An Isoform of the Eukaryotic Translation Elongation Factor 1A (eEF1a) Acts as a Pro-Viral Factor Required for Tomato Spotted Wilt Virus Disease in Nicotiana benthamiana

The tripartite genome of the negative-stranded RNA virus Tomato spotted wilt orthotospovirus (TSWV) is assembled, together with two viral proteins, the nucleocapsid protein and the RNA-dependent RNA polymerase, into infectious ribonucleoprotein complexes (RNPs). These two viral proteins are, together, essential for viral replication and transcription, yet our knowledge on the host factors supporting these two processes remains limited. To fill this knowledge gap, the protein composition of viral RNPs collected from TSWV-infected Nicotiana benthamiana plants, and of those collected from a reconstituted TSWV replicon system in the yeast Saccharomyces cerevisiae, was analysed. RNPs obtained from infected plant material were enriched for plant proteins implicated in (i) sugar and phosphate transport and (ii) responses to cellular stress. In contrast, the yeast-derived viral RNPs primarily contained proteins implicated in RNA processing and ribosome biogenesis. The latter suggests that, in yeast, the translational machinery is recruited to these viral RNPs. To examine whether one of these cellular proteins is important for a TSWV infection, the corresponding N. benthamiana genes were targeted for virus-induced gene silencing, and these plants were subsequently challenged with TSWV. This approach revealed four host factors that are important for systemic spread of TSWV and disease symptom development.

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Molecular Co-Chaperone SGT1 Is Critical for Cell-to-Cell Movement and Systemic Infection of Tomato Spotted Wilt Virus in Nicotiana benthamiana

Viruses ◽

10.3390/v10110647 ◽

2018 ◽

Vol 10 (11) ◽

pp. 647 ◽

Cited By ~ 2

Author(s):

Xin Qian ◽

Qing Xiang ◽

Tongqing Yang ◽

Hongyu Ma ◽

Xin Ding ◽

...

Keyword(s):

Virus Infection ◽

Nicotiana Benthamiana ◽

Tomato Spotted Wilt Virus ◽

Rna Virus ◽

Cell Movement ◽

Systemic Infection ◽

Plant Viruses ◽

Host Factors ◽

Tomato Spotted Wilt ◽

Wilt Virus

Tospovirus is a tripartite negative stranded RNA virus and is considered as one of the most devastating plant viruses. Successful virus infection in plant requires many host factors. To date, very few host factors have been identified as important in Tospovirus infection in plants. We reported earlier that NSm protein encoded by Tomato spotted wilt virus (TSWV), a type species of the genus Orthotospovirus, plays critical roles in viral cell-to-cell and long-distance movement. In this study, we determined that molecular co-chaperone NbSGT1 interacted with TSWV NSm in Nicotiana benthamiana. TSWV infection significantly upregulated the expression of NbSGT1 gene and transient overexpression of NbSGT1 in N. benthamiana leaves accelerated TSWV infection. In contrast, silencing the NbSGT1 gene expression using a virus-induced gene silencing (VIGS) approach strongly inhibited TSWV NSm cell-to-cell movement, as well as TSWV local and systemic infection in N. benthamiana plants. Furthermore, NbSGT1 was found to regulate the infection of both American and Euro/Asia type tospoviruses in N. benthamiana plant. Collectively, our findings presented in this paper and the results published previously indicated that molecular co-chaperone NbSGT1 plays important roles in modulating both positive stranded and tripartite negative stranded RNA virus infection in plants.

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Reprogramming mRNA Expression in Response to Defect in RNA Polymerase III Assembly in the Yeast Saccharomyces cerevisiae

International Journal of Molecular Sciences ◽

10.3390/ijms22147298 ◽

2021 ◽

Vol 22 (14) ◽

pp. 7298

Author(s):

Izabela Rudzińska ◽

Małgorzata Cieśla ◽

Tomasz W. Turowski ◽

Alicja Armatowska ◽

Ewa Leśniewska ◽

...

