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Lin, Jinzhong, Anders Fuglsang, Anders Lynge Kjeldsen, Kaiyan Sun, Yuvaraj Bhoobalan-Chitty, and Xu Peng. "DNA targeting by subtype I-D CRISPR–Cas shows type I and type III features." Nucleic Acids Research 48, no. 18 (September 22, 2020): 10470–78. http://dx.doi.org/10.1093/nar/gkaa749.
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Abstract Prokaryotic CRISPR–Cas immune systems are classified into six types based on their effector complexes which cleave dsDNA specifically (types I, II and V), ssRNA exclusively (type VI) or both ssRNA via a ruler mechanism and ssDNA unspecifically (type III). To date, no specific cleavage of ssDNA target has been reported for CRISPR–Cas. Here, we demonstrate dual dsDNA and ssDNA cleavage activities of a subtype I-D system which carries a type III Cas10-like large subunit, Cas10d. In addition to a specific dsDNA cleavage activity dependent on the HD domain of Cas10d, the helicase Cas3′ and a compatible protospacer adjacent motif (PAM), the subtype I-D effector complex can cleave ssDNA that is complementary in sequence to the crRNA. Significantly, the ssDNA cleavage sites occur at 6-nt intervals and the cleavage is catalysed by the backbone subunit Csc2 (Cas7), similar to the periodic cleavage of ssRNA by the backbone subunit of type III effectors. The typical type I cleavage of dsDNA combined with the exceptional 6-nt spaced cleavage of ssDNA and the presence of a type III like large subunit provide strong evidence for the subtype I-D system being an evolutionary intermediate between type I and type III CRISPR–Cas systems.
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Roner, Michael R., and Bradley G. Steele. "Features of the mammalian orthoreovirus 3 Dearing l1 single-stranded RNA that direct packaging and serotype restriction." Journal of General Virology 88, no. 12 (December 1, 2007): 3401–12. http://dx.doi.org/10.1099/vir.0.83209-0.
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A series of recombinant mammalian orthoreoviruses (mammalian orthoreovirus 3 Dearing, MRV-3De) were generated that express an MRV-3De λ3–CAT fusion protein. Individual viruses contain L1CAT double-stranded (ds) RNAs that range in length from a minimum of 1020 bp to 4616 bp. The engineered dsRNAs were generated from in vitro-transcribed single-stranded (ss) RNAs and incorporated into infectious virus particles by using reverse genetics. In addition to defining the sequences required for these ssRNAs to be ‘identified’ as l1 ssRNAs, the individual nucleotides in these regions that ‘mark’ each ssRNA as originating from mammalian orthoreovirus 1 Lang (MRV-1La), mammalian orthoreovirus 2 D5/Jones (MRV-2Jo) or MRV-3De have been identified. A C at position 81 in the MRV-1La 5′ 129 nt sequence was able to be replaced with a U, as normally present in MRV-3De; this toggled the activity of the MRV-1La ssRNA to that of an MRV-3De 5′ l1. RNA secondary-structure predictions for the 5′ 129 nt of both the biologically active MRV-3De l1 ssRNA and the U81-MRV-3De-restored MRV-1La 5′ ssRNA predicted a common structure.
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Li, Jiacheng, Tong Luo, Yao He, Hui Liu, ZhiWei Deng, Jiaqi Bu, Xi Long, Shian Zhong, and Yanjing Yang. "Discovery of the Rnase activity of CRISPR–Cas12a and its distinguishing cleavage efficiency on various substrates." Chemical Communications 58, no. 15 (2022): 2540–43. http://dx.doi.org/10.1039/d1cc06295f.
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LbCas12a bound to ssDNA (a) or ssRNA (b) target exhibits different activities to different substrates. a. The order of cleavage speed: hairpin DNA > short ssDNA > hairpin RNA > linear RNA; b. The order of cleavage speed: hairpin DNA > hairpin RNA > short ssDNA. TS means targeted strand. Substrates are all single strands with different secondary structures (hairpin and linear).
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Kolundžija, Sandra, Dong-Qiang Cheng, and Federico M. Lauro. "RNA Viruses in Aquatic Ecosystems through the Lens of Ecological Genomics and Transcriptomics." Viruses 14, no. 4 (March 28, 2022): 702. http://dx.doi.org/10.3390/v14040702.
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Massive amounts of data from nucleic acid sequencing have changed our perspective about diversity and dynamics of marine viral communities. Here, we summarize recent metatranscriptomic and metaviromic studies targeting predominantly RNA viral communities. The analysis of RNA viromes reaffirms the abundance of lytic (+) ssRNA viruses of the order Picornavirales, but also reveals other (+) ssRNA viruses, including RNA bacteriophages, as important constituents of extracellular RNA viral communities. Sequencing of dsRNA suggests unknown diversity of dsRNA viruses. Environmental metatranscriptomes capture the dynamics of ssDNA, dsDNA, ssRNA, and dsRNA viruses simultaneously, unravelling the full complexity of viral dynamics in the marine environment. RNA viruses are prevalent in large size fractions of environmental metatranscriptomes, actively infect marine unicellular eukaryotes larger than 3 µm, and can outnumber bacteriophages during phytoplankton blooms. DNA and RNA viruses change abundance on hourly timescales, implying viral control on a daily temporal basis. Metatranscriptomes of cultured protists host a diverse community of ssRNA and dsRNA viruses, often with multipartite genomes and possibly persistent intracellular lifestyles. We posit that RNA viral communities might be more diverse and complex than formerly anticipated and that the influence they exert on community composition and global carbon flows in aquatic ecosystems may be underestimated.
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Vidhyasagar, Venkatasubramanian, Yujiong He, Manhong Guo, Tanu Talwar, Ravi Shankar Singh, Manisha Yadav, George Katselis, Franco J. Vizeacoumar, Kiven E. Lukong, and Yuliang Wu. "Biochemical characterization of INTS3 and C9ORF80, two subunits of hNABP1/2 heterotrimeric complex in nucleic acid binding." Biochemical Journal 475, no. 1 (January 2, 2018): 45–60. http://dx.doi.org/10.1042/bcj20170351.
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Human nucleic acid-binding protein 1 and 2 (hNABP1 and hNABP2, also known as hSSB2 and hSSB1 respectively) form two separate and independent complexes with two identical proteins, integrator complex subunit 3 (INTS3) and C9ORF80. We and other groups have demonstrated that hNABP1 and 2 are single-stranded (ss) DNA- and RNA-binding proteins, and function in DNA repair; however, the function of INTS3 and C9OFR80 remains elusive. In the present study, we purified recombinant proteins INTS3 and C9ORF80 to near homogeneity. Both proteins exist as a monomer in solution; however, C9ORF80 exhibits anomalous behavior on SDS–PAGE and gel filtration because of 48% random coil present in the protein. Using electrophoretic mobility shift assay (EMSA), INTS3 displays higher affinity toward ssRNA than ssDNA, and C9ORF80 binds ssDNA but not ssRNA. Neither of them binds dsDNA, dsRNA, or RNA : DNA hybrid. INTS3 requires minimum of 30 nucleotides, whereas C9OFR80 requires 20 nucleotides for its binding, which increased with the increasing length of ssDNA. Interestingly, our GST pulldown results suggest that the N-terminus of INTS3 is involved in protein–protein interaction, while EMSA implies that the C-terminus is required for nucleic acid binding. Furthermore, we purified the INTS3–hNABP1/2–C9ORF80 heterotrimeric complex. It exhibits weaker binding compared with the individual hNABP1/2; interestingly, the hNABP1 complex prefers ssDNA, whereas hNABP2 complex prefers ssRNA. Using reconstituted heterotrimeric complex from individual proteins, EMSA demonstrates that INTS3, but not C9ORF80, affects the nucleic acid-binding ability of hNABP1 and hNABP2, indicating that INTS3 might regulate hNABP1/2's biological function, while the role of C9ORF80 remains unknown.
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Karlowicz, Anna, and Michal R. Szymanski. "Exog Displays 5′-Exonuclease Activity on Both ssDNA and ssRNA." Biophysical Journal 116, no. 3 (February 2019): 76a. http://dx.doi.org/10.1016/j.bpj.2018.11.452.
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Tourís-Otero, Fernando, José Martínez-Costas, Vikram N. Vakharia, and Javier Benavente. "Characterization of the nucleic acid-binding activity of the avian reovirus non-structural protein σNS." Journal of General Virology 86, no. 4 (April 1, 2005): 1159–69. http://dx.doi.org/10.1099/vir.0.80491-0.
