scholarly journals Direct RNA nanopore sequencing of full-length coronavirus genomes provides novel insights into structural variants and enables modification analysis

2018 ◽  
Author(s):  
Adrian Viehweger ◽  
Sebastian Krautwurst ◽  
Kevin Lamkiewicz ◽  
Ramakanth Madhugiri ◽  
John Ziebuhr ◽  
...  
Keyword(s):  
Rna Sequencing ◽  
Rna Virus ◽  
Consensus Sequence ◽  
Full Length ◽  
Viral Rna ◽  
Genome Replication ◽  
Nanopore Sequencing ◽  
Human Coronavirus ◽  
Viral Rnas ◽  
Virus Genomes

Sequence analyses of RNA virus genomes remain challenging due to the exceptional genetic plasticity of these viruses. Because of high mutation and recombination rates, genome replication by viral RNA-dependent RNA polymerases leads to populations of closely related viruses, so-called 'quasispecies'. Standard (short-read) sequencing technologies are ill-suited to reconstruct large numbers of full-length haplotypes of (i) RNA virus genomes and (ii) subgenome-length (sg) RNAs comprised of noncontiguous genome regions. Here, we used a full-length, direct RNA sequencing (DRS) approach based on nanopores to characterize viral RNAs produced in cells infected with a human coronavirus. Using DRS, we were able to map the longest (~26 kb) contiguous read to the viral reference genome. By combining Illumina and nanopore sequencing, we reconstructed a highly accurate consensus sequence of the human coronavirus (HCoV) 229E genome (27.3 kb). Furthermore, using long reads that did not require an assembly step, we were able to identify, in infected cells, diverse and novel HCoV-229E sg RNAs that remain to be characterized. Also, the DRS approach, which circumvents reverse transcription and amplification of RNA, allowed us to detect methylation sites in viral RNAs. Our work paves the way for haplotype-based analyses of viral quasispecies by demonstrating the feasibility of intra-sample haplotype separation. Even though several technical challenges remain to be addressed to exploit the potential of the nanopore technology fully, our work illustrates that direct RNA sequencing may significantly advance genomic studies of complex virus populations, including predictions on long-range interactions in individual full-length viral RNA haplotypes.

scholarly journals Suppression of Viral RNA Recombination by a Host Exoribonuclease

2006 ◽  
Vol 80 (6) ◽  
pp. 2631-2640 ◽  
Author(s):  
Chi-Ping Cheng ◽  
Elena Serviene ◽  
Peter D. Nagy
Keyword(s):  
Rna Virus ◽  
Viral Rna ◽  
Rna Turnover ◽  
Rna Recombination ◽  
Viral Rnas ◽  
Yeast Strains ◽  
Genome Wide ◽  
Host Genes

ABSTRACT RNA viruses of humans, animals, and plants evolve rapidly due to mutations and RNA recombination. A previous genome-wide screen in Saccharomyces cerevisiae, a model host, identified five host genes, including XRN1, encoding a 5′-3′ exoribonuclease, whose absence led to an ∼10- to 50-fold enhancement of RNA recombination in Tomato bushy stunt virus (E. Serviene, N. Shapka, C. P. Cheng, T. Panavas, B. Phuangrat, J. Baker, and P. D. Nagy, Proc. Natl. Acad. Sci. USA 102:10545-10550, 2005). In this study, we found abundant 5′-truncated viral RNAs in xrn1Δ mutant strains but not in the parental yeast strains, suggesting that these RNAs might serve as recombination substrates promoting RNA recombination in xrn1Δ mutant yeast. This model is supported by data showing that an enhanced level of viral recombinant accumulation occurred when two different 5′-truncated viral RNAs were expressed in the parental and xrn1Δ mutant yeast strains or electroporated into plant protoplasts. Moreover, we demonstrate that purified Xrn1p can degrade the 5′-truncated viral RNAs in vitro. Based on these findings, we propose that Xrn1p can suppress viral RNA recombination by rapidly removing the 5′-truncated RNAs, the substrates of recombination, and thus reducing the chance for recombination to occur in the parental yeast strain. In addition, we show that the 5′-truncated viral RNAs are generated by host endoribonucleases. Accordingly, overexpression of the Ngl2p endoribonuclease led to an increased accumulation of cleaved viral RNAs in vivo and in vitro. Altogether, this paper establishes that host ribonucleases and host-mediated viral RNA turnover play major roles in RNA virus recombination and evolution.

