scholarly journals Antiviral RISC mainly targets viral mRNA but not genomic RNA of tospovirus

2021 ◽  
Vol 17 (7) ◽  
pp. e1009757
Author(s):  
Hao Hong ◽  
Chunli Wang ◽  
Ying Huang ◽  
Min Xu ◽  
Jiaoling Yan ◽  
...  
Keyword(s):  
Tobacco Rattle Virus ◽  
Plant Viruses ◽  
Genomic Rna ◽  
In Planta ◽  
Viral Mrnas ◽  
Genomic Rnas ◽  
N Protein ◽  
Viral Nucleocapsid

Antiviral RNA silencing/interference (RNAi) of negative-strand (-) RNA plant viruses (NSVs) has been studied less than for single-stranded, positive-sense (+)RNA plant viruses. From the latter, genomic and subgenomic mRNA molecules are targeted by RNAi. However, genomic RNA strands from plant NSVs are generally wrapped tightly within viral nucleocapsid (N) protein to form ribonucleoproteins (RNPs), the core unit for viral replication, transcription and movement. In this study, the targeting of the NSV tospoviral genomic RNA and mRNA molecules by antiviral RNA-induced silencing complexes (RISC) was investigated, in vitro and in planta. RISC fractions isolated from tospovirus-infected N. benthamiana plants specifically cleaved naked, purified tospoviral genomic RNAs in vitro, but not genomic RNAs complexed with viral N protein. In planta RISC complexes, activated by a tobacco rattle virus (TRV) carrying tospovirus NSs or Gn gene fragments, mainly targeted the corresponding viral mRNAs and hardly genomic (viral and viral-complementary strands) RNA assembled into RNPs. In contrast, for the (+)ssRNA cucumber mosaic virus (CMV), RISC complexes, activated by TRV carrying CMV 2a or 2b gene fragments, targeted CMV genomic RNA. Altogether, the results indicated that antiviral RNAi primarily targets tospoviral mRNAs whilst their genomic RNA is well protected in RNPs against RISC-mediated cleavage. Considering the important role of RNPs in the replication cycle of all NSVs, the findings made in this study are likely applicable to all viruses belonging to this group.

scholarly journals In vitro trackable assembly of RNA-specific nucleocapsids of the respiratory syncytial virus

2019 ◽  
Vol 295 (3) ◽  
pp. 883-895 ◽  
Author(s):  
Yunrong Gao ◽  
Dongdong Cao ◽  
Hyunjun Max Ahn ◽  
Anshuman Swain ◽  
Shaylan Hill ◽  
...  
Keyword(s):  
Respiratory Syncytial Virus ◽  
Rna Synthesis ◽  
Genomic Rnas ◽  
Viral Rnas ◽  
N Protein ◽  
Syncytial Virus ◽  
Nucleoprotein N ◽  
Negative Sense ◽  
Transcription And Replication

The templates for transcription and replication by respiratory syncytial virus (RSV) polymerase are helical nucleocapsids (NCs), formed by viral RNAs that are encapsidated by the nucleoprotein (N). Proper NC assembly is vital for RSV polymerase to engage the RNA template for RNA synthesis. Previous studies of NCs or nucleocapsid-like particles (NCLPs) from RSV and other nonsegmented negative-sense RNA viruses have provided insights into the overall NC architecture. However, in these studies, the RNAs were either random cellular RNAs or average viral genomic RNAs. An in-depth mechanistic understanding of NCs has been hampered by lack of an in vitro assay that can track NC or NCLP assembly. Here we established a protocol to obtain RNA-free N protein (N0) and successfully demonstrated the utility of a new assay for tracking assembly of N with RNA oligonucleotides into NCLPs. We discovered that the efficiency of the NCLP (N–RNA) assembly depends on the length and sequence of the RNA incorporated into NCLPs. This work provides a framework to generate purified N0 and incorporate it with RNA into NCLPs in a controllable manner. We anticipate that our assay for in vitro trackable assembly of RSV-specific nucleocapsids may enable in-depth mechanistic analyses of this process.