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The avian reovirus non-structural protein σNS has previously been shown to bind single-stranded (ss) RNA in vitro in a sequence-independent manner. The results of the present study further reveal that σNS binds poly(A), poly(U) and ssDNA, but not poly(C), poly(G) or duplex nucleic acids, suggesting that σNS has some nucleotide-sequence specificity for ssRNA binding. The current findings also show that σNS is present in large ribonucleoprotein complexes in the cytoplasm of avian reovirus-infected cells, indicating that it exists in intimate association with ssRNAs in vivo. Removal of RNA from the complexes generates a σNS protein form that sediments between 4·5 and 7 S, suggesting that RNA-free σNS associates into small oligomers. Expression and purification of recombinant σNS in insect cells allowed us to generate specific antibodies and to perform a variety of assays. The results of these assays revealed that: (i) RNA-free σNS exists as homodimers and homotrimers; (ii) the minimum RNA size for σNS binding is between 10 and 20 nt; (iii) σNS does not have a preference for viral mRNA sequences; and (iv) its RNA-binding activity is conformation-dependent. Baculovirus expression of point and deletion σNS mutants in insect cells showed that the five conserved basic amino acids that are important for RNA binding and ribonucleoprotein-complex formation are dispersed throughout the entire σNS sequence, suggesting that this protein binds ssRNA through conformational domains. Finally, the properties of the avian reovirus protein σNS are compared with those of its mammalian reovirus counterpart.
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Rūmnieks, Jānis, Ilva Liekniņa, Gints Kalniņš, Mihails Šišovs, Ināra Akopjana, Jānis Bogans, and Kaspars Tārs. "Three-dimensional structure of 22 uncultured ssRNA bacteriophages: Flexibility of the coat protein fold and variations in particle shapes." Science Advances 6, no. 36 (September 2020): eabc0023. http://dx.doi.org/10.1126/sciadv.abc0023.
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The single-stranded RNA (ssRNA) bacteriophages are among the simplest known viruses with small genomes and exceptionally high mutation rates. The number of ssRNA phage isolates has remained very low, but recent metagenomic studies have uncovered an immense variety of distinct uncultured ssRNA phages. The coat proteins (CPs) in these genomes are particularly diverse, with notable variation in length and often no recognizable similarity to previously known viruses. We recombinantly expressed metagenome-derived ssRNA phage CPs to produce virus-like particles and determined the three-dimensional structure of 22 previously uncharacterized ssRNA phage capsids covering nine distinct CP types. The structures revealed substantial deviations from the previously known ssRNA phage CP fold, uncovered an unusual prolate particle shape, and revealed a previously unseen dsRNA binding mode. These data expand our knowledge of the evolution of viral structural proteins and are of relevance for applications such as ssRNA phage–based vaccine design.
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Bermúdez-Cruz, Rosa Ma, Jaime García-Mena, and Cecilia Montañez. "Polynucleotide phosphorylase binds to ssRNA with same affinity as to ssDNA." Biochimie 84, no. 4 (April 2002): 321–28. http://dx.doi.org/10.1016/s0300-9084(02)01385-8.
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Deng, Yong-Jie, Lei Feng, Huan Zhou, Xiang Xiao, Feng-Ping Wang, and Xi-Peng Liu. "NanoRNase from Aeropyrum pernix shows nuclease activity on ssDNA and ssRNA." DNA Repair 65 (May 2018): 54–63. http://dx.doi.org/10.1016/j.dnarep.2018.03.005.
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Cánovas-Márquez, José Tomás, Sebastian Falk, Francisco E. Nicolás, Subramanian Padmanabhan, Rubén Zapata-Pérez, Álvaro Sánchez-Ferrer, Eusebio Navarro, and Victoriano Garre. "A ribonuclease III involved in virulence of Mucorales fungi has evolved to cut exclusively single-stranded RNA." Nucleic Acids Research 49, no. 9 (April 20, 2021): 5294–307. http://dx.doi.org/10.1093/nar/gkab238.
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Abstract Members of the ribonuclease III (RNase III) family regulate gene expression by processing double-stranded RNA (dsRNA). This family includes eukaryotic Dicer and Drosha enzymes that generate small dsRNAs in the RNA interference (RNAi) pathway. The fungus Mucor lusitanicus, which causes the deadly infection mucormycosis, has a complex RNAi system encompassing a non-canonical RNAi pathway (NCRIP) that regulates virulence by degrading specific mRNAs. In this pathway, Dicer function is replaced by R3B2, an atypical class I RNase III, and small single-stranded RNAs (ssRNAs) are produced instead of small dsRNA as Dicer-dependent RNAi pathways. Here, we show that R3B2 forms a homodimer that binds to ssRNA and dsRNA molecules, but exclusively cuts ssRNA, in contrast to all known RNase III. The dsRNA cleavage inability stems from its unusual RNase III domain (RIIID) because its replacement by a canonical RIIID allows dsRNA processing. A crystal structure of R3B2 RIIID resembles canonical RIIIDs, despite the low sequence conservation. However, the groove that accommodates dsRNA in canonical RNases III is narrower in the R3B2 homodimer, suggesting that this feature could be responsible for the cleavage specificity for ssRNA. Conservation of this activity in R3B2 proteins from other mucormycosis-causing Mucorales fungi indicates an early evolutionary acquisition.
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Nurkiyanova, Kulpash M., Eugene V. Ryabov, Natalia O. Kalinina, Yongchang Fan, Igor Andreev, Alexander G. Fitzgerald, Peter Palukaitis, and Michael Taliansky. "Umbravirus-encoded movement protein induces tubule formation on the surface of protoplasts and binds RNA incompletely and non-cooperatively." Journal of General Virology 82, no. 10 (October 1, 2001): 2579–88. http://dx.doi.org/10.1099/0022-1317-82-10-2579.
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Various functions of the cell-to-cell movement protein (MP) of Groundnut rosette virus (GRV) were analysed. The GRV ORF4-encoded protein was shown by immunofluorescence microscopy to generate tubular structures that protrude from the surface of the protoplast. The protein encoded by ORF4 was assessed also for RNA-binding properties. This protein was tagged at its C terminus with six histidine residues, produced in Escherichia coli using an expression vector and purified by affinity chromatography. Gel retardation analysis demonstrated that, in contrast to many other viral MPs, including the 3a MP of Cucumber mosaic virus (CMV), the ORF4-encoded protein bound non-cooperatively to viral ssRNA and formed complexes of low protein:RNA ratios. Competition binding experiments showed that the ORF4-encoded protein bound to both ssRNA and ssDNA without sequence specificity, but did not bind to dsDNA. UV cross-linking and nitrocellulose membrane-retention assays confirmed that both the GRV and the CMV MPs formed complexes with ssRNA and that these complexes showed similar stability in NaCl. Probing the MP–RNA complexes by atomic force microscopy demonstrated that the ORF4-encoded protein bound RNA incompletely, leaving protein-free RNA segments of varying length, while the CMV 3a protein formed highly packed complexes. The significance of the two properties of limited RNA binding and tubule formation of the umbraviral MP is discussed.
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Tomaru, Yuji, and Kei Kimura. "Novel Protocol for Estimating Viruses Specifically Infecting the Marine Planktonic Diatoms." Diversity 12, no. 6 (June 4, 2020): 225. http://dx.doi.org/10.3390/d12060225.
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Since their discovery, at least 15 diatom viruses have been isolated and characterised using a culture method with two cycles of extinction dilution. However, the method is time consuming and laborious, and it isolates only the most dominant virus in a water sample. Recent studies have suggested inter-species host specificity of diatom viruses. Here, we describe a new protocol to estimate previously unrecognised host-virus relationships. Host cell cultures after inoculation of natural sediment pore water samples were obtained before complete lysis. The proliferated viral genomes in the host cells were amplified using degenerate primer pairs targeting protein replication regions of single-stranded RNA (ssRNA) and single-stranded DNA (ssDNA) viruses, and then sequenced. Diverse ssRNA virus types within known diatom virus group were detected from inoculated Chaetoceros tenuissimus and C. setoensis cells. A previously unknown ssDNA virus type was detected in inoculated C. tenuissimus cells, but not in C. setoensis cells. Despite the possible protocol biases, for example non-specific adsorptions of virions onto the host cells, the present method helps to estimate the viruses infectious to a single host species. Further improvements to this protocol targeting the proliferated viral genomes might reveal unexpected diatom–virus ecological relationships.
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Fillmore, G. C., H. Lin, and J. K. K. Li. "Localization of the Single-Stranded RNA-Binding Domains of Bluetongue Virus Nonstructural Protein NS2." Journal of Virology 76, no. 2 (January 15, 2002): 499–506. http://dx.doi.org/10.1128/jvi.76.2.499-506.2002.
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ABSTRACT The S2 gene of bluetongue virus, serotype 17, has been cloned, and the nonstructural protein NS2 has been expressed. Synthetic peptides matching regions within the amino acid sequence of NS2 were used to map three single-stranded RNA (ssRNA)-binding regions within the protein. A prokaryotic expression system was then used to generate a series of deletion mutants with the ssRNA-binding domains of NS2 removed, singly and in different combinations. These truncated proteins were expressed on a large scale and purified to near homogeneity. The affinity of each truncated protein towards ssRNA was then assayed by electrophoretic mobility shift assays. As a result, the three ssRNA-binding domains of BTV nonstructural protein NS2 have been conclusively localized, and removal of these three domains completely abrogates the ability of NS2 to bind to ssRNA.
