Compositional analysis of Sindbis virus ribonucleoproteins reveals an extensive co-opting of key nuclear RNA-binding proteins

The expansion of tropical mosquito habitats and associated arboviruses is a risk for human health, and it thus becomes fundamental to identify new antiviral strategies. In this study we employ a new approach to elucidate the composition of the ribonucleoproteins (RNPs) of a prototypical arbovirus called Sindbis (SINV). SINV RNPs contain 453 cellular and 6 viral proteins, many of these proteins are nuclear in uninfected cells and redistribute to the cytoplasm upon infection. These findings suggest that SINV RNAs act as 'spiderwebs', capturing host factors required for viral replication and gene expression in the cytoplasm. Functional perturbation of several of these host proteins causes profound effects in virus infection, as illustrated here with the tRNA ligase complex. Moreover, inhibition of viral RNP components with available drugs hampers the infection of a wide range of viruses, opening new avenues for the development of broad-spectrum therapies.

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The SARS-CoV-2 RNA interactome

10.1101/2020.11.02.364497 ◽

2020 ◽

Author(s):

Sungyul Lee ◽

Young-suk Lee ◽

Yeon Choi ◽

Ahyeon Son ◽

Youngran Park ◽

...

Keyword(s):

Life Cycle ◽

Rna Binding ◽

Rna Binding Proteins ◽

Rna Virus ◽

Host Factors ◽

Therapeutic Interventions ◽

Host Proteins ◽

Viral Rnas ◽

Comprehensive List ◽

Evolutionarily Conserved

AbstractSARS-CoV-2 is an RNA virus whose success as a pathogen relies on its ability to repurpose host RNA-binding proteins (RBPs) to form its own RNA interactome. Here, we developed and applied a robust ribonucleoprotein capture protocol to uncover the SARS-CoV-2 RNA interactome. We report 109 host factors that directly bind to SARS-CoV-2 RNAs including general antiviral factors such as ZC3HAV1, TRIM25, and PARP12. Applying RNP capture on another coronavirus HCoV-OC43 revealed evolutionarily conserved interactions between viral RNAs and host proteins. Network and transcriptome analyses delineated antiviral RBPs stimulated by JAK-STAT signaling and proviral RBPs responsible for hijacking multiple steps of the mRNA life cycle. By knockdown experiments, we further found that these viral-RNA-interacting RBPs act against or in favor of SARS-CoV-2. Overall, this study provides a comprehensive list of RBPs regulating coronaviral replication and opens new avenues for therapeutic interventions.

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Network-Guided Discovery of Influenza Virus Replication Host Factors

mBio ◽

10.1128/mbio.02002-18 ◽

2018 ◽

Vol 9 (6) ◽

Cited By ~ 14

Author(s):

Emily E. Ackerman ◽

Eiryo Kawakami ◽

Manami Katoh ◽

Tokiko Watanabe ◽

Shinji Watanabe ◽

...

Keyword(s):