scholarly journals Significantly Improved Recovery of Recombinant Sonchus Yellow Net Rhabdovirus by Expressing the Negative-Strand Genomic RNA

Viruses ◽  
2020 ◽  
Vol 12 (12) ◽  
pp. 1459
Author(s):  
Xiaonan Ma ◽  
Zhenghe Li
Keyword(s):  
Matrix Protein ◽  
Plant Viruses ◽  
Biologically Active ◽  
Messenger Rnas ◽  
Genomic Rna ◽  
Genomic Rnas ◽  
Double Stranded Rna ◽  
Core Proteins ◽  
Negative Sense

Generation of recombinant negative-stranded RNA viruses (NSVs) from plasmids involves in vivo reconstitution of biologically active nucleocapsids and faces a unique antisense problem where the negative-sense viral genomic RNAs can hybridize to viral messenger RNAs. To overcome this problem, a positive-sense RNA approach has been devised through expression of viral antigenomic (ag)RNA and core proteins for assembly of antigenomic nucleocapsids. Although this detour strategy works for many NSVs, the process is still inefficient. Using Sonchus yellow net rhabdovirus (SYNV) as a model; here, we develop a negative-sense genomic RNA-based approach that increased rescue efficiency by two orders of magnitude compared to the conventional agRNA approach. The system relied on suppression of double-stranded RNA induced antiviral responses by co-expression of plant viruses-encoded RNA silencing suppressors or animal viruses-encoded double-stranded RNA antagonists. With the improved approach, we were able to recover a highly attenuated SYNV mutant with a deletion in the matrix protein gene which otherwise could not be rescued via the agRNA approach. Reverse genetics analyses of the generated mutant virus provided insights into SYNV virion assembly and morphogenesis. This approach may potentially be applicable to other NSVs of plants or animals.

The role of a fadA ortholog in the growth and development of Colletotrichum graminicola in vitro and in planta

2008 ◽  
Vol 45 (6) ◽  
pp. 973-983 ◽  
Author(s):  
C. Venard ◽  
S. Kulshrestha ◽  
J. Sweigard ◽  
E. Nuckles ◽  
L. Vaillancourt
Keyword(s):  
Growth And Development ◽  
Colletotrichum Graminicola ◽  
In Planta

scholarly journals The TUTase URT1 connects decapping activators and prevents the accumulation of excessively deadenylated mRNAs to avoid siRNA biogenesis

2020 ◽  
Author(s):  
Hélène Scheer ◽  
Caroline de Almeida ◽  
Emilie Ferrier ◽  
Quentin Simonnot ◽  
Laure Poirier ◽  
...  
Keyword(s):  
Rna Sequencing ◽  
Growth And Development ◽  
Molecular Mechanisms ◽  
Tail Length ◽  
Mrna Degradation ◽  
In Planta ◽  
Rna Helicases ◽  
Eukaryotic Mrnas

AbstractUridylation is a widespread modification destabilizing eukaryotic mRNAs. Yet, molecular mechanisms underlying TUTase-mediated mRNA degradation remain mostly unresolved. Here, we report that the Arabidopsis TUTase URT1 participates in a molecular network connecting several translational repressors/decapping activators. URT1 directly interacts with DECAPPING 5 (DCP5), the Arabidopsis ortholog of human LSM14 and yeast Scd6, and this interaction connects URT1 to additional decay factors like DDX6/Dhh1-like RNA helicases. Nanopore direct RNA sequencing reveals a global role of URT1 in shaping poly(A) tail length, notably by preventing the accumulation of excessively deadenylated mRNAs. Based on in vitro and in planta data, we propose a model that explains how URT1 could reduce the accumulation of oligo(A)-tailed mRNAs both by favoring their degradation and because 3’ terminal uridines intrinsically hinder deadenylation. Importantly, preventing the accumulation of excessively deadenylated mRNAs avoids the biogenesis of illegitimate siRNAs that silence endogenous mRNAs and perturb Arabidopsis growth and development.

scholarly journals Unique Secreted in Xylem Genes in Banana-Infecting Endophytic Fusarium Oxysporum