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Park, Hyeong-Jun, Hae Li Ko, Dong-Hoon Won, Da-Bin Hwang, Yoo-Sub Shin, Hye-Won Kwak, Hye-Jung Kim, Jun-Won Yun, and Jae-Hwan Nam. "Comprehensive Analysis of the Safety Profile of a Single-Stranded RNA Nano-Structure Adjuvant." Pharmaceutics 11, no. 9 (September 7, 2019): 464. http://dx.doi.org/10.3390/pharmaceutics11090464.
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Adjuvants enhance the efficacy of vaccines by stimulating immune response-related gene expression and pathways. Although some adjuvants have been approved for commercial use in human vaccines (e.g., Alum, MF59, and AS03), they might elicit adverse side effects, such as autoimmune diseases. Recently, we developed a novel single-stranded RNA (ssRNA) nano-structure adjuvant, which can stimulate both Th1 and Th2 responses. In this study, we evaluated the safety and toxicological profiles of this ssRNA nano-structure adjuvant in vitro and in vivo. Mice were intramuscularly immunized with the ssRNA nano-structure adjuvant three times, once every 2 weeks. The results indicate no significant differences in hematological and serum biochemistry parameters between the ssRNA-treated groups and the control group. From a histopathological perspective, no evidence of tissue damage was found in any group. The levels of IgE and anti-nuclear antibodies, which are markers of autoimmune disease, were not different between the ssRNA-treated groups and the control group. The findings of this study suggest that the ssRNA nano-structure can be used as a safe adjuvant to increase vaccine efficacies.
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Callanan, J., S. R. Stockdale, A. Shkoporov, L. A. Draper, R. P. Ross, and C. Hill. "Expansion of known ssRNA phage genomes: From tens to over a thousand." Science Advances 6, no. 6 (February 2020): eaay5981. http://dx.doi.org/10.1126/sciadv.aay5981.
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The first sequenced genome was that of the 3569-nucleotide single-stranded RNA (ssRNA) bacteriophage MS2. Despite the recent accumulation of vast amounts of DNA and RNA sequence data, only 12 representative ssRNA phage genome sequences are available from the NCBI Genome database (June 2019). The difficulty in detecting RNA phages in metagenomic datasets raises questions as to their abundance, taxonomic structure, and ecological importance. In this study, we iteratively applied profile hidden Markov models to detect conserved ssRNA phage proteins in 82 publicly available metatranscriptomic datasets generated from activated sludge and aquatic environments. We identified 15,611 nonredundant ssRNA phage sequences, including 1015 near-complete genomes. This expansion in the number of known sequences enabled us to complete a phylogenetic assessment of both sequences identified in this study and known ssRNA phage genomes. Our expansion of these viruses from two environments suggests that they have been overlooked within microbiome studies.
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Turgeon, Nathalie, Marie-Josée Toulouse, Bruno Martel, Sylvain Moineau, and Caroline Duchaine. "Comparison of Five Bacteriophages as Models for Viral Aerosol Studies." Applied and Environmental Microbiology 80, no. 14 (May 2, 2014): 4242–50. http://dx.doi.org/10.1128/aem.00767-14.
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ABSTRACTBacteriophages are perceived to be good models for the study of airborne viruses because they are safe to use, some of them display structural features similar to those of human and animal viruses, and they are relatively easy to produce in large quantities. Yet, only a few studies have investigated them as models. It has previously been demonstrated that aerosolization, environmental conditions, and sampling conditions affect viral infectivity, but viral infectivity is virus dependent. Thus, several virus models are likely needed to study their general behavior in aerosols. The aim of this study was to compare the effects of aerosolization and sampling on the infectivity of five tail-less bacteriophages and two pathogenic viruses: MS2 (a single-stranded RNA [ssRNA] phage of theLeviviridaefamily), Φ6 (a segmented double-stranded RNA [dsRNA] phage of theCystoviridaefamily), ΦX174 (a single-stranded DNA [ssDNA] phage of theMicroviridaefamily), PM2 (a double-stranded DNA [dsDNA] phage of theCorticoviridaefamily), PR772 (a dsDNA phage of theTectiviridaefamily), human influenza A virus H1N1 (an ssRNA virus of theOrthomyxoviridaefamily), and the poultry virus Newcastle disease virus (NDV; an ssRNA virus of theParamyxoviridaefamily). Three nebulizers and two nebulization salt buffers (with or without organic fluid) were tested, as were two aerosol sampling devices, a liquid cyclone (SKC BioSampler) and a dry cyclone (National Institute for Occupational Safety and Health two-stage cyclone bioaerosol sampler). The presence of viruses in collected air samples was detected by culture and quantitative PCR (qPCR). Our results showed that these selected five phages behave differently when aerosolized and sampled. RNA phage MS2 and ssDNA phage ΦX174 were the most resistant to aerosolization and sampling. The presence of organic fluid in the nebulization buffer protected phages PR772 and Φ6 throughout the aerosolization and sampling with dry cyclones. In this experimental setup, the behavior of the influenza virus resembled that of phages PR772 and Φ6, while the behavior of NDV was closer to that of phages MS2 and ΦX174. These results provide critical information for the selection of appropriate phage models to mimic the behavior of specific human and animal viruses in aerosols.
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Warwick, Timothy, Ralf P. Brandes, and Matthias S. Leisegang. "Computational Methods to Study DNA:DNA:RNA Triplex Formation by lncRNAs." Non-Coding RNA 9, no. 1 (January 21, 2023): 10. http://dx.doi.org/10.3390/ncrna9010010.
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Long non-coding RNAs (lncRNAs) impact cell function via numerous mechanisms. In the nucleus, interactions between lncRNAs and DNA and the consequent formation of non-canonical nucleic acid structures seems to be particularly relevant. Along with interactions between single-stranded RNA (ssRNA) and single-stranded DNA (ssDNA), such as R-loops, ssRNA can also interact with double-stranded DNA (dsDNA) to form DNA:DNA:RNA triplexes. A major challenge in the study of DNA:DNA:RNA triplexes is the identification of the precise RNA component interacting with specific regions of the dsDNA. As this is a crucial step towards understanding lncRNA function, there exist several computational methods designed to predict these sequences. This review summarises the recent progress in the prediction of triplex formation and highlights important DNA:DNA:RNA triplexes. In particular, different prediction tools (Triplexator, LongTarget, TRIPLEXES, Triplex Domain Finder, TriplexFFP, TriplexAligner and Fasim-LongTarget) will be discussed and their use exemplified by selected lncRNAs, whose DNA:DNA:RNA triplex forming potential was validated experimentally. Collectively, these tools revealed that DNA:DNA:RNA triplexes are likely to be numerous and make important contributions to gene expression regulation.
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Marin-Esteban, Viviana, Mubashira Abdul, Dominique Charron, Alain Haziot, and Nuala Mooney. "Dendritic Cells Differentiated in the Presence of a Single-Stranded Viral RNA Sequence Conserve Their Ability To Activate CD4 T Lymphocytes but Lose Their Capacity for Th1 Polarization." Clinical and Vaccine Immunology 15, no. 6 (April 9, 2008): 954–62. http://dx.doi.org/10.1128/cvi.00428-07.
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ABSTRACT Monocyte-derived dendritic cells (DCs) differentiate in the presence of Toll-like-receptor (TLR) ligands in the course of ongoing infections. A single-stranded RNA (ssRNA) sequence, corresponding to the sequence of the U5 region of human immunodeficiency virus type 1 RNA, was used to mimic viral activation of TLR7 in human DCs. We determined the effector potential of DCs differentiated in the presence of this ssRNA molecule (ssRNA-DCs). ssRNA-DCs phenotypically resembled mature DCs. In contrast, their capacity to allostimulate naive CD4+ T cells resembled that of conventional immature DCs and could be increased by TLR4 stimulation. Th1 polarization of CD4+ T cells and production of interleukin 12p70 (IL-12p70) by ssRNA-DCs were selectively abrogated in response to a late TLR4, but not in response to a CD40, maturation signal. Inhibition of p38 mitogen-activated protein kinase partially restored IL-12p70 secretion but did not restore Th1 polarization, whereas addition of exogenous IL-12 led to recovery of Th1 polarization. In contrast to lipopolysaccharide, ssRNA induced IL-12p70 production at the very earliest stages of DC differentiation, indicating a particular role for TLR7 in monocyte-derived DCs recently engaged in differentiation. These data demonstrate generation of phenotypically mature DCs with the ability to expand CD4+ T lymphocytes lacking Th1/2-polarizing capacity.
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Pal, Arumay, and Yaakov Levy. "Structure, stability and specificity of the binding of ssDNA and ssRNA with proteins." PLOS Computational Biology 15, no. 4 (April 1, 2019): e1006768. http://dx.doi.org/10.1371/journal.pcbi.1006768.
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Zaitsev, Eugene N., and Stephen C. Kowalczykowski. "A novel pairing process promoted by Escherichia coli RecA protein: inverse DNA and RNA strand exchange." Genes & Development 14, no. 6 (March 15, 2000): 740–49. http://dx.doi.org/10.1101/gad.14.6.740.