Influenza Virus ◽

Viral Replication ◽

Virus Replication ◽

Drug Targets ◽

Antiviral Drug ◽

Viral Proteins ◽

Host Factors ◽

Ppi Network ◽

Host Proteins ◽

Influenza Virus Replication

ABSTRACTThe positions of host factors required for viral replication within a human protein-protein interaction (PPI) network can be exploited to identify drug targets that are robust to drug-mediated selective pressure. Host factors can physically interact with viral proteins, be a component of virus-regulated pathways (where proteins do not interact with viral proteins), or be required for viral replication but unregulated by viruses. Here, we demonstrate a method of combining human PPI networks with virus-host PPI data to improve antiviral drug discovery for influenza viruses by identifying target host proteins. Analysis shows that influenza virus proteins physically interact with host proteins in network positions significant for information flow, even after the removal of known abundance-degree bias within PPI data. We have isolated a subnetwork of the human PPI network that connects virus-interacting host proteins to host factors that are important for influenza virus replication without physically interacting with viral proteins. The subnetwork is enriched for signaling and immune processes distinct from those associated with virus-interacting proteins. Selecting proteins based on subnetwork topology, we performed an siRNA screen to determine whether the subnetwork was enriched for virus replication host factors and whether network position within the subnetwork offers an advantage in prioritization of drug targets to control influenza virus replication. We found that the subnetwork is highly enriched for target host proteins—more so than the set of host factors that physically interact with viral proteins. Our findings demonstrate that network positions are a powerful predictor to guide antiviral drug candidate prioritization.IMPORTANCEIntegrating virus-host interactions with host protein-protein interactions, we have created a method using these established network practices to identify host factors (i.e., proteins) that are likely candidates for antiviral drug targeting. We demonstrate that interaction cascades between host proteins that directly interact with viral proteins and host factors that are important to influenza virus replication are enriched for signaling and immune processes. Additionally, we show that host proteins that interact with viral proteins are in network locations of power. Finally, we demonstrate a new network methodology to predict novel host factors and validate predictions with an siRNA screen. Our results show that integrating virus-host proteins interactions is useful in the identification of antiviral drug target candidates.

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TDP-43 dysfunction restricts dendritic complexity by inhibiting CREB activation and altering gene expression

10.1101/2019.12.12.874735 ◽

2019 ◽

Cited By ~ 1

Author(s):

Josiah J. Herzog ◽

Mugdha Deshpande ◽

Weijin Xu ◽

Reazur Rahman ◽

Hannah Suib ◽

...

Keyword(s):

Neurodegenerative Diseases ◽

Rna Binding ◽

Rna Binding Proteins ◽

Dendritic Morphology ◽

New Approach ◽

Creb Activation ◽

Dendritic Branching ◽

Transcriptional Output ◽

Dendritic Complexity ◽

Lateral Sclerosis

AbstractAmyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are two related neurodegenerative diseases that present with similar TDP-43 pathology in patient tissue. TDP-43 is an RNA-binding protein and forms aggregates in neurons of ALS and FTD patients as well as in a subset of patients diagnosed with other neurodegenerative diseases. Despite our understanding that TDP-43 is essential for many aspects of RNA metabolism, it remains obscure how TDP-43 dysfunction contributes to neurodegeneration. Interestingly, several neurological disorders display altered dendritic morphology and complexity, which are thought to precede neurodegeneration. In this study, we used TRIBE (targets of RNA-binding proteins identified by editing) as a new approach to identify signaling pathways that regulate dendritic branching downstream of TDP-43. We found that TDP-43 targets are enriched for pathways that signal to the CREB transcription factor. We further found that TDP-43 dysfunction inhibits CREB activation and CREB transcriptional output, and restoring CREB signaling rescued defects in dendritic branching. Our data therefore provide a novel mechanism by which TDP-43 dysfunction interferes with dendritic branching, and define new pathways for therapeutic intervention in neurodegenerative diseases.

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Picornaviruses and RNA Metabolism: Local and Global Effects of Infection

Journal of Virology ◽

10.1128/jvi.02088-17 ◽

2019 ◽

Vol 93 (21) ◽

Cited By ~ 3

Author(s):

Autumn C. Holmes ◽

Bert L. Semler

Keyword(s):