Proceedings ◽  
2020 ◽  
Vol 36 (1) ◽  
pp. 180
Author(s):  
Rebecca Lyons ◽  
Elizabeth Czislowski ◽  
Isabel Zeil-Rolfe ◽  
Shubhdeep Kaur ◽  
Zhendong Liu ◽  
...  
Keyword(s):  
Fusarium Oxysporum ◽  
Effector Proteins ◽  
In Planta ◽  
Banana Industry ◽  
Fusarium Wilt Of Banana ◽  
Race 4 ◽  
Fusarium Oxysporum Species Complex ◽  
Six Genes

Members of the Fusarium oxysporum species complex include pathogenic and non-pathogenic isolates and infect a broad range of plant species. F. oxysporum f. sp. cubense (Foc) causes the destructive Fusarium wilt of banana, and the recently emerged Foc tropical race 4 strain threatens the global banana industry. Secreted in xylem (SIX) genes encode for F. oxysporum effector proteins that are associated with virulence in pathogenic F. oxysporum, however they have rarely been reported from non-pathogenic F. oxysporum isolates. Our recent survey of asymptomatic banana plants grown in Foc-infested fields in Queensland and northern NSW revealed that diverse Fusarium spp, including F. oxysporum, reside in the plant roots and pseudostem without causing obvious damage to the plant. Intriguingly, we amplified SIX genes from several of the putative endophytic F. oxysporum isolates identified in the survey and found that they differ in their profile to known Foc SIX genes. To study the role of the endophytic F. oxysporum isolates in planta and the biological function of their SIX genes in more detail, we will re-inoculate cultivated and wild diploid banana lines with the endophytic F. oxysporum strains under glasshouse conditions to assess if they are non-pathogenic on banana. Secondly, we will determine whether the endophytic F. oxysporum SIX genes are expressed in planta and/or in vitro and look at the transcriptome changes occurring in the host following infection. Finally, endophytic F. oxysporum strains transformed with GFP will be used to investigate the extent of fungal colonisation in the plant.

scholarly journals SARS-CoV-2 nucleocapsid protein forms condensates with viral genomic RNA

PLoS Biology ◽  
2021 ◽  
Vol 19 (10) ◽  
pp. e3001425
Author(s):  
Amanda Jack ◽  
Luke S. Ferro ◽  
Michael J. Trnka ◽  
Eddie Wehri ◽  
Amrut Nadgir ◽  
...  
Keyword(s):  
Small Molecules ◽  
Mammalian Cells ◽  
Underlying Mechanism ◽  
Host Cells ◽  
Genomic Rna ◽  
N Protein ◽  
Infected Cells ◽  
Rna Genome ◽  
Viral Genomic Rna

The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection causes Coronavirus Disease 2019 (COVID-19), a pandemic that seriously threatens global health. SARS-CoV-2 propagates by packaging its RNA genome into membrane enclosures in host cells. The packaging of the viral genome into the nascent virion is mediated by the nucleocapsid (N) protein, but the underlying mechanism remains unclear. Here, we show that the N protein forms biomolecular condensates with viral genomic RNA both in vitro and in mammalian cells. While the N protein forms spherical assemblies with homopolymeric RNA substrates that do not form base pairing interactions, it forms asymmetric condensates with viral RNA strands. Cross-linking mass spectrometry (CLMS) identified a region that forms interactions between N proteins in condensates, and truncation of this region disrupts phase separation. We also identified small molecules that alter the formation of N protein condensates and inhibit the proliferation of SARS-CoV-2 in infected cells. These results suggest that the N protein may utilize biomolecular condensation to package the SARS-CoV-2 RNA genome into a viral particle.

scholarly journals Nucleocapsid Protein Recruitment to Replication-Transcription Complexes Plays a Crucial Role in Coronaviral Life Cycle

2019 ◽  
Vol 94 (4) ◽  
Author(s):  
Yingying Cong ◽  
Mustafa Ulasli ◽  
Hein Schepers ◽  
Mario Mauthe ◽  
Philip V’kovski ◽  
...  
Keyword(s):  
Life Cycle ◽  
Rna Synthesis ◽  
Genomic Rna ◽  
Protein Variant ◽  
N Protein ◽  
Transcription Complexes ◽  
Protein Recruitment ◽  
N Proteins