Keyword(s):

Mrna Expression ◽

Ribosome Biogenesis ◽

Rna Polymerase Iii ◽

Mrna Levels ◽

Rna Seq ◽

Yeast Saccharomyces Cerevisiae ◽

Protein Coding ◽

General Transcription Factor ◽

Pol I ◽

Pol Iii

The coordinated transcription of the genome is the fundamental mechanism in molecular biology. Transcription in eukaryotes is carried out by three main RNA polymerases: Pol I, II, and III. One basic problem is how a decrease in tRNA levels, by downregulating Pol III efficiency, influences the expression pattern of protein-coding genes. The purpose of this study was to determine the mRNA levels in the yeast mutant rpc128-1007 and its overdose suppressors, RBS1 and PRT1. The rpc128-1007 mutant prevents assembly of the Pol III complex and functionally mimics similar mutations in human Pol III, which cause hypomyelinating leukodystrophies. We applied RNAseq followed by the hierarchical clustering of our complete RNA-seq transcriptome and functional analysis of genes from the clusters. mRNA upregulation in rpc128-1007 cells was generally stronger than downregulation. The observed induction of mRNA expression was mostly indirect and resulted from the derepression of general transcription factor Gcn4, differently modulated by suppressor genes. rpc128-1007 mutation, regardless of the presence of suppressors, also resulted in a weak increase in the expression of ribosome biogenesis genes. mRNA genes that were downregulated by the reduction of Pol III assembly comprise the proteasome complex. In summary, our results provide the regulatory links affected by Pol III assembly that contribute differently to cellular fitness.

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Voltage-Dependent Anion Channel 1 Interacts with Ribonucleoprotein Complexes To Enhance Infectious Bursal Disease Virus Polymerase Activity

Journal of Virology ◽

10.1128/jvi.00584-17 ◽

2017 ◽

Vol 91 (16) ◽

Cited By ~ 9

Author(s):

Chunyan Han ◽

Xiangwei Zeng ◽

Shuai Yao ◽

Li Gao ◽

Lizhou Zhang ◽

...

Keyword(s):

Rna Polymerase ◽

Disease Virus ◽

Infectious Bursal Disease Virus ◽

Viral Proteins ◽

Polymerase Activity ◽

Infectious Bursal Disease ◽

Cellular Factor ◽

Ribonucleoprotein Complexes ◽

Viral Yield ◽

Bursal Disease

ABSTRACTInfectious bursal disease virus (IBDV) is a double-stranded RNA (dsRNA) virus. Segment A contains two overlapping open reading frames (ORFs), which encode viral proteins VP2, VP3, VP4, and VP5. Segment B contains one ORF and encodes the viral RNA-dependent RNA polymerase, VP1. IBDV ribonucleoprotein complexes are composed of VP1, VP3, and dsRNA and play a critical role in mediating viral replication and transcription during the virus life cycle. In the present study, we identified a cellular factor, VDAC1, which was upregulated during IBDV infection and found to mediate IBDV polymerase activity. VDAC1 senses IBDV infection by interacting with viral proteins VP1 and VP3. This association is caused by RNA bridging, and all three proteins colocalize in the cytoplasm. Furthermore, small interfering RNA (siRNA)-mediated downregulation ofVDAC1resulted in a reduction in viral polymerase activity and a subsequent decrease in viral yield. Moreover, overexpression of VDAC1 enhanced IBDV polymerase activity. We also found that the viral protein VP3 can replace segment A to execute polymerase activity. A previous study showed that mutations in the C terminus of VP3 directly influence the formation of VP1-VP3 complexes. Our immunoprecipitation experiments demonstrated that protein-protein interactions between VDAC1 and VP3 and between VDAC1 and VP1 play a role in stabilizing the interaction between VP3 and VP1, further promoting IBDV polymerase activity.IMPORTANCEThe cellular factor VDAC1 controls the entry and exit of mitochondrial metabolites and plays a pivotal role during intrinsic apoptosis by mediating the release of many apoptogenic molecules. Here we identify a novel role of VDAC1, showing that VDAC1 interacts with IBDV ribonucleoproteins (RNPs) and facilitates IBDV replication by enhancing IBDV polymerase activity through its ability to stabilize interactions in RNP complexes. To our knowledge, this is the first report that VDAC1 is specifically involved in regulating IBDV RNA polymerase activity, providing novel insight into virus-host interactions.