Host Cell ◽

Rna Binding ◽

Rna Binding Proteins ◽

Foot And Mouth Disease ◽

Rna Metabolism ◽

Cell Nuclei ◽

Cell Functions ◽

Mouth Disease Virus ◽

Wide Range ◽

Positive Strand Rna

ABSTRACT Due to the limiting coding capacity for members of the Picornaviridae family of positive-strand RNA viruses, their successful replication cycles require complex interactions with host cell functions. These interactions span from the down-modulation of many aspects of cellular metabolism to the hijacking of specific host functions used during viral translation, RNA replication, and other steps of infection by picornaviruses, such as human rhinovirus, coxsackievirus, poliovirus, foot-and-mouth disease virus, enterovirus D-68, and a wide range of other human and nonhuman viruses. Although picornaviruses replicate exclusively in the cytoplasm of infected cells, they have extensive interactions with host cell nuclei and the proteins and RNAs that normally reside in this compartment of the cell. This review will highlight some of the more recent studies that have revealed how picornavirus infections impact the RNA metabolism of the host cell posttranscriptionally and how they usurp and modify host RNA binding proteins as well as microRNAs to potentiate viral replication.

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Host Factors Associated with the Sindbis Virus RNA-Dependent RNA Polymerase: Role for G3BP1 and G3BP2 in Virus Replication

Journal of Virology ◽

10.1128/jvi.01983-09 ◽

2010 ◽

Vol 84 (13) ◽

pp. 6720-6732 ◽

Cited By ~ 77

Author(s):

Ileana M. Cristea ◽

Heather Rozjabek ◽

Kelly R. Molloy ◽

Sophiya Karki ◽

Laura L. White ◽

...

Keyword(s):

Rna Polymerase ◽

Sindbis Virus ◽

Specific Treatment ◽

Host Factors ◽

Host Proteins ◽

Rna Dependent Rna Polymerase ◽

Replication Process ◽

Flag Epitope ◽

Factors Associated ◽

Host Interaction

ABSTRACT Sindbis virus (SINV) is the prototype member of the Alphavirus genus, whose members cause severe human diseases for which there is no specific treatment. To ascertain host factors important in the replication of the SINV RNA genome, we generated a SINV expressing nsP4, the viral RNA-dependent RNA polymerase, with an in-frame 3×Flag epitope tag. Proteomic analysis of nsP4-containing complexes isolated from cells infected with the tagged virus revealed 29 associated host proteins. Of these, 10 proteins were associated only at a later time of infection (12 h), 14 were associated both early and late, and five were isolated only at the earlier time (6 h postinfection). These results demonstrate the dynamic nature of the virus-host interaction that occurs over the course of infection and suggest that different host proteins may be required for the multiple functions carried out by nsP4. Two related proteins found in association with nsP4 at both times of infection, GTPase-activating protein (SH3 domain) binding protein 1 (G3BP1) and G3BP2 were also previously identified as associated with SINV nsP2 and nsP3. We demonstrate a likely overlapping role for these host factors in limiting SINV replication events. The present study also identifies 10 host factors associated with nsP4 6 h after infection that were not found to be associated with nsP2 or nsP3. These factors are candidates for playing important roles in the RNA replication process. Identifying host factors essential for replication should lead to new strategies to interrupt alphavirus replication.

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STAG proteins promote cohesin ring loading at R-loops

10.1101/2021.02.20.432055 ◽

2021 ◽

Author(s):

Hayley Porter ◽

Yang Li ◽

Wazeer Varsally ◽

Maria Victoria Neguembor ◽

Manuel Beltran ◽

...

Keyword(s):

Functional Data ◽

Cancer Biology ◽

Rna Binding ◽

Rna Binding Proteins ◽

Biological Processes ◽

Loop Formation ◽

Core Complex ◽

Wide Range ◽

Processing Mechanisms ◽

Pan Cancer

ABSTRACTMost studies of cohesin function consider the Stromalin Antigen (STAG/SA) proteins as core complex members given their ubiquitous interaction with the cohesin ring. Here, we provide functional data to support the notion that the SA subunit is not a mere passenger in this structure, but instead plays a key role in cohesins localization to diverse biological processes and promotes loading of the complex at these sites. We show that in cells acutely depleted for RAD21, SA proteins remain bound to chromatin and interact with CTCF, as well as a wide range of RNA binding proteins involved in multiple RNA processing mechanisms. Accordingly, SA proteins interact with RNA and are localised to endogenous R-loops where they act to suppress R-loop formation. Our results place SA proteins on chromatin upstream of the cohesin complex and reveal a role for SA in cohesin loading at R-loops which is independent of NIPBL, the canonical cohesin loader. We propose that SA takes advantage of this structural R-loop platform to link cohesin loading and chromatin structure with diverse genome functions. Since SA proteins are pan-cancer targets, and R-loops play an increasingly prevalent role in cancer biology, our results have important implications for the mechanistic understanding of SA proteins in cancer and disease.