ABSTRACT Coronavirus (CoV) nucleocapsid (N) proteins are key for incorporating genomic RNA into progeny viral particles. In infected cells, N proteins are present at the replication-transcription complexes (RTCs), the sites of CoV RNA synthesis. It has been shown that N proteins are important for viral replication and that the one of mouse hepatitis virus (MHV), a commonly used model CoV, interacts with nonstructural protein 3 (nsp3), a component of the RTCs. These two aspects of the CoV life cycle, however, have not been linked. We found that the MHV N protein binds exclusively to nsp3 and not other RTC components by using a systematic yeast two-hybrid approach, and we identified two distinct regions in the N protein that redundantly mediate this interaction. A selective N protein variant carrying point mutations in these two regions fails to bind nsp3 in vitro, resulting in inhibition of its recruitment to RTCs in vivo. Furthermore, in contrast to the wild-type N protein, this N protein variant impairs the stimulation of genomic RNA and viral mRNA transcription in vivo and in vitro, which in turn leads to impairment of MHV replication and progeny production. Altogether, our results show that N protein recruitment to RTCs, via binding to nsp3, is an essential step in the CoV life cycle because it is critical for optimal viral RNA synthesis. IMPORTANCE CoVs have long been regarded as relatively harmless pathogens for humans. Severe respiratory tract infection outbreaks caused by severe acute respiratory syndrome CoV and Middle East respiratory syndrome CoV, however, have caused high pathogenicity and mortality rates in humans. These outbreaks highlighted the relevance of being able to control CoV infections. We used a model CoV, MHV, to investigate the importance of the recruitment of N protein, a central component of CoV virions, to intracellular platforms where CoVs replicate, transcribe, and translate their genomes. By identifying the principal binding partner at these intracellular platforms and generating a specific mutant, we found that N protein recruitment to these locations is crucial for promoting viral RNA synthesis. Moreover, blocking this recruitment strongly inhibits viral infection. Thus, our results explain an important aspect of the CoV life cycle and reveal an interaction of viral proteins that could be targeted in antiviral therapies.

scholarly journals Cooperation, Competition, and Specialized Metabolism in a Simplified Root Nodule Microbiome

mBio ◽  
2020 ◽  
Vol 11 (4) ◽  
Author(s):  
Bridget L. Hansen ◽  
Rita de Cassia Pessotti ◽  
Monika S. Fischer ◽  
Alyssa Collins ◽  
Laila El-Hifnawi ◽  
...  
Keyword(s):  
Potential Role ◽  
Root Nodule ◽  
Root Nodules ◽  
Imaging Mass Spectrometry ◽  
In Planta ◽  
Content Type ◽  
Specialized Metabolites ◽  
Brevibacillus Brevis

ABSTRACT Microbiomes associated with various plant structures often contain members with the potential to make specialized metabolites, e.g., molecules with antibacterial, antifungal, or siderophore activities. However, when and where microbes associated with plants produce specialized metabolites, and the potential role of these molecules in mediating intramicrobiome interactions, is not well understood. Root nodules of legume plants are organs devoted to hosting symbiotic bacteria that fix atmospheric nitrogen and have recently been shown to harbor a relatively simple accessory microbiome containing members with the ability to produce specialized metabolites in vitro. On the basis of these observations, we sought to develop a model nodule microbiome system for evaluating specialized microbial metabolism in planta. Starting with an inoculum derived from field-grown Medicago sativa nodules, serial passaging through gnotobiotic nodules yielded a simplified accessory community composed of four members: Brevibacillus brevis, Paenibacillus sp., Pantoea agglomerans, and Pseudomonas sp. Some members of this community exhibited clear cooperation in planta, while others were antagonistic and capable of disrupting cooperation between other partners. Using matrix-assisted laser desorption ionization–imaging mass spectrometry, we found that metabolites associated with individual taxa had unique distributions, indicating that some members of the nodule community were spatially segregated. Finally, we identified two families of molecules produced by B. brevis in planta as the antibacterial tyrocidines and a novel set of gramicidin-type molecules, which we term the britacidins. Collectively, these results indicate that in addition to nitrogen fixation, legume root nodules are likely also sites of active antimicrobial production.