scholarly journals Uncoupled Expression of p33 and p92 Permits Amplification of Tomato Bushy Stunt Virus RNAs

1998 ◽  
Vol 72 (7) ◽  
pp. 5845-5851 ◽  
Author(s):  
Sara K. Oster ◽  
Baodong Wu ◽  
K. Andrew White
Keyword(s):  
Viral Genome ◽  
Stop Codon ◽  
Rna Virus ◽  
Rna Replication ◽  
Tomato Bushy Stunt Virus ◽  
Viral Rna ◽  
Rna Amplification ◽  
Viral Rnas ◽  
Reading Frame ◽  
Translational Enhancer

ABSTRACT Tomato bushy stunt virus (TBSV) is a plus-sense RNA virus which encodes a 33-kDa protein in its 5′-most open reading frame (ORF). Readthrough of the amber stop codon of the p33 ORF results in the production of a 92-kDa fusion protein. Both of these products are expressed directly from the viral genome and are suspected to be involved in viral RNA replication. We have investigated further the roles of these proteins in the amplification of viral RNAs by using a complementation system in which p33 and p92 are expressed from different viral RNAs. Our results indicate that (i) both of these proteins are necessary for viral RNA amplification; (ii) translation of these proteins can be uncoupled while maintaining amplification of viral RNAs; (iii) if compatibility requirements exist between p33 and p92, they are not exceptionally strict; and (iv) the C-terminal ∼6% of p33 is necessary for its functional activity. Interestingly, no complementation was observed when a p33-encoding replicon containing a deletion of a 3′-located segment, region 3.5, was tested. However, when 5′-capped transcripts of the same replicon were analyzed, complementation allowing for RNA amplification was observed. This ability to compensate functionally for the absence of region 3.5 by the addition of a 5′ cap suggests that this RNA segment may act as a translational enhancer for the expression of virally encoded products.

scholarly journals Harnessing host ROS-generating machinery for the robust genome replication of a plant RNA virus

2017 ◽  
Vol 114 (7) ◽  
pp. E1282-E1290 ◽  
Author(s):  
Kiwamu Hyodo ◽  
Kenji Hashimoto ◽  
Kazuyuki Kuchitsu ◽  
Nobuhiro Suzuki ◽  
Tetsuro Okuno
Keyword(s):  
Mosaic Virus ◽  
Rna Virus ◽  
Plant Stress ◽  
Rna Replication ◽  
Plant Viruses ◽  
Brome Mosaic Virus ◽  
Viral Rna ◽  
Genome Replication ◽  
Dependent Manner ◽  
Positive Strand Rna

As sessile organisms, plants have to accommodate to rapid changes in their surrounding environment. Reactive oxygen species (ROS) act as signaling molecules to transduce biotic and abiotic stimuli into plant stress adaptations. It is established that a respiratory burst oxidase homolog B of Nicotiana benthamiana (NbRBOHB) produces ROS in response to microbe-associated molecular patterns to inhibit pathogen infection. Plant viruses are also known as causative agents of ROS induction in infected plants; however, the function of ROS in plant–virus interactions remains obscure. Here, we show that the replication of red clover necrotic mosaic virus (RCNMV), a plant positive-strand RNA [(+)RNA] virus, requires NbRBOHB-mediated ROS production. The RCNMV replication protein p27 plays a pivotal role in this process, redirecting the subcellular localization of NbRBOHB and a subgroup II calcium-dependent protein kinase of N. benthamiana (NbCDPKiso2) from the plasma membrane to the p27-containing intracellular aggregate structures. p27 also induces an intracellular ROS burst in an RBOH-dependent manner. NbCDPKiso2 was shown to be an activator of the p27-triggered ROS accumulations and to be required for RCNMV replication. Importantly, this RBOH-derived ROS is essential for robust viral RNA replication. The need for RBOH-derived ROS was demonstrated for the replication of another (+)RNA virus, brome mosaic virus, suggesting that this characteristic is true for plant (+)RNA viruses. Collectively, our findings revealed a hitherto unknown viral strategy whereby the host ROS-generating machinery is diverted for robust viral RNA replication.