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Cofactor-Independent RNA Editing by a Synthetic S-Type PPR Protein

Synthetic Biology ◽

10.1093/synbio/ysab034 ◽

2021 ◽

Author(s):

Kalia Bernath-Levin ◽

Jason Schmidberger ◽

Suvi Honkanen ◽

Bernard Gutmann ◽

Yueming Kelly Sun ◽

...

Keyword(s):

Rna Editing ◽

Rna Processing ◽

Tandem Repeats ◽

Rna Binding ◽

Rna Binding Proteins ◽

Screening Method ◽

Pentatricopeptide Repeat ◽

Wide Range ◽

Ppr Proteins ◽

P Type

ABSTRACT Pentatricopeptide repeat (PPR) proteins are RNA-binding proteins that are attractive tools for RNA processing in synthetic biology applications given their modular structure and ease of design. Several distinct types of motifs have been described from natural PPR proteins, but almost all work so far with synthetic PPR proteins has focused on the most widespread P-type motifs. We have investigated synthetic PPR proteins based on tandem repeats of the more compact S-type PPR motif found in plant organellar RNA editing factors, and particularly prevalent in the lycophyte Selaginella. With the aid of a novel plate-based screening method we show that synthetic S-type PPR proteins are easy to design, bind with high affinity and specificity, and are functional in a wide range of pH, salt and temperature conditions. We find that they outperform a synthetic P-type PPR scaffold in many situations. We designed an S-type editing factor to edit an RNA target in E. coli and demonstrate that it edits effectively without requiring any additional cofactors to be added to the system. These qualities make S-type PPR scaffolds ideal for developing new RNA processing tools.

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Network-Guided Discovery of Influenza Virus Replication Host Factors

10.1101/344861 ◽

2018 ◽

Cited By ~ 1

Author(s):

Emily E. Ackerman ◽

Eiryo Kawakami ◽

Manami Katoh ◽

Tokiko Watanabe ◽

Shinji Watanabe ◽

...

Keyword(s):

Influenza Virus ◽

Virus Replication ◽

Drug Targets ◽

Antiviral Drug ◽

Viral Proteins ◽

Host Factors ◽

Host Protein ◽

Ppi Network ◽

Host Proteins ◽

Influenza Virus Replication

ABSTRACTThe position of host factors required for viral replication within a human protein-protein interaction (PPI) network can be exploited to identify drug targets that are robust to drug-mediated selective pressure. Host factors can physically interact with viral proteins, be a component of pathways regulated by viruses (where proteins themselves do not interact with viral proteins) or be required for viral replication but unregulated by viruses. Here, we demonstrate a method of combining a human PPI network with virus-host protein interaction data to improve antiviral drug discovery for influenza viruses by identifying target host proteins. Network analysis shows that influenza virus proteins physically interact with host proteins in network positions significant for information flow. We have isolated a subnetwork of the human PPI network which connects virus-interacting host proteins to host factors that are important for influenza virus replication without physically interacting with viral proteins. The subnetwork is enriched for signaling and immune processes. Selecting proteins based on network topology within the subnetwork, we performed an siRNA screen to determine if the subnetwork was enriched for virus replication host factors and if network position within the subnetwork offers an advantage in prioritization of drug targets to control influenza virus replication. We found that the subnetwork is highly enriched for target host proteins – more so than the set of host factors that physically interact with viral proteins. Our findings demonstrate that network positions are a powerful predictor to guide antiviral drug candidate prioritization.IMPORTANCEIntegrating virus-host interactions with host protein-protein interactions, we have created a method using these established network practices to identify host factors (i.e. proteins) that are likely candidates for antiviral drug targeting. We demonstrate that interaction cascades between host proteins that directly interact with viral proteins and host factors that are important to influenza replication are enriched for signaling and immune processes. Additionally, we show that host proteins that interact with viral proteins are in network locations of power. Finally, we demonstrate a new network methodology to predict novel host factors and validate predictions with an siRNA screen. Our results show that integrating virus-host proteins interactions is useful in the identification of antiviral drug target candidates.