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Traditionally, recombination reactions promoted by RecA-like proteins initiate by forming a nucleoprotein filament on a single-stranded DNA (ssDNA), which then pairs with homologous double-stranded DNA (dsDNA). In this paper, we describe a novel pairing process that occurs in an unconventional manner: RecA protein polymerizes along dsDNA to form an active nucleoprotein filament that can pair and exchange strands with homologous ssDNA. Our results demonstrate that this “inverse” reaction is a unique, highly efficient DNA strand exchange reaction that is not due to redistribution of RecA protein from dsDNA to the homologous ssDNA partner. Finally, we demonstrate that the RecA protein–dsDNA filament can also pair and promote strand exchange with ssRNA. This inverse RNA strand exchange reaction is likely responsible for R-loop formation that is required for recombination-dependent DNA replication.
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Boyce, Mark, and Polly Roy. "Recovery of Infectious Bluetongue Virus from RNA." Journal of Virology 81, no. 5 (December 6, 2006): 2179–86. http://dx.doi.org/10.1128/jvi.01819-06.
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ABSTRACT Bluetongue virus (BTV) is an insect-vectored emerging pathogen of ruminants with the potential for devastating economic impact on European agriculture. BTV and many other members of the Reoviridae have remained stubbornly refractory to the development of methods for the rescue of infectious virus from cloned nucleic acid (reverse genetics). Partially disassembled virus particles are transcriptionally active, synthesizing viral transcripts in the cytoplasm of infected cells, in essence delivering viral nucleic acids in situ. With the goal of generating a reverse-genetics system for BTV, we examined the possibility of recovering infectious BTV by the transfection of BSR cells with BTV transcripts (single-stranded RNA [ssRNA]) synthesized in vitro using BTV core particles. Following transfection, viral-protein synthesis was detected by immunoblotting, and confocal examination of the cells showed a punctate cytoplasmic distribution of inclusion bodies similar to that seen in infected cells. Viral double-stranded RNA (dsRNA) was isolated from ssRNA-transfected cells, demonstrating that replication of the ssRNA had occurred. Additionally, infectious virus was present in the medium of transfected cells, as demonstrated by the passage of infectivity in BSR cells. Infectivity was sensitive to single-strand-specific RNase A, and cotransfection of genomic BTV dsRNA with transcribed ssRNA demonstrated that the ssRNA species, rather than dsRNA, were the active components. We conclude that it is possible to recover infectious BTV wholly from ssRNA, which suggests a means for establishing helper virus-independent reverse-genetics systems for members of the Reoviridae.
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Wu, Jianyan, Jia Li, Xiang Mao, Weiwu Wang, Zhaobang Cheng, Yijun Zhou, Xueping Zhou, and Xiaorong Tao. "Viroplasm Protein P9-1 ofRice Black-Streaked Dwarf VirusPreferentially Binds to Single-Stranded RNA in Its Octamer Form, and the Central Interior Structure Formed by This Octamer Constitutes the Major RNA Binding Site." Journal of Virology 87, no. 23 (September 25, 2013): 12885–99. http://dx.doi.org/10.1128/jvi.02264-13.
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The P9-1 protein ofRice black-streaked dwarf virus(RBSDV) is an essential part of the viroplasm. However, little is known about its nature or biological function in the viroplasm. In this study, the structure and function of P9-1 were analyzed forin vitrobinding to nucleic acids. We found that the P9-1 protein preferentially bound to single-stranded versus double-stranded nucleic acids; however, the protein displayed no preference for RBSDV versus non-RBSDV single-stranded ssRNA (ssRNA). A gel mobility shift assay revealed that the RNA gradually shifted as increasing amounts of P9-1 were added, suggesting that multiple subunits of P9-1 bind to ssRNA. By using discontinuous blue native gel and chromatography analysis, we found that the P9-1 protein was capable of forming dimers, tetramers, and octamers. Strikingly, we demonstrated that P9-1 preferentially bound to ssRNA in the octamer, rather than the dimer, form. Deletion of the C-terminal arm resulted in P9-1 no longer forming octamers; consequently, the deletion mutant protein bound to ssRNA with significantly lower affinity and with fewer copies bound per ssRNA. Alanine substitution analysis revealed that electropositive amino acids among residues 25 to 44 are important for RNA binding and map to the central interior structure that was formed only by P9-1 octamers. Collectively, our findings provide novel insights into the structure and function of RBSDV viroplasm protein P9-1 binding to RNA.
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Wang, Minghong, Yong Wang, Xiangzhong Sun, Jiasen Cheng, Yanping Fu, Huiquan Liu, Daohong Jiang, Said A. Ghabrial, and Jiatao Xie. "Characterization of a Novel Megabirnavirus from Sclerotinia sclerotiorum Reveals Horizontal Gene Transfer from Single-Stranded RNA Virus to Double-Stranded RNA Virus." Journal of Virology 89, no. 16 (June 10, 2015): 8567–79. http://dx.doi.org/10.1128/jvi.00243-15.
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ABSTRACTMycoviruses have been detected in all major groups of filamentous fungi, and their study represents an important branch of virology. Here, we characterized a novel double-stranded RNA (dsRNA) mycovirus,Sclerotiniasclerotiorummegabirnavirus1(SsMBV1), in an apparently hypovirulent strain (SX466) ofSclerotinia sclerotiorum. Two similarly sized dsRNA segments (L1- and L2-dsRNA), the genome of SsMBV1, are packaged in rigid spherical particles purified from strain SX466. The full-length cDNA sequence of L1-dsRNA/SsMBV1 comprises two large open reading frames (ORF1 and ORF2), which encode a putative coat protein and an RNA-dependent RNA polymerase (RdRp), respectively. Phylogenetic analysis of the RdRp domain clearly indicates that SsMBV1 is related toRosellinia necatrixmegabirnavirus 1 (RnMBV1). L2-dsRNA/SsMBV1 comprises two nonoverlapping ORFs (ORFA and ORFB) encoding two hypothetical proteins with unknown functions. The 5′-terminal regions of L1- and L2-dsRNA/SsMBV1 share strictly conserved sequences and form stable stem-loop structures. Although L2-dsRNA/SsMBV1 is dispensable for replication, genome packaging, and pathogenicity of SsMBV1, it enhances transcript accumulation of L1-dsRNA/SsMBV1 and stability of virus-like particles (VLPs). Interestingly, a conserved papain-like protease domain similar to a multifunctional protein (p29) ofCryphonectriahypovirus 1 was detected in the ORFA-encoded protein of L2-dsRNA/SsMBV1. Phylogenetic analysis based on the protease domain suggests that horizontal gene transfer may have occurred from a single-stranded RNA (ssRNA) virus (hypovirus) to a dsRNA virus, SsMBV1. Our results reveal that SsMBV1 has a slight impact on the fundamental biological characteristics of its host regardless of the presence or absence of L2-dsRNA/SsMBV1.IMPORTANCEMycoviruses are widespread in all major fungal groups, and they possess diverse genomes of mostly ssRNA and dsRNA and, recently, circular ssDNA. Here, we have characterized a novel dsRNA virus (Sclerotinia sclerotiorummegabirnavirus 1 [SsMBV1]) that was isolated from an apparently hypovirulent strain, SX466, ofSclerotinia sclerotiorum. Although SsMBV1 is phylogenetically related to RnMBV1, SsMBV1 is markedly distinct from other reported megabirnaviruses with two features of VLPs and conserved domains. Our results convincingly showed that SsMBV1 is viable in the absence of L2-dsRNA/SsMBV1 (a potential large satellite-like RNA or genuine genomic virus component). More interestingly, we detected a conserved papain-like protease domain that commonly exists in ssRNA viruses, including members of the familiesPotyviridaeandHypoviridae. Phylogenetic analysis based on the protease domain suggests that horizontal gene transfer might have occurred from an ssRNA virus to a dsRNA virus, which may provide new insights into the evolutionary history of dsRNA and ssRNA viruses.
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Decrey, Loïc, Shinobu Kazama, and Tamar Kohn. "Ammonia as anIn SituSanitizer: Influence of Virus Genome Type on Inactivation." Applied and Environmental Microbiology 82, no. 16 (June 3, 2016): 4909–20. http://dx.doi.org/10.1128/aem.01106-16.