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Bop1 Is a Mouse WD40 Repeat Nucleolar Protein Involved in 28S and 5.8S rRNA Processing and 60S Ribosome Biogenesis

Molecular and Cellular Biology ◽

10.1128/mcb.20.15.5516-5528.2000 ◽

2000 ◽

Vol 20 (15) ◽

pp. 5516-5528 ◽

Cited By ~ 117

Author(s):

Žaklina Strezoska ◽

Dimitri G. Pestov ◽

Lester F. Lau

Keyword(s):

Ribosome Biogenesis ◽

Ribosomal Subunit ◽

Weight Fraction ◽

Rnase A ◽

Mutant Form ◽

Rrna Processing ◽

Ribonucleoprotein Complexes ◽

Nuclear Extracts ◽

Mouse Tissues ◽

Mouse Cells

ABSTRACT We have identified and characterized a novel mouse protein, Bop1, which contains WD40 repeats and is highly conserved through evolution. bop1 is ubiquitously expressed in all mouse tissues examined and is upregulated during mid-G1 in serum-stimulated fibroblasts. Immunofluorescence analysis shows that Bop1 is localized predominantly to the nucleolus. In sucrose density gradients, Bop1 from nuclear extracts cosediments with the 50S-80S ribonucleoprotein particles that contain the 32S rRNA precursor. RNase A treatment disrupts these particles and releases Bop1 into a low-molecular-weight fraction. A mutant form of Bop1, Bop1Δ, which lacks 231 amino acids in the N- terminus, is colocalized with wild-type Bop1 in the nucleolus and in ribonucleoprotein complexes. Expression of Bop1Δ leads to cell growth arrest in the G1phase and results in a specific inhibition of the synthesis of the 28S and 5.8S rRNAs without affecting 18S rRNA formation. Pulse-chase analyses show that Bop1Δ expression results in a partial inhibition in the conversion of the 36S to the 32S pre-rRNA and a complete inhibition of the processing of the 32S pre-rRNA to form the mature 28S and 5.8S rRNAs. Concomitant with these defects in rRNA processing, expression of Bop1Δ in mouse cells leads to a deficit in the cytosolic 60S ribosomal subunits. These studies thus identify Bop1 as a novel, nonribosomal mammalian protein that plays a key role in the formation of the mature 28S and 5.8S rRNAs and in the biogenesis of the 60S ribosomal subunit.

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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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NSvc4 Encoded by Rice Stripe Virus Targets Host Chloroplasts to Suppress Chloroplast-Mediated Defense

Viruses ◽

10.3390/v14010036 ◽

2021 ◽

Vol 14 (1) ◽

pp. 36

Author(s):

Zongdi Li ◽

Chenyang Li ◽

Shuai Fu ◽

Yu Liu ◽

Yi Xu ◽

...

Keyword(s):

Nicotiana Benthamiana ◽

Actin Filaments ◽

Movement Protein ◽

Infectious Clone ◽

Rice Stripe Virus ◽

Potato Virus X ◽

Host Factors ◽

Ros Production ◽

Rsv Infection ◽

Detailed Function

Our previous research found that NSvc4, the movement protein of rice stripe virus (RSV), could localize to the actin filaments, endoplasmic reticulum, plasmodesmata, and chloroplast, but the roles of NSvc4 played in the chloroplast were opaque. Here, we confirm the accumulation of NSvc4 in the chloroplasts and the N-terminal 1–73 amino acids of NSvc4 are sufficient to localize to chloroplasts. We provide evidence to show that chloroplast-localized NSvc4 can impair the chloroplast-mediated immunity. Expressing NSvc4 in Nicotiana benthamiana leaves results in the decreased expression of defense-related genes NbPR1, NbPR2, and NbWRKY12 and the inhibition of chloroplast-derived ROS production. In addition, generation of an infectious clone of potato virus X (PVX) carrying NSvc4 facilitates PVX infection in N. benthamiana plants. Moreover, we identify two chloroplast-related host factors, named NbGAPDH-A and NbPsbQ1, both of which can interact with NSvc4. Knockdown of NbGAPDH-A or NbPsbQ1 can both promote RSV infection. Our results decipher a detailed function of NSvc4 in the chloroplast.