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Human DICER helicase domain recruits PKR and modulates its antiviral activity

PLoS Pathogens ◽

10.1371/journal.ppat.1009549 ◽

2021 ◽

Vol 17 (5) ◽

pp. e1009549

Author(s):

Thomas C. Montavon ◽

Morgane Baldaccini ◽

Mathieu Lefèvre ◽

Erika Girardi ◽

Béatrice Chane-Woon-Ming ◽

...

Keyword(s):

Rna Silencing ◽

Mammalian Cells ◽

Sindbis Virus ◽

Rna Binding ◽

Semliki Forest Virus ◽

Rna Binding Proteins ◽

Helicase Domain ◽

Type I ◽

Dependent Manner ◽

Rna Helicases

The antiviral innate immune response mainly involves type I interferon (IFN) in mammalian cells. The contribution of the RNA silencing machinery remains to be established, but several recent studies indicate that the ribonuclease DICER can generate viral siRNAs in specific conditions. It has also been proposed that type I IFN and RNA silencing could be mutually exclusive antiviral responses. In order to decipher the implication of DICER during infection of human cells with alphaviruses such as the Sindbis virus and Semliki forest virus, we determined its interactome by proteomics analysis. We show that DICER specifically interacts with several double-stranded RNA binding proteins and RNA helicases during viral infection. In particular, proteins such as DHX9, ADAR-1 and the protein kinase RNA-activated (PKR) are enriched with DICER in virus-infected cells. We demonstrate that the helicase domain of DICER is essential for this interaction and that its deletion confers antiviral properties to this protein in an RNAi-independent, PKR-dependent, manner.

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Substrate Specificity of the TRAMP Nuclear Surveillance Complexes

10.1101/2020.03.04.976274 ◽

2020 ◽

Author(s):

Clémentine Delan-Forino ◽

Christos Spanos ◽

Juri Rappsilber ◽

David Tollervey

Keyword(s):

Gene Expression ◽

Mass Spectrometry ◽

Binding Sites ◽

Rna Binding ◽

Rna Binding Proteins ◽

Rna Binding Protein ◽

Rna Helicase ◽

Nuclear Rna ◽

Rna Exosome

ABSTRACTDuring nuclear surveillance in yeast, the RNA exosome functions together with the TRAMP complexes. These include the DEAH-box RNA helicase Mtr4 together with an RNA-binding protein (Air1 or Air2) and a poly(A) polymerase (Trf4 or Trf5). To better determine how RNA substrates are targeted, we analyzed protein and RNA interactions for TRAMP components. Mass spectrometry identified three distinct TRAMP complexes formed in vivo. These complexes preferentially assemble on different classes of transcripts. Unexpectedly, on many substrates, including pre-rRNAs and pre-mRNAs, binding specificity was apparently conferred by Trf4 and Trf5. Clustering of mRNAs by TRAMP association showed co-enrichment for mRNAs with functionally related products, supporting the significance of surveillance in regulating gene expression. We compared binding sites of TRAMP components with multiple nuclear RNA binding proteins, revealing preferential colocalization of subsets of factors. TRF5 deletion reduced Mtr4 recruitment and increased RNA abundance for mRNAs specifically showing high Trf5 binding.

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