scholarly journals Temperature controlled high-throughput magnetic tweezers show striking difference in activation energies of replicating viral RNA-dependent RNA polymerases

2020 ◽  
Author(s):  
M. Seifert ◽  
P. van Nies ◽  
F.S. Papini ◽  
J.J. Arnold ◽  
M.M. Poranen ◽  
...  
Keyword(s):  
High Throughput ◽  
Single Molecule ◽  
Rna Virus ◽  
Magnetic Tweezers ◽  
Activation Energies ◽  
Viral Rna ◽  
Controlled Study ◽  
Striking Difference ◽  
Genome Replication ◽  
Temperature Controlled

AbstractRNA virus survival depends on efficient viral genome replication, which is performed by the viral RNA dependent RNA polymerase (RdRp). The recent development of high throughput magnetic tweezers has enabled the simultaneous observation of dozens of viral RdRp elongation traces on kilobases long templates, and this has shown that RdRp nucleotide addition kinetics is stochastically interrupted by rare pauses of 1-1000 s duration, of which the short-lived ones (1-10 s) are the temporal signature of a low fidelity catalytic pathway. We present a simple and precise temperature controlled system for magnetic tweezers to characterize the replication kinetics temperature dependence between 25°C and 45°C of RdRps from three RNA viruses, i.e. the double-stranded RNA bacteriophage Φ6, and the positive-sense single-stranded RNA poliovirus (PV) and human rhinovirus C (HRV-C). We found that Φ6 RdRp is largely temperature insensitive, while PV and HRV-C RdRps replication kinetics are activated by temperature. Furthermore, the activation energies we measured for PV RdRp catalytic state corroborate previous estimations from ensemble pre-steady state kinetic studies, further confirming the catalytic origin of the short pauses and their link to temperature independent RdRp fidelity. This work will enable future temperature controlled study of biomolecular complex at the single molecule level.

scholarly journals KHNYN is essential for ZAP-mediated restriction of HIV-1 containing clustered CpG dinucleotides

2019 ◽  
Author(s):  
Mattia Ficarelli ◽  
Harry Wilson ◽  
Rui Pedro Galão ◽  
Stuart J D Neil ◽  
Chad M Swanson
Keyword(s):  
Infectious Virus ◽  
Rna Virus ◽  
Virus Production ◽  
Innate Immune ◽  
Viral Rna ◽  
Antiviral Protein ◽  
Cpg Dinucleotides ◽  
Endonuclease Domain ◽  
Virus Genomes ◽  
Hiv 1

AbstractCpG dinucleotides are suppressed in most vertebrate RNA viruses, including HIV-1, and introducing CpGs into RNA virus genomes inhibits their replication. The zinc-finger antiviral protein (ZAP) binds regions of viral RNA containing CpGs and targets them for degradation. ZAP does not have enzymatic activity and recruits other cellular proteins to inhibit viral replication. Here we show that KHNYN, a protein with no previously known function, interacts with ZAP. KHNYN overexpression selectively inhibits HIV-1 containing clustered CpG dinucleotides and this requires ZAP and its cofactor TRIM25. KHNYN requires both its KH-like domain and NYN endonuclease domain for antiviral activity. Crucially, depletion of KHNYN eliminated the deleterious effect of CpG dinucleotides on HIV-1 RNA abundance and infectious virus production indicating that KHNYN is required for this antiviral pathway. Overall, we have identified KHNYN as a novel ZAP cofactor that is essential for innate immune destruction of CpG containing viral RNA.

scholarly journals Full-length genome sequence of segmented RNA virus from ticks was obtained using small RNA sequencing data

BMC Genomics ◽  
2020 ◽  
Vol 21 (1) ◽  
Author(s):  
Xiaofeng Xu ◽  
Jinlong Bei ◽  
Yibo Xuan ◽  
Jiayuan Chen ◽  
Defu Chen ◽  
...  
Keyword(s):  
Rna Sequencing ◽  
Genome Sequence ◽  
Small Rna ◽  
Rna Viruses ◽  
Rna Virus ◽  
Full Length ◽  
Small Rna Sequencing ◽  
Sequencing Data ◽  
Geographical Regions ◽  
Full Length Genome