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Release of the ribosome biogenesis factor Bud23 from small subunit precursors in yeast

RNA ◽

10.1261/rna.079025.121 ◽

2021 ◽

pp. rna.079025.121

Author(s):

Joshua J Black ◽

Arlen W Johnson

Keyword(s):

Binding Site ◽

Ribosomal Proteins ◽

Ribosome Biogenesis ◽

Assembly Line ◽

Small Subunit ◽

Nucleotide Hydrolysis ◽

Ribonucleoprotein Complexes ◽

Intermediate States ◽

Ssu Processome

Ribosomes are the universally conserved ribonucleoprotein complexes that synthesize proteins. The two subunits of the eukaryotic ribosome are produced through a quasi-independent assembly-line-like pathway involving the hierarchical actions of numerous trans-acting biogenesis factors and the incorporation of ribosomal proteins. The factors work together to shape the nascent subunits through a series of intermediate states into their functional architectures. The earliest intermediate of the small subunit (SSU or 40S) is the SSU Processome which is subsequently transformed into the pre-40S intermediate. This transformation is, in part, facilitated by the binding of the methyltransferase Bud23. How Bud23 is released from the resultant pre-40S is not known. The ribosomal proteins Rps0, Rps2, and Rps21, termed the Rps0-cluster proteins, and several biogenesis factors are known to bind the pre-40S around the time that Bud23 is released, suggesting that one or more of these factors induce Bud23 release. Here, we systematically examined the requirement of these factors for the release of Bud23 from pre-40S particles. We found that the Rps0-cluster proteins are needed but not sufficient for Bud23 release. The atypical kinase/ATPase Rio2 shares a binding site with Bud23 and is thought to be recruited to pre-40S after the Rps0-cluster proteins. Depletion of Rio2 prevented the release of Bud23 from the pre-40S. More importantly, the addition of recombinant Rio2 to pre-40S particles affinity-purified from Rio2-depleted cells was sufficient for Bud23 release in vitro. The ability of Rio2 to displace Bud23 was independent of nucleotide hydrolysis. We propose a novel role for Rio2 in which its binding to the pre-40S actively displaces Bud23 from the pre-40S, and we suggest a model in which the binding of the Rps0-cluster proteins and Rio2 promote the release of Bud23.

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Requirements for the nuclear export of the small ribosomal subunit

Journal of Cell Science ◽

10.1242/jcs.115.14.2985 ◽

2002 ◽

Vol 115 (14) ◽

pp. 2985-2995 ◽

Cited By ~ 4

Author(s):

Terence I. Moy ◽

Pamela A. Silver

Keyword(s):

Nuclear Export ◽

Ribosome Biogenesis ◽

Large Scale ◽

Ribosomal Subunit ◽

Small Subunit ◽

Temperature Sensitive ◽

Yeast Saccharomyces Cerevisiae ◽

Small Ribosomal Subunit ◽

Factors Affecting ◽

The Stability

Eukaryotic ribosome biogenesis requires multiple steps of nuclear transport because ribosomes are assembled in the nucleus while protein synthesis occurs in the cytoplasm. Using an in situ RNA localization assay in the yeast Saccharomyces cerevisiae, we determined that efficient nuclear export of the small ribosomal subunit requires Yrb2, a factor involved in Crm1-mediated export. Furthermore, in cells lacking YRB2, the stability and abundance of the small ribosomal subunit is decreased in comparison with the large ribosomal subunit. To identify additional factors affecting small subunit export, we performed a large-scale screen of temperature-sensitive mutants. We isolated new alleles of several nucleoporins and Ran-GTPase regulators. Together with further analysis of existing mutants,we show that nucleoporins previously shown to be defective in ribosomal assembly are also defective in export of the small ribosomal subunit.

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