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ABSTRACTTreatment of human excreta and animal manure (HEAM) is key in controlling the spread of persistent enteric pathogens, such as viruses. The extent of virus inactivation during HEAM storage and treatment appears to vary with virus genome type, although the reasons for this variability are not clear. Here, we investigated the inactivation of viruses of different genome types under conditions representative of HEAM storage or mesophilic digestion. The goals were to characterize the influence of HEAM solution conditions on inactivation and to determine the potential mechanisms involved. Specifically, eight viruses representing the four viral genome types (single-stranded RNA [ssRNA], double-stranded RNA [dsRNA], single-stranded DNA [ssDNA], and double-stranded DNA [dsDNA]) were exposed to synthetic solutions with well-controlled temperature (20 to 35°C), pH (8 to 9), and ammonia (NH3) concentrations (0 to 40 mmol liter−1). DNA and dsRNA viruses were considerably more resistant than ssRNA viruses, resulting in up to 1,000-fold-longer treatment times to reach a 4-log inactivation. The apparently slower inactivation of DNA viruses was rationalized by the higher stability of DNA than that of ssRNA in HEAM. Pushing the system toward harsher pH (>9) and temperature (>35°C) conditions, such as those encountered in thermophilic digestion and alkaline treatments, led to more consistent inactivation kinetics among ssRNA and other viruses. This suggests that the dependence of inactivation on genome type disappeared in favor of protein-mediated inactivation mechanisms common to all viruses. Finally, we recommend the use of MS2 as a conservative indicator to assess the inactivation of ssRNA viruses and the stable ΦX174 or dsDNA phages as indicators for persistent viruses.IMPORTANCEViruses are among the most environmentally persistent pathogens. They can be present in high concentrations in human excreta and animal manure (HEAM). Therefore, appropriate treatment of HEAM is important prior to its reuse or discharge into the environment. Here, we investigated the factors that determine the persistence of viruses in HEAM, and we determined the main mechanisms that lead to their inactivation. Unlike other organisms, viruses can have four different genome types (double- or single-stranded RNA or DNA), and the viruses studied herein represent all four types. Genome type appeared to be the major determinant for persistence. Single-stranded RNA viruses are the most labile, because this genome type is susceptible to degradation in HEAM. In contrast, the other genome types are more stable; therefore, inactivation is slower and mainly driven by the degradation of viral proteins. Overall, this study allows us to better understand the behavior of viruses in HEAM.
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Rajendran, K. S., and Peter D. Nagy. "Characterization of the RNA-Binding Domains in the Replicase Proteins of Tomato Bushy Stunt Virus." Journal of Virology 77, no. 17 (September 1, 2003): 9244–58. http://dx.doi.org/10.1128/jvi.77.17.9244-9258.2003.
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ABSTRACT Tomato bushy stunt virus (TBSV), a tombusvirus with a nonsegmented, plus-stranded RNA genome, codes for two essential replicase proteins. The sequence of one of the replicase proteins, namely p33, overlaps with the N-terminal domain of p92, which contains the signature motifs of RNA-dependent RNA polymerases (RdRps) in its nonoverlapping C-terminal portion. In this work, we demonstrate that both replicase proteins bind to RNA in vitro based on gel mobility shift and surface plasmon resonance measurements. We also show evidence that the binding of p33 to single-stranded RNA (ssRNA) is stronger than binding to double-stranded RNA (dsRNA), ssDNA, or dsDNA in vitro. Competition experiments with ssRNA revealed that p33 binds to a TBSV-derived sequence with higher affinity than to other nonviral ssRNA sequences. Additional studies revealed that p33 could bind to RNA in a cooperative manner. Using deletion derivatives of the Escherichia coli-expressed recombinant proteins in gel mobility shift and Northwestern assays, we demonstrate that p33 and the overlapping domain of p92, based on its sequence identity with p33, contain an arginine- and proline-rich RNA-binding motif (termed RPR, which has the sequence RPRRRP). This motif is highly conserved among tombusviruses and related carmoviruses, and it is similar to the arginine-rich motif present in the Tat trans-activator protein of human immunodeficiency virus type 1. We also find that the nonoverlapping C-terminal domain of p92 contains additional RNA-binding regions. Interestingly, the location of one of the RNA-binding domains in p92 is similar to the RNA-binding domain of the NS5B RdRp protein of hepatitis C virus.
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Abid, Muniba, Muhammad A. U. Khan, Sehrish Mushtaq, Sohaib Afzaal, and Muhammad S. Haider. "A COMPREHENSIVE REVIEW ON MYCOVIRUSES AS BIOLOGICAL CONTROL AGENT." World Journal of Biology and Biotechnology 3, no. 2 (August 15, 2018): 187. http://dx.doi.org/10.33865/wjb.003.02.0146.
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Mycoviruses are viruses that infect fungi. They have been reported in all major taxa of fungi. Generally extracellular phase is not present in mycoviruses and are transmitted during cell division intracellularly in sporogenesis, and/or hyphal anastomosis. The genome of mycoviruses has dsRNA, but a number of positive- or negative-strand ssRNA and ssDNA viruses have been isolated and characterized. There is an increasing interest for use of mycoviruses as novel biocontrol agent as they reduce the virulence of their phytopathogeinc fungal hosts. Mycoviruses can also infect endophytic fungi and encode killer toxins. Better understanding of mycoviruses assembly, function, and evolution have been done through their structural analyses. Recent report on virus taxonomy suggested that mycoviruses genome mostly consists of double-stranded RNA (dsRNA), and about 30% of mycoviruses genome is composed of a positive, single-stranded RNA (+ssRNA). Recently a mycovirus that is related to Gemini viruses has been reported. Scientists have reported mycoviruses in Chytridiomycota, Zygomycota, Ascomycota, Deuteromycota, and Basidiomycota. Many mycoviruses and their fungal hosts still remain unknown recently developed metagenomics approaches will be useful for detecting and identifying new mycoviruses
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Wang, Wenqing, Xianhong Wang, Chunyan Tu, Mengmeng Yang, Jun Xiang, Liping Wang, Ni Hong, Lifeng Zhai, and Guoping Wang. "Novel Mycoviruses Discovered from a Metatranscriptomics Survey of the Phytopathogenic Alternaria Fungus." Viruses 14, no. 11 (November 18, 2022): 2552. http://dx.doi.org/10.3390/v14112552.
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Alternaria fungus can cause notable diseases in cereals, ornamental plants, vegetables, and fruits around the world. To date, an increasing number of mycoviruses have been accurately and successfully identified in this fungus. In this study, we discovered mycoviruses from 78 strains in 6 species of the genus Alternaria, which were collected from 10 pear production areas using high-throughput sequencing technology. Using the total RNA-seq, we detected the RNA-dependent RNA polymerase of 19 potential viruses and the coat protein of two potential viruses. We successfully confirmed these viruses using reverse transcription polymerase chain reaction with RNA as the template. We identified 12 mycoviruses that were positive-sense single-stranded RNA (+ssRNA) viruses, 5 double-strand RNA (dsRNA) viruses, and 4 negative single-stranded RNA (−ssRNA) viruses. In these viruses, five +ssRNA and four −ssRNA viruses were novel mycoviruses classified into diverse the families Botourmiaviridae, Deltaflexivirus, Mymonaviridea, and Discoviridae. We identified a novel −ssRNA mycovirus isolated from an A. tenuissima strain HB-15 as Alternaria tenuissima negative-stranded RNA virus 2 (AtNSRV2). Additionally, we characterized a novel +ssRNA mycovirus isolated from an A. tenuissima strain SC-8 as Alternaria tenuissima deltaflexivirus 1 (AtDFV1). According to phylogenetic and sequence analyses, we determined that AtNSRV2 was related to the viruses of the genus Sclerotimonavirus in the family Mymonaviridae. We also found that AtDFV1 was related to the virus family Deltaflexivirus. This study is the first to use total RNA sequencing to characterize viruses in Alternaria spp. These results expand the number of Alternaria viruses and demonstrate the diversity of these mycoviruses.
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Nishizawa, Toyohiko. "Marine fish pathogenic ssRNA viruses." Uirusu 46, no. 1 (1996): 67–72. http://dx.doi.org/10.2222/jsv.46.67.
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Eruera, Alice-Roza, Alice M. McSweeney, Geena M. McKenzie-Goldsmith, and Vernon K. Ward. "Protein Nucleotidylylation in +ssRNA Viruses." Viruses 13, no. 8 (August 5, 2021): 1549. http://dx.doi.org/10.3390/v13081549.
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Nucleotidylylation is a post-transcriptional modification important for replication in the picornavirus supergroup of RNA viruses, including members of the Caliciviridae, Coronaviridae, Picornaviridae and Potyviridae virus families. This modification occurs when the RNA-dependent RNA polymerase (RdRp) attaches one or more nucleotides to a target protein through a nucleotidyl-transferase reaction. The most characterized nucleotidylylation target is VPg (viral protein genome-linked), a protein linked to the 5′ end of the genome in Caliciviridae, Picornaviridae and Potyviridae. The nucleotidylylation of VPg by RdRp is a critical step for the VPg protein to act as a primer for genome replication and, in Caliciviridae and Potyviridae, for the initiation of translation. In contrast, Coronaviridae do not express a VPg protein, but the nucleotidylylation of proteins involved in replication initiation is critical for genome replication. Furthermore, the RdRp proteins of the viruses that perform nucleotidylylation are themselves nucleotidylylated, and in the case of coronavirus, this has been shown to be essential for viral replication. This review focuses on nucleotidylylation within the picornavirus supergroup of viruses, including the proteins that are modified, what is known about the nucleotidylylation process and the roles that these modifications have in the viral life cycle.
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Usui, Kimihito, Norikazu Ichihashi, and Tetsuya Yomo. "A design principle for a single-stranded RNA genome that replicates with less double-strand formation." Nucleic Acids Research 43, no. 16 (July 21, 2015): 8033–43. http://dx.doi.org/10.1093/nar/gkv742.
