major challenge of malaria postgenomic biology is to understand how the 5,300 predicted proteins coexist and interact to perform the essential tasks that define the complex life cycle of the parasite. One approach to assign function to these proteins is by identifying their physiological partners. Here we describe the use of tandem affinity purification (TAP) and mass spectrometry for identification of native protein interactions and purification of protein complexes in P. falciparum. Transgenic parasites were generated which express the translation elongation factor PfEF-1β harboring a C-terminal PTP tag which consists of the protein C epitope, a tobacco etch virus protease cleavage site, and two protein A domains. Purification of PfEF-1β-PTP from crude extracts followed by mass spectrometric analysis revealed, in addition to the tagged protein itself, the presence of the native PfEF-1β, the G-protein PfEF-1α, and two new proteins that we named PfEF-1γ and PfEF-1δ based on their homology to other eukaryotic γ and δ translation elongation factor subunits. These data, which constitute the first application of TAP for purification of a protein complex under native conditions in P. falciparum, revealed that the translation elongation complex in this organism contains at least two subunits of PfEF-1β. The success of this approach will set the stage for a systematic analysis of protein interactions in this important human pathogen.

scholarly journals Systematic profiling of protein complex dynamics reveals DNA-PK phosphorylation of IFI16 en route to herpesvirus immunity

2021 ◽  
Vol 7 (25) ◽  
pp. eabg6680
Author(s):  
Joshua L. Justice ◽  
Michelle A. Kennedy ◽  
Josiah E. Hutton ◽  
Dawei Liu ◽  
Bokai Song ◽  
...  
Keyword(s):  
Dna Damage ◽  
Protein Interactions ◽  
De Novo ◽  
Complex Dynamics ◽  
Nuclear Periphery ◽  
Global Dynamics ◽  
Herpes Simplex Virus 1 ◽  
Time Resolved ◽  
Simplex Virus ◽  
Hsv 1

Dynamically shifting protein-protein interactions (PPIs) regulate cellular responses to viruses and the resulting immune signaling. Here, we use thermal proximity coaggregation (TPCA) mass spectrometry to characterize the on-off behavior of PPIs during infection with herpes simplex virus 1 (HSV-1), a virus with an ancient history of coevolution with hosts. Advancing the TPCA analysis to infer associations de novo, we build a time-resolved portrait of thousands of host-host, virus-host, and virus-virus PPIs. We demonstrate that, early in infection, the DNA sensor IFI16 recruits the active DNA damage response kinase, DNA-dependent protein kinase (DNA-PK), to incoming viral DNA at the nuclear periphery. We establish IFI16 T149 as a substrate of DNA-PK upon viral infection or DNA damage. This phosphorylation promotes IFI16-driven cytokine responses. Together, we characterize the global dynamics of PPIs during HSV-1 infection, uncovering the co-regulation of IFI16 and DNA-PK functions as a missing link in immunity to herpesvirus infection.

scholarly journals Antiviral Properties of the LSD1 Inhibitor SP-2509

2020 ◽  
Vol 94 (19) ◽  
Author(s):  
Mitchell R. Harancher ◽  
Jessica E. Packard ◽  
Shane P. Cowan ◽  
Neal A. DeLuca ◽  
Jill A. Dembowski
Keyword(s):  
Gene Expression ◽  
Life Cycle ◽  
Dna Replication ◽  
Viral Replication ◽  
Virus Production ◽  
Coupled Processes ◽  
Viral Dna ◽  
Viral Dna Replication ◽  
Pol Ii ◽  
Hsv 1