Abstract Background In 2014, a novel tick-borne virus of the Flaviviridae family was first reported in the Mogiana region of Brazil and named the Mogiana tick virus (MGTV). Thereafter, the Jingmen tick virus (JMTV), Kindia tick virus (KITV), and Guangxi tick virus (GXTV)—evolutionarily related to MGTV—were reported. Results In the present study, we used small RNA sequencing (sRNA-seq) to detect viruses in ticks and discovered a new MGTV strain in Amblyomma testudinarium ticks collected in China’s Yunnan Province in 2016. We obtained the full-length genome sequence of this MGTV strain Yunnan2016 (GenBank: MT080097, MT080098, MT080099 and MT080100) and recommended it for its inclusion in the NCBI RefSeq database for future studies on MGTV, JMTV, KITV and GXTV. Phylogenetic analysis showed that MGTV, JMTV, KITV and GXTV are monophyletic and belong to a MGTV group. Furthermore, this MGTV group of viruses may be phylogenetically related to geographical regions that were formerly part of the supercontinents Gondwana and Laurasia. Conclusions To the best of our knowledge, this is the first study in which 5′ and 3′ sRNAs were used to generate full-length genome sequences of, but not limited to, RNA viruses. We also demonstrated the feasibility of using the sRNA-seq based method for the detection of viruses in pooled two and even possible one small ticks. MGTV may preserve the characteristic of ancient RNA viruses, which can be used to study the origin and evolution of RNA viruses. In addition, MGTV can be used as novel species for studies in phylogeography.

scholarly journals Crystal Structure of the Measles Virus Nucleoprotein Core in Complex with an N-Terminal Region of Phosphoprotein

2015 ◽  
Vol 90 (6) ◽  
pp. 2849-2857 ◽  
Author(s):  
Sergey G. Guryanov ◽  
Lassi Liljeroos ◽  
Prasad Kasaragod ◽  
Tommi Kajander ◽  
Sarah J. Butcher
Keyword(s):  
Measles Virus ◽  
Matrix Protein ◽  
Hydrophobic Interactions ◽  
Rna Virus ◽  
Viral Rna ◽  
Genome Replication ◽  
Tail Domain ◽  
Human Pathogen ◽  
Nucleoprotein N ◽  
Insight Into

ABSTRACTThe enveloped negative-stranded RNA virus measles virus (MeV) is an important human pathogen. The nucleoprotein (N0) assembles with the viral RNA into helical ribonucleocapsids (NC) which are, in turn, coated by a helical layer of the matrix protein. The viral polymerase complex uses the NC as its template. The N0assembly onto the NC and the activity of the polymerase are regulated by the viral phosphoprotein (P). In this study, we pulled down an N01-408fragment lacking most of its C-terminal tail domain by several affinity-tagged, N-terminal P fragments to map the N0-binding region of P to the first 48 amino acids. We showed biochemically and using P mutants the importance of the hydrophobic interactions for the binding. We fused an N0binding peptide, P1-48, to the C terminus of an N021-408fragment lacking both the N-terminal peptide and the C-terminal tail of N protein to reconstitute and crystallize the N0-P complex. We solved the X-ray structure of the resulting N0-P chimeric protein at a resolution of 2.7 Å. The structure reveals the molecular details of the conserved N0-P interface and explains how P chaperones N0, preventing both self-assembly of N0and its binding to RNA. Finally, we propose a model for a preinitiation complex for RNA polymerization.IMPORTANCEMeasles virus is an important, highly contagious human pathogen. The nucleoprotein N binds only to viral genomic RNA and forms the helical ribonucleocapsid that serves as a template for viral replication. We address how N is regulated by another protein, the phosphoprotein (P), to prevent newly synthesized N from binding to cellular RNA. We describe the atomic model of an N-P complex and compare it to helical ribonucleocapsid. We thus provide insight into how P chaperones N and helps to start viral RNA synthesis. Our results provide a new insight into mechanisms of paramyxovirus replication. New data on the mechanisms of phosphoprotein chaperone action allows better understanding of virus genome replication and nucleocapsid assembly. We describe a conserved structural interface for the N-P interaction which could be a target for drug development to treat not only measles but also potentially other paramyxovirus diseases.