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Abstract Single-stranded RNA (ssRNA) is the simplest form of genetic molecule and constitutes the genome in some viruses and presumably in primitive life-forms. However, an innate and unsolved problem regarding the ssRNA genome is formation of inactive double-stranded RNA (dsRNA) during replication. Here, we addressed this problem by focusing on the secondary structure. We systematically designed RNAs with various structures and observed dsRNA formation during replication using an RNA replicase (Qβ replicase). From the results, we extracted a simple rule regarding ssRNA genome replication with less dsRNA formation (less GC number in loops) and then designed an artificial RNA that encodes a domain of the β-galactosidase gene based on this rule. We also obtained evidence that this rule governs the natural genomes of all bacterial and most fungal viruses presently known. This study revealed one of the structural design principles of an ssRNA genome that replicates continuously with less dsRNA formation.
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Jacobson, David R., Dustin B. McIntosh, Mark J. Stevens, Michael Rubinstein, and Omar A. Saleh. "Single-stranded nucleic acid elasticity arises from internal electrostatic tension." Proceedings of the National Academy of Sciences 114, no. 20 (May 1, 2017): 5095–100. http://dx.doi.org/10.1073/pnas.1701132114.
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Understanding of the conformational ensemble of flexible polyelectrolytes, such as single-stranded nucleic acids (ssNAs), is complicated by the interplay of chain backbone entropy and salt-dependent electrostatic repulsions. Molecular elasticity measurements are sensitive probes of the statistical conformation of polymers and have elucidated ssNA conformation at low force, where electrostatic repulsion leads to a strong excluded volume effect, and at high force, where details of the backbone structure become important. Here, we report measurements of ssDNA and ssRNA elasticity in the intermediate-force regime, corresponding to 5- to 100-pN forces and 50–85% extension. These data are explained by a modified wormlike chain model incorporating an internal electrostatic tension. Fits to the elastic data show that the internal tension decreases with salt, from >5 pN under 5 mM ionic strength to near zero at 1 M. This decrease is quantitatively described by an analytical model of electrostatic screening that ascribes to the polymer an effective charge density that is independent of force and salt. Our results thus connect microscopic chain physics to elasticity and structure at intermediate scales and provide a framework for understanding flexible polyelectrolyte elasticity across a broad range of relative extensions.
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Umer, Muhammad, Jiwen Liu, Huafeng You, Chuan Xu, Kaili Dong, Ni Luo, Linghong Kong, et al. "Genomic, Morphological and Biological Traits of the Viruses Infecting Major Fruit Trees." Viruses 11, no. 6 (June 4, 2019): 515. http://dx.doi.org/10.3390/v11060515.
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Banana trees, citrus fruit trees, pome fruit trees, grapevines, mango trees, and stone fruit trees are major fruit trees cultured worldwide and correspond to nearly 90% of the global production of woody fruit trees. In light of the above, the present manuscript summarizes the viruses that infect the major fruit trees, including their taxonomy and morphology, and highlights selected viruses that significantly affect fruit production, including their genomic and biological features. The results showed that a total of 163 viruses, belonging to 45 genera classified into 23 families have been reported to infect the major woody fruit trees. It is clear that there is higher accumulation of viruses in grapevine (80/163) compared to the other fruit trees (each corresponding to less than 35/163), while only one virus species has been reported infecting mango. Most of the viruses (over 70%) infecting woody fruit trees are positive-sense single-stranded RNA (+ssRNA), and the remainder belong to the -ssRNA, ssRNA-RT, dsRNA, ssDNA and dsDNA-RT groups (each corresponding to less than 8%). Most of the viruses are icosahedral or isometric (79/163), and their diameter ranges from 16 to 80 nm with the majority being 25–30 nm. Cross-infection has occurred in a high frequency among pome and stone fruit trees, whereas no or little cross-infection has occurred among banana, citrus and grapevine. The viruses infecting woody fruit trees are mostly transmitted by vegetative propagation, grafting, and root grafting in orchards and are usually vectored by mealybug, soft scale, aphids, mites or thrips. These viruses cause adverse effects in their fruit tree hosts, inducing a wide range of symptoms and significant damage, such as reduced yield, quality, vigor and longevity.
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Xu, Yang, Guangbao Yao, Chen Gong, Xiaodong Qi, and Hao Yan. "Abstract 320: Combined RNA nanostructure and 5FU for enhanced pancreatic cancer treatment through immunochemotherapy of targeting TAM2 deletion in TME." Cancer Research 82, no. 12_Supplement (June 15, 2022): 320. http://dx.doi.org/10.1158/1538-7445.am2022-320.
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Abstract Pancreatic ductal adenocarcinoma (PDA) is one of the most immune-resistant tumor types. Single-agent immune modulators targeting immune checkpoint blockade and multi-modal therapies including target immunotherapy have been proven to be clinically ineffective. Intensive research to explore novel targeted therapeutic strategies is undoubtedly required. Recently, nucleic acid nanotechnology has emerged as a promising approach for cancer treatment as it is highly programmable. Our previous results demonstrated that a replicable single-strand RNA origami (RNA-OG) technology stimulates a potent innate response primarily through a Toll-like receptor (TLR3) pathway to activate NK- and CD8-dependent antitumor immunity and counteract the peritoneal immunosuppressive environment in a murine peritoneal color cancer model. In this study, inspired by the robust innate immune suppressor cell types including myeloid-derived suppressor cells (MDSC) and tumor-associated macrophages M2 (TAM2) in the tumor microenvironment of pancreatic cancer, we designed a combined single-strand RNA (ssRNA) origami integrated with 5FU immunochemotherapeutic strategy to efficiently target pancreatic cancer cells and TAM in tumor microenvironments. The ssRNA is designed to fold from a long ssRNA molecule in a programmable manner. In pancreatic cancer cell lines, the ssRNA combined with 5FU significantly inhibited pancreatic cancer cell growth by growth curve and colony formation assay. We found that the combined ssRNA with 5FU dramatically induced pancreatic cancer cell death using pancreatic cell line and Raw264.7 in 2D cultures compared with 5FU. In 2D culture pancreatic cancer and Raw264.7 cell lines, 90% of the RNA origami was internalized in Raw264.7 cells within an hour. We also found that ssRNA origami triggered Raw264.7 cell death. In the xenograft model, ssRNA origami used with 5FU demonstrated high antitumor efficacy against pancreatic cancer. The Arg1 expression, which is the marker of TAM M2 and MDSC, was analyzed in xenograft tumor tissues using immunohistochemistry. We found that ssRNA implemented alongside 5FU significantly decreases the amount of Arg 1 positive TAM2 and MDSC cells in xenograft tumor tissue. We hypothesize that the ssRNA origami induced cell death of TAM M2 and MDSC cells in tumor microenvironments used alongside 5FU to target pancreatic cancer cells synergistically increases antitumor efficacy based on immunochemotherapy. Our integrated strategy is a promising approach to synergistically kill and remove the innate immune-suppressor cells in the tumor microenvironment, improving the therapeutic efficacy against pancreatic cancer. Citation Format: Yang Xu, Guangbao Yao, Chen Gong, Xiaodong Qi, Hao Yan. Combined RNA nanostructure and 5FU for enhanced pancreatic cancer treatment through immunochemotherapy of targeting TAM2 deletion in TME [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 320.
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Kannoly, Sherin, Yongping Shao, and Ing-Nang Wang. "Rethinking the Evolution of Single-Stranded RNA (ssRNA) Bacteriophages Based on Genomic Sequences and Characterizations of Two R-Plasmid-Dependent ssRNA Phages, C-1 and Hgal1." Journal of Bacteriology 194, no. 18 (July 20, 2012): 5073–79. http://dx.doi.org/10.1128/jb.00929-12.
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ABSTRACTWe have sequenced and characterized two R-plasmid-dependent single-stranded RNA bacteriophages (RPD ssRNA phages), C-1 and Hagl1. Phage C-1 requires a conjugative plasmid of the IncC group, while Hgal1 requires the IncH group. Both the adsorption rate constants and one-step growth curves are determined for both phages. We also empirically confirmed the lysis function of the predicted lysis genes. Genomic sequencing and phylogenetic analyses showed that both phages belong to theLevivirusgroup and are most closely related to another IncP-plasmid-dependent ssRNA phage, PRR1. Furthermore, our result strongly suggests that the stereotypical bauplans of genome organization found inLevivirusandAlloleviviruspredate phage specialization for conjugative plasmids, suggesting that the utilization of conjugative plasmids for cell attachment and entry comprises independent evolutionary events for these two main clades of ssRNA phages. Our result is also consistent with findings of a previous study, making theLevivirus-like genome organization ancestral and theAllolevivirus-like genome derived. To obtain a deeper insight into the evolution of ssRNA phages, more phages specializing for various conjugative plasmids and infecting different bacterial species would be needed.