scholarly journals Hantavirus N Protein Exhibits Genus-Specific Recognition of the Viral RNA Panhandle

2006 ◽  
Vol 80 (22) ◽  
pp. 11283-11292 ◽  
Author(s):  
M. A. Mir ◽  
B. Brown ◽  
B. Hjelle ◽  
W. A. Duran ◽  
A. T. Panganiban
Keyword(s):  
Viral Rna ◽  
Genomic Rna ◽  
N Protein ◽  
High Affinity ◽  
The Andes ◽  
The Family ◽  
Viral Genomic Rna ◽  
Negative Sense

ABSTRACT A key genomic characteristic that helps define Hantavirus as a genus of the family Bunyaviridae is the presence of distinctive terminal complementary nucleotides that promote the folding of the viral genomic segments into “panhandle” hairpin structures. The hantavirus nucleocapsid protein (N protein), which is encoded by the smallest of the three negative-sense genomic RNA segments, undergoes in vivo and in vitro trimerization. Trimeric hantavirus N protein specifically recognizes the panhandle structure formed by complementary base sequence of 5′ and 3′ ends of viral genomic RNA. N protein trimers from the Andes, Puumala, Prospect Hill, Seoul, and Sin Nombre viruses recognize their individual homologous panhandles as well as other hantavirus panhandles with high affinity. In contrast, these hantavirus N proteins bind with markedly reduced affinity to the panhandles from the genera Bunyavirus, Tospovirus, and Phlebovirus or Nairovirus. Interactions between most hantavirus N and heterologous hantavirus viral RNA panhandles are mediated by the nine terminal conserved nucleotides of the panhandle, whereas Sin Nombre virus N requires the first 23 nucleotides for high-affinity binding. Trimeric hantavirus N complexes undergo a prominent conformational change while interacting with panhandles from members of the genus Hantavirus but not while interacting with panhandles from viruses of other genera of the family Bunyaviridae. These data indicate that high-affinity interactions between trimeric N and hantavirus panhandles are conserved within the genus Hantavirus.

scholarly journals The Role of Male Reproductive Organs in the Transmission of African Swine Fever—Implications for Transmission

Viruses ◽  
2021 ◽  
Vol 14 (1) ◽  
pp. 31
Author(s):  
Hanna Roszyk ◽  
Kati Franzke ◽  
Angele Breithaupt ◽  
Paul Deutschmann ◽  
Jutta Pikalo ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Mononuclear Cells ◽  
African Swine Fever ◽  
Cell Types ◽  
Reproductive Organs ◽  
Viral Mrnas ◽  
Boar Semen ◽  
Male Reproductive Organs

African swine fever (ASF) has evolved from an exotic animal disease to a threat to global pig production. An important avenue for the wide-spread transmission of animal diseases is their dissemination through boar semen used for artificial insemination. In this context, we investigated the role of male reproductive organs in the transmission of ASF. Mature domestic boars and adolescent wild boars, inoculated with different ASF virus strains, were investigated by means of virological and pathological methods. Additionally, electron microscopy was employed to investigate in vitro inoculated sperm. The viral genome, antigens and the infectious virus could be found in all gonadal tissues and accessory sex glands. The viral antigen and viral mRNAs were mainly found in mononuclear cells of the respective tissues. However, some other cell types, including Leydig, endothelial and stromal cells, were also found positive. Using RNAScope, p72 mRNA could be found in scattered halo cells of the epididymal duct epithelium, which could point to the disruption of the barrier. No direct infection of spermatozoa was observed by immunohistochemistry, or electron microscopy. Taken together, our results strengthen the assumption that ASFV can be transmitted via boar semen. Future studies are needed to explore the excretion dynamics and transmission efficiency.

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