ABSTRACT Lysine-specific demethylase 1 (LSD1) targets cellular proteins, including histone H3, p53, E2F, and Dnmt1, and is involved in the regulation of gene expression, DNA replication, the cell cycle, and the DNA damage response. LSD1 catalyzes demethylation of histone H3K9 associated with herpes simplex virus 1 (HSV-1) immediate early (IE) promoters and is necessary for IE gene expression, viral DNA replication, and reactivation from latency. We previously found that LSD1 associates with HSV-1 replication forks and replicating viral DNA, suggesting that it may play a direct role in viral replication or coupled processes. We investigated the effects of the LSD1 inhibitor SP-2509 on the HSV-1 life cycle. Unlike previously investigated LSD1 inhibitors tranylcypromine (TCP) and OG-L002, which covalently attach to the LSD1 cofactor flavin adenine dinucleotide (FAD) to inhibit demethylase activity, SP-2509 has previously been shown to inhibit LSD1 protein-protein interactions. We found that SP-2509 does not inhibit HSV-1 IE gene expression or transcription factor and RNA polymerase II (Pol II) association with viral DNA prior to the onset of replication. However, SP-2509 does inhibit viral DNA replication, late gene expression, and virus production. We used EdC labeling of nascent viral DNA to image aberrant viral replication compartments that form in the presence of SP-2509. Treatment resulted in the formation of small replication foci that colocalize with replication proteins but are defective for Pol II recruitment. Taken together, these data highlight a potential new role for LSD1 in the regulation of HSV-1 DNA replication and gene expression after the onset of DNA replication. IMPORTANCE Treatment of HSV-1-infected cells with SP-2509 blocked viral DNA replication, gene expression after the onset of DNA replication, and virus production. These data support a potential new role for LSD1 in the regulation of viral DNA replication and successive steps in the virus life cycle, and further highlight the promising potential to utilize LSD1 inhibition as an antiviral approach.

scholarly journals Viral DNA synthesis in nonpermissive rat F-111 cells and its role in neoplastic transformation by polyomavirus.

1989 ◽  
Vol 9 (2) ◽  
pp. 648-658 ◽  
Author(s):  
D L Hacker ◽  
M M Fluck
Keyword(s):  
Dna Synthesis ◽  
Viral Genome ◽  
De Novo ◽  
Neoplastic Transformation ◽  
T Antigen ◽  
Large T Antigen ◽  
Wild Type ◽  
Viral Dna ◽  
Supercoiled Form ◽  
The Difference

We have investigated the occurrence and role of polyomavirus DNA synthesis in neoplastic transformation by this virus. We show that after infection of Fischer rat F-111 cells at 37 degrees C, there is two- to threefold increase in the level of viral DNA as compared with the input signal, with a peak observed between 5 and 7 days postinfection. Viral DNA synthesis is about 10 times higher at 33 degrees C and increases up to 15 days postinfection. Most of the viral DNA produced is supercoiled (form I DNA). On the basis of in situ hybridization, it appears that viral replication is restricted to a small fraction of the population. At the lower temperature, more cells are permissive for viral DNA synthesis and the level of synthesis per permissive cell is higher. The DNA synthesis observed is large T-antigen dependent, and the increase in viral DNA synthesis at 33 degrees C is paralleled by an increase in the expression of this viral protein. When large T antigen is inactivated, the half-life of de novo-synthesized viral DNA is less than 12 h, suggesting that large T antigen may be responsible for the stability of the viral genomes as well as their synthesis. Surprisingly, at early times postinfection (0 to 48 h), when the essential function of large T antigen in transformation is expressed (as demonstrated in shift-up experiments with tsa mutants), the level of large T antigen is below the detection level and is at least 10-fold lower than the levels observed in permissive infections at the start of viral DNA synthesis. The difference in viral DNA at 37 and 33 degrees C allowed us to study its effect on transformation. Although an increase in transformation frequency is observed in wild-type A2 infections carried at 33 degrees C (frequencies two to three times higher than at 37 degrees C), this increase appears to be unrelated to the increase in viral DNA synthesis. Furthermore, the overall level of viral DNA and large T antigen in F-111 cells may not affect the integration of the viral genome, since the patterns of integration in cells transformed by wild-type A2 at 33 and 37 degrees C appear similar. The results are compatible with a role for large T antigen in integration-transformation which is not simply to amplify the viral genome to enhance the probability of its integration.

scholarly journals The SMC5/6 Complex Interacts with the Papillomavirus E2 Protein and Influences Maintenance of Viral Episomal DNA