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Smith, Stephanie A., and James H. Morrissey. "Size Matters: Differential Effects of RNA and Polyphosphate on Blood Clotting." Blood 112, no. 11 (November 16, 2008): 3074. http://dx.doi.org/10.1182/blood.v112.11.3074.3074.
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Abstract Introduction: Recently, both inorganic polyphosphate (Smith et al., PNAS103:903-8, 2006) and RNA (Kannemeier et al., PNAS104:6388–93, 2007) have been proposed as (patho)physiologic activators of the contact pathway in blood clotting. We also recently showed that polyphosphate of the size secreted by human platelets (approximately 75-mer) acts at two other points in the blood clotting cascade: it accelerates factor V activation and it enhances fibrin clot structure (Smith & Morrissey, Blood, in press). We now compare the ability of RNA and polyphosphate of varying chain lengths to modulate the blood clotting cascade at these three critical points: initiation, factor V activation, and fibrin polymerization. Methods: Polyphosphate was size-fractionated and its procoagulant activities were compared to those of polyinosinic acid, a synthetic singlestranded RNA (ssRNA); polyinosinic acid:polycytidylic acid, a synthetic double-stranded RNA (dsRNA); yeast tRNA or kaolin. Clotting assays were performed using purified fibrinogen, pooled normal plasma, or factor V-deficient plasma to which factor Va was added. Clotting was initiated by CaCl2 (contact pathway), factor Xa, or thrombin. Results: Long-chain polyphosphate (100-mer to 800-mer) triggered the contact pathway with a potency similar to kaolin and was about 30-fold more potent than ssRNA and some 3000- fold more potent than dsRNA or tRNA. Medium-chain polyphosphate (20-mer to 100-mer) and ssRNA both shortened factor Xa clotting times with similar potency, but dsRNA and yeast tRNA were without effect. Replacing plasma factor V with Va blocked the ability of either polyphosphate or ssRNA to shorten factor Xa clotting times, suggesting that both polymers accelerate factor V activation. And finally, when purified fibrinogen was clotted with thrombin, adding either ssRNA or polyphosphate yielded fibrin clots that were about threefold more turbid, indicating that both polymers enhance fibrin clot formation, while dsRNA and yeast tRNA were without effect. (We previously documented that polyphosphate enhances fibrin turbidity by dramatically increasing fibril diameter.) Interestingly, ssRNA significantly shortened the thrombin clotting time of purified fibrinogen, while polyphosphate had no effect on thrombin clotting times. Furthermore, the ability of polyphosphate to enhance fibrin clot structure was calcium-dependent, while ssRNA enhancement of fibrin clotting by thrombin was metal ion-independent. Conclusions: This study shows that RNA modulates critical downstream clotting functions in addition to its previously identified role in triggering the contact pathway. Long-chain polyphosphate (i.e., the size that accumulate in microorganisms, but not the size secreted by platelets) is substantially more potent than RNA in triggering the contact pathway of blood clotting. We therefore propose that polyphosphate may play an important role in host responses to pathogens by triggering the contact pathway. Polyphosphate of the size secreted by platelets had similar potency to ssRNA in accelerating factor V activation. And finally, polyphosphate of the size secreted by platelets had similar potency to RNA in enhancing fibrin clot structure, although the metal ion-dependencies of the two differed, as did their effects on thrombin clotting time. In general, ssRNA was far more potent than dsRNA or tRNA in modulating the blood clotting system.
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Lal, Avantika, Mariana Galvao Ferrarini, and Andreas J. Gruber. "Investigating the Human Host—ssRNA Virus Interaction Landscape Using the SMEAGOL Toolbox." Viruses 14, no. 7 (June 29, 2022): 1436. http://dx.doi.org/10.3390/v14071436.
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Viruses have evolved numerous mechanisms to exploit the molecular machinery of their host cells, including the broad spectrum of host RNA-binding proteins (RBPs). However, the RBP interactomes of most viruses are largely unknown. To shed light on the interaction landscape of RNA viruses with human host cell RBPs, we have analysed 197 single-stranded RNA (ssRNA) viral genome sequences and found that the majority of ssRNA virus genomes are significantly enriched or depleted in motifs for specific human RBPs, suggesting selection pressure on these interactions. To facilitate tailored investigations and the analysis of genomes sequenced in future, we have released our methodology as a fast and user-friendly computational toolbox named SMEAGOL. Our resources will contribute to future studies of specific ssRNA virus—host cell interactions and support the identification of antiviral drug targets.
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Isogai, Masamichi, and Nobuyuki Yoshikawa. "Mapping the RNA-binding domain on the Apple chlorotic leaf spot virus movement protein." Journal of General Virology 86, no. 1 (January 1, 2005): 225–29. http://dx.doi.org/10.1099/vir.0.80493-0.
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The RNA-binding properties of the cell-to-cell movement protein (MP) of Apple chlorotic leaf spot virus were analysed. MP was expressed in Escherichia coli and was used in UV-crosslinking analysis, using a digoxigenin–UTP-labelled RNA probe and gel-retardation analysis. The analyses demonstrated that MP bound cooperatively to single-stranded RNA (ssRNA). When analysed for NaCl dependence of the RNA-binding activity, the majority of the MP could bind ssRNA even in binding buffer with 1 M NaCl. Furthermore, competition binding experiments showed that the MP bound preferentially to ssRNA and single-stranded DNA without sequence specificity. MP deletion mutants were used to identify the RNA-binding domain by UV-crosslinking analysis. Amino acid residues 82–126 and 127–287 potentially contain two independently active, single-stranded nucleic acid-binding domains.
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Wong, Wei. "ssRNA stimulates Piezo1 and serotonin synthesis." Science Signaling 13, no. 648 (September 8, 2020): eabe6352. http://dx.doi.org/10.1126/scisignal.abe6352.
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Kazlauskas, Darius, Anisha Dayaram, Simona Kraberger, Sharyn Goldstien, Arvind Varsani, and Mart Krupovic. "Evolutionary history of ssDNA bacilladnaviruses features horizontal acquisition of the capsid gene from ssRNA nodaviruses." Virology 504 (April 2017): 114–21. http://dx.doi.org/10.1016/j.virol.2017.02.001.
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Li, Yuting, Siwei Li, Yumeng Zhao, Tao Zhou, Xuehong Wu, and Can Zhao. "Six Novel Mycoviruses Containing Positive Single-Stranded RNA and Double-Stranded RNA Genomes Co-Infect a Single Strain of the Rhizoctonia solani AG-3 PT." Viruses 14, no. 4 (April 14, 2022): 813. http://dx.doi.org/10.3390/v14040813.
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Six novel mycoviruses that collectively represent the mycovirome of Rhizoctonia solani anastomosis group (AG)-3 PT strain ZJ-2H, which causes potato black scurf, were identified through metatranscriptome sequencing and putatively designated as Rhizoctonia solani fusarivirus 4 [RsFV4, positive single-stranded RNA (+ssRNA)], Rhizoctonia solani fusarivirus 5 (RsFV5, +ssRNA), Rhizoctonia solani mitovirus 40 (RsMV40, +ssRNA), Rhizoctonia solani partitivirus 10 [RsPV10, double-stranded RNA (dsRNA)], Rhizoctonia solani partitivirus 11 (RsPV11, dsRNA), and Rhizoctonia solani RNA virus 11 (RsRV11, dsRNA). Whole genome sequences of RsFV4, RsMV40, RsPV10, RsPV11, and RsRV11, as well as a partial genome sequence of RsFV5, were obtained. The 3’- and 5’- untranslated regions of the five mycoviruses with complete genome sequences were folded into stable stem-loop or panhandle secondary structures. RsFV4 and RsFV5 are most closely related to Rhizoctonia solani fusarivirus 1 (RsFV1), however, the first open reading frame (ORF) of RsFV4 and RsFV5 encode a hypothetical protein that differs from the first ORF of RsFV1, which encodes a helicase. We confirmed that RsPV10 and RsPV11 assemble into the spherical virus particles (approximately 30 nm in diameter) that were extracted from strain ZJ-2H. This is the first report that +ssRNA and dsRNA viruses co-infect a single strain of R. solani AG-3 PT.
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Hill, Sam R., Reidun Twarock, and Eric C. Dykeman. "The impact of local assembly rules on RNA packaging in a T = 1 satellite plant virus." PLOS Computational Biology 17, no. 8 (August 24, 2021): e1009306. http://dx.doi.org/10.1371/journal.pcbi.1009306.
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The vast majority of viruses consist of a nucleic acid surrounded by a protective icosahedral protein shell called the capsid. During viral infection of a host cell, the timing and efficiency of the assembly process is important for ensuring the production of infectious new progeny virus particles. In the class of single-stranded RNA (ssRNA) viruses, the assembly of the capsid takes place in tandem with packaging of the ssRNA genome in a highly cooperative co-assembly process. In simple ssRNA viruses such as the bacteriophage MS2 and small RNA plant viruses such as STNV, this cooperative process results from multiple interactions between the protein shell and sites in the RNA genome which have been termed packaging signals. Using a stochastic assembly algorithm which includes cooperative interactions between the protein shell and packaging signals in the RNA genome, we demonstrate that highly efficient assembly of STNV capsids arises from a set of simple local rules. Altering the local assembly rules results in different nucleation scenarios with varying assembly efficiencies, which in some cases depend strongly on interactions with RNA packaging signals. Our results provide a potential simple explanation based on local assembly rules for the ability of some ssRNA viruses to spontaneously assemble around charged polymers and other non-viral RNAs in vitro.