scholarly journals Ebolavirus polymerase uses an unconventional genome replication mechanism

2019 ◽  
Vol 116 (17) ◽  
pp. 8535-8543 ◽  
Author(s):  
Laure R. Deflubé ◽  
Tessa N. Cressey ◽  
Adam J. Hume ◽  
Judith Olejnik ◽  
Elaine Haddock ◽  
...  
Keyword(s):  
Rna Viruses ◽  
Rna Virus ◽  
Replication Initiation ◽  
Genome Replication ◽  
Terminal Nucleotide ◽  
Nucleotide Addition ◽  
Replication Strategy ◽  
Virus Genomes ◽  
High Degree

Most nonsegmented negative strand (NNS) RNA virus genomes have complementary 3′ and 5′ terminal nucleotides because the promoters at the 3′ ends of the genomes and antigenomes are almost identical to each other. However, according to published sequences, both ends of ebolavirus genomes show a high degree of variability, and the 3′ and 5′ terminal nucleotides are not complementary. If correct, this would distinguish the ebolaviruses from other NNS RNA viruses. Therefore, we investigated the terminal genomic and antigenomic nucleotides of three different ebolavirus species, Ebola (EBOV), Sudan, and Reston viruses. Whereas the 5′ ends of ebolavirus RNAs are highly conserved with the sequence ACAGG-5′, the 3′ termini are variable and are typically 3′-GCCUGU, ACCUGU, or CCUGU. A small fraction of analyzed RNAs had extended 3′ ends. The majority of 3′ terminal sequences are consistent with a mechanism of nucleotide addition by hairpin formation and back-priming. Using single-round replicating EBOV minigenomes, we investigated the effect of the 3′ terminal nucleotide on viral replication and found that the EBOV polymerase initiates replication opposite the 3′-CCUGU motif regardless of the identity of the 3′ terminal nucleotide(s) and of the position of this motif relative to the 3′ end. Deletion or mutation of the first residue of the 3′-CCUGU motif completely abolished replication initiation, suggesting a crucial role of this nucleotide in directing initiation. Together, our data show that ebolaviruses have evolved a unique replication strategy among NNS RNA viruses resulting in 3′ overhangs. This could be a mechanism to avoid antiviral recognition.

scholarly journals Temperature controlled high-throughput magnetic tweezers show striking difference in activation energies of replicating viral RNA-dependent RNA polymerases

2020 ◽  
Vol 48 (10) ◽  
pp. 5591-5602 ◽  
Author(s):  
Mona Seifert ◽  
Pauline van Nies ◽  
Flávia S Papini ◽  
Jamie J Arnold ◽  
Minna M Poranen ◽  
...  
Keyword(s):  
High Throughput ◽  
Single Molecule ◽  
Rna Virus ◽  
Magnetic Tweezers ◽  
Activation Energies ◽  
Viral Rna ◽  
Controlled Study ◽  
Striking Difference ◽  
Genome Replication ◽  
Temperature Controlled

Abstract RNA virus survival depends on efficient viral genome replication, which is performed by the viral RNA dependent RNA polymerase (RdRp). The recent development of high throughput magnetic tweezers has enabled the simultaneous observation of dozens of viral RdRp elongation traces on kilobases long templates, and this has shown that RdRp nucleotide addition kinetics is stochastically interrupted by rare pauses of 1–1000 s duration, of which the short-lived ones (1–10 s) are the temporal signature of a low fidelity catalytic pathway. We present a simple and precise temperature controlled system for magnetic tweezers to characterize the replication kinetics temperature dependence between 25°C and 45°C of RdRps from three RNA viruses, i.e. the double-stranded RNA bacteriophage Φ6, and the positive-sense single-stranded RNA poliovirus (PV) and human rhinovirus C (HRV-C). We found that Φ6 RdRp is largely temperature insensitive, while PV and HRV-C RdRps replication kinetics are activated by temperature. Furthermore, the activation energies we measured for PV RdRp catalytic state corroborate previous estimations from ensemble pre-steady state kinetic studies, further confirming the catalytic origin of the short pauses and their link to temperature independent RdRp fidelity. This work will enable future temperature controlled study of biomolecular complex at the single molecule level.

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