2018 ◽  
Vol 92 (15) ◽  
Author(s):  
Peris Bentley ◽  
Min Jie Alvin Tan ◽  
Alison A. McBride ◽  
Elizabeth A. White ◽  
Peter M. Howley
Keyword(s):  
Cervical Cancer ◽  
High Risk ◽  
Protein Interactions ◽  
Persistent Infection ◽  
Viral Genome ◽  
Host Protein ◽  
Cellular Proteins ◽  
Genome Maintenance ◽  
Viral Dna ◽  
E2 Protein

ABSTRACTThe papillomavirus E2 protein executes numerous essential functions related to viral transcription, replication of viral DNA, and viral genome maintenance. Because E2 lacks enzymatic activity, many of these functions are mediated by interactions with host cellular proteins. Unbiased proteomics approaches have successfully identified a number of E2-host protein interactions. We have extended such studies and have identified and validated the cellular proteins structural maintenance of chromosome 5 (SMC5) and SMC6 as interactors of the viral E2 protein. These two proteins make up the core components of the SMC5/6 complex. The SMC5/6 complex is a member of the conserved structural maintenance of chromosomes (SMC) family of proteins, which are essential for genome maintenance. We have examined the role of SMC5/6 in various E2 functions. Our data suggest that SMC6 is not required for E2-mediated transcriptional activation, E1/E2-mediated transient replication, or differentiation-dependent amplification of viral DNA. Our data, however, suggest a role for SMC5/6 in viral genome maintenance.IMPORTANCEThe high-risk human papillomaviruses (HPVs) are the etiological cause of cervical cancer and the most common sexually transmitted infection. While the majority of infections may be asymptomatic or cause only benign lesions, persistent infection with the oncogenic high-risk HPV types may lead to serious diseases, such as cervical cancer, anogenital carcinoma, or head and neck oropharyngeal squamous cell carcinoma. The identification of virus-host protein interactions provides insights into the mechanisms of viral DNA persistence, viral genome replication, and cellular transformation. Elucidating the mechanism of early events in the virus replication cycle as well as of integration of viral DNA into host chromatin may present novel antiviral strategies and targets for counteracting persistent infection. The E2 protein is an important viral regulatory protein whose functions are mediated through interactions with host cell proteins. Here we explore the interaction of E2 with SMC5/6 and the functional consequences.

scholarly journals A SARS-CoV-2 – host proximity interactome

2020 ◽  
Author(s):  
Payman Samavarchi-Tehrani ◽  
Hala Abdouni ◽  
James D.R. Knight ◽  
Audrey Astori ◽  
Reuben Samson ◽  
...  
Keyword(s):  
Host Cell ◽  
Protein Interactions ◽  
Affinity Purification ◽  
Drug Repurposing ◽  
Viral Proteins ◽  
Host Protein ◽  
Virus Protein ◽  
Host Proteins ◽  
Biochemical Purification ◽  
Cell Functions

AbstractViral replication is dependent on interactions between viral polypeptides and host proteins. Identifying virus-host protein interactions can thus uncover unique opportunities for interfering with the virus life cycle via novel drug compounds or drug repurposing. Importantly, many viral-host protein interactions take place at intracellular membranes and poorly soluble organelles, which are difficult to profile using classical biochemical purification approaches. Applying proximity-dependent biotinylation (BioID) with the fast-acting miniTurbo enzyme to 27 SARS-CoV-2 proteins in a lung adenocarcinoma cell line (A549), we detected 7810 proximity interactions (7382 of which are new for SARS-CoV-2) with 2242 host proteins (results available at covid19interactome.org). These results complement and dramatically expand upon recent affinity purification-based studies identifying stable host-virus protein complexes, and offer an unparalleled view of membrane-associated processes critical for viral production. Host cell organellar markers were also subjected to BioID in parallel, allowing us to propose modes of action for several viral proteins in the context of host proteome remodelling. In summary, our dataset identifies numerous high confidence proximity partners for SARS-CoV-2 viral proteins, and describes potential mechanisms for their effects on specific host cell functions.

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