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Baker, David G., Tyson A. Woods, Niranjan B. Butchi, Timothy M. Morgan, R. Travis Taylor, Piyanate Sunyakumthorn, Piyali Mukherjee, Kirk J. Lubick, Sonja M. Best, and Karin E. Peterson. "Toll-like receptor 7 suppresses virus replication in neurons but does not affect viral pathogenesis in a mouse model of Langat virus infection." Journal of General Virology 94, no. 2 (February 1, 2013): 336–47. http://dx.doi.org/10.1099/vir.0.043984-0.
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Toll-like receptor 7 (TLR7) recognizes guanidine-rich viral ssRNA and is an important mediator of peripheral immune responses to several ssRNA viruses. However, the role that TLR7 plays in regulating the innate immune response to ssRNA virus infections in specific organs such as the central nervous system (CNS) is not as clear. This study examined the influence of TLR7 on the neurovirulence of Langat virus (LGTV), a ssRNA tick-borne flavivirus. TLR7 deficiency did not substantially alter the onset or incidence of LGTV-induced clinical disease; however, it did significantly affect virus levels in the CNS with a log10 increase in virus titres in brain tissue from TLR7-deficient mice. This difference in virus load was also observed following intracranial inoculation, indicating a direct effect of TLR7 deficiency on regulating virus replication in the brain. LGTV-induced type I interferon responses in the CNS were not dependent on TLR7, being higher in TLR7-deficient mice compared with wild-type controls. In contrast, induction of pro-inflammatory cytokines including tumour necrosis factor, CCL3, CCL4 and CXCL13 were dependent on TLR7. Thus, although TLR7 is not essential in controlling LGTV pathogenesis, it is important in controlling virus infection in neurons in the CNS, possibly by regulating neuroinflammatory responses.
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Bang, Yoo-Jin, Yun-Hee Kim, Yu-Sun Lee, Jae-Yong Kim, Hyo-Jung Park, Hae-Li Ko, Sang-In Park, Kyung-Ah Hwang, Hun Kim, and Jae-Hwan Nam. "Development of inactivated subunit influenza vaccine endowing with IgA induction and protection against heterologous strain." Journal of Immunology 204, no. 1_Supplement (May 1, 2020): 245.11. http://dx.doi.org/10.4049/jimmunol.204.supp.245.11.
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Abstract Vaccination is known to be the best way to prevent and control seasonal influenza infections. Among the various available influenza vaccines, an inactivated vaccine shows improved protective effects associated with greater safety. However, since the influenza virus is continuously evolving, its response to inactivated vaccines becomes increasingly difficult to predict, leading to complete or partial loss of protection against the virus. In addition, immunogenicity is lower than other types and Th2-biased immune responses have been reported. In this study, we have investigated the role of the single-stranded RNA (ssRNA) adjuvant derived from the intergenic region of internal ribosome entry site of Cricket Paralysis virus in seasonal inactivated subunit influenza vaccine (ISIV). We found that the ssRNA adjuvant stimulated balanced cellular (indicated by IgG2a, IFN-γ, IL-2, and TNF-α) and humoral (indicated by IgG1 and HI) responses, along with mucosal (indicated by IgA) immune response. Moreover, the ssRNA adjuvant formulated ISIV enhanced viral clearance and improved lung pathology after homologous and even heterologous influenza virus infections. The proportion of memory CD4+ and CD8+ T cells, important for long term immunity, was also observed to increase. Therefore, the ssRNA adjuvant formulated ISIV is effective in inducing humoral and cellular immune responses, cross protection, and long-term immunity.
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Wilson, David P., and Danielle A. Roof. "Viral Phrenology." Viruses 13, no. 11 (October 30, 2021): 2191. http://dx.doi.org/10.3390/v13112191.
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We introduce Viral Phrenology, a new scheme for understanding the genomic composition of spherical viruses based on the locations of their structural protrusions. We used icosahedral point arrays to classify 135 distinct viral capsids collected from over 600 capsids available in the VIPERdb. Using gauge points of point arrays, we found 149 unique structural protrusions. We then show how to use the locations of these protrusions to determine the genetic composition of the virus. We then show that ssDNA, dsDNA, dsRNA and ssRNA viruses use different arrangements for distributing their protrusions. We also found that Triangulation number is also partially dependent on the structural protrusions. This analysis begins to tie together Baltimore Classification and Triangulation number using point arrays.
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Benzaria, Samira, Dorothée Bardiot, Tony Bouisset, Clément Counor, Céline Rabeson, Claire Pierra, Richard Storer, et al. "2′-C-Methyl Branched Pyrimidine Ribonucleoside Analogues: Potent Inhibitors of RNA Virus Replication." Antiviral Chemistry and Chemotherapy 18, no. 4 (August 2007): 225–42. http://dx.doi.org/10.1177/095632020701800406.
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RNA viruses are the agents of numerous widespread and often severe diseases. Their unique RNA-dependent RNA polymerase (RDRP) is essential for replication and, thus, constitutes a valid target for the development of selective chemotherapeutic agents. In this regard, we have investigated sugar-modified ribonucleoside analogues as potential inhibitors of the RDRP. Title compounds retain ‘natural’ pyrimidine bases, but possess a β-methyl substituent at the 2′-position of the D- or L-ribose moiety. Evaluation against a broad range of RNA viruses, either single-stranded positive (ssRNA), single-stranded negative (ssRNA−) or double-stranded (dsRNA), revealed potent activities for D-2′- C-methyl-cytidine and -uridine against ssRNA+, and dsRNA viruses. None of the L-enantiomers were active. Moreover, the 5′-triphosphates of the active D-enantiomers were found to inhibit the bovine virus diarrhoea virus polymerase. Thus, the 2′-methyl branching of natural pyrimidine ribonucleosides transforms physiological molecules into potent, broad-spectrum antiviral agents that merit further development.
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Burkhardt, Christiane, Po-Yu Sung, Cristina C. Celma, and Polly Roy. "Structural constraints in the packaging of bluetongue virus genomic segments." Journal of General Virology 95, no. 10 (October 1, 2014): 2240–50. http://dx.doi.org/10.1099/vir.0.066647-0.
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The mechanism used by bluetongue virus (BTV) to ensure the sorting and packaging of its 10 genomic segments is still poorly understood. In this study, we investigated the packaging constraints for two BTV genomic segments from two different serotypes. Segment 4 (S4) of BTV serotype 9 was mutated sequentially and packaging of mutant ssRNAs was investigated by two newly developed RNA packaging assay systems, one in vivo and the other in vitro. Modelling of the mutated ssRNA followed by biochemical data analysis suggested that a conformational motif formed by interaction of the 5′ and 3′ ends of the molecule was necessary and sufficient for packaging. A similar structural signal was also identified in S8 of BTV serotype 1. Furthermore, the same conformational analysis of secondary structures for positive-sense ssRNAs was used to generate a chimeric segment that maintained the putative packaging motif but contained unrelated internal sequences. This chimeric segment was packaged successfully, confirming that the motif identified directs the correct packaging of the segment.
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Bukrejewska, Malgorzata, Urszula Derewenda, Malwina Radwanska, Daniel A. Engel, and Zygmunt S. Derewenda. "Crystal structures of the methyltransferase and helicase from the ZIKA 1947 MR766 Uganda strain." Acta Crystallographica Section D Structural Biology 73, no. 9 (August 15, 2017): 767–74. http://dx.doi.org/10.1107/s2059798317010737.
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Two nonstructural proteins encoded byZika virusstrain MR766 RNA, a methyltransferase and a helicase, were crystallized and their structures were solved and refined at 2.10 and 2.01 Å resolution, respectively. The NS5 methyltransferase contains a boundS-adenosyl-L-methionine (SAM) co-substrate. The NS3 helicase is in the apo form. Comparison with published crystal structures of the helicase in the apo, nucleotide-bound and single-stranded RNA (ssRNA)-bound states suggests that binding of ssRNA to the helicase may occur through conformational selection rather than induced fit.
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Hanhijärvi, Kalle J., Gabija Ziedaite, Dennis H. Bamford, Edward Hæggström, and Minna M. Poranen. "Single-molecule measurements of viral ssRNA packaging." RNA 23, no. 1 (November 1, 2016): 119–29. http://dx.doi.org/10.1261/rna.057471.116.
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Mamasakhlisov, Yevgeni, Shura Hayryan, Vladimir Morozov, and Chin-Kun Hu. "Kinetics of the long ssRNA: Steady state." EPL (Europhysics Letters) 106, no. 4 (May 1, 2014): 48007. http://dx.doi.org/10.1209/0295-5075/106/48007.
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