scholarly journals Reovirus RNA recombination is sequence directed and generates internally deleted defective genome segments during passage

2021 ◽  
Author(s):  
Sydni Caet Smith ◽  
Jennifer Gribble ◽  
Julia R. Diller ◽  
Michelle A. Wiebe ◽  
Timothy W. Thoner ◽  
...  
Keyword(s):  
Cultured Cells ◽  
Rna Virus ◽  
Serial Passage ◽  
Viral Population ◽  
Rna Recombination ◽  
Chronic Infections ◽  
Rna Sequences ◽  
Rna Molecules ◽  
Mammalian Orthoreovirus

For viruses with segmented genomes, genetic diversity is generated by genetic drift, reassortment, and recombination. Recombination produces RNA populations distinct from full-length gene segments and can influence viral population dynamics, persistence, and host immune responses. Viruses in the Reoviridae family, including rotavirus and mammalian orthoreovirus (reovirus), have been reported to package segments containing rearrangements or internal deletions. Rotaviruses with RNA segments containing rearrangements have been isolated from immunocompromised and immunocompetent children and in vitro following serial passage at relatively high multiplicity. Reoviruses that package small, defective RNA segments have established chronic infections in cells and in mice. However, the mechanism and extent of Reoviridae RNA recombination are undefined. Towards filling this gap in knowledge, we determined the titers and RNA segment profiles for reovirus and rotavirus following serial passage in cultured cells. The viruses exhibited occasional titer reductions characteristic of interference. Reovirus strains frequently accumulated segments that retained 5′ and 3′ terminal sequences and featured large internal deletions, while similarly fragmented segments were rarely detected in rotavirus populations. Using next-generation RNA-sequencing to analyze RNA molecules packaged in purified reovirus particles, we identified distinct recombination sites within individual viral genome segments. Recombination junctions were frequently but not always characterized by short direct sequence repeats upstream and downstream that spanned junction sites. Taken together, these findings suggest that reovirus accumulates defective gene segments featuring internal deletions during passage and undergoes sequence-directed recombination at distinct sites. IMPORTANCE Viruses in the Reoviridae family include important pathogens of humans and other animals and have segmented RNA genomes. Recombination in RNA virus populations can facilitate novel host exploration and increased disease severity. The extent, patterns, and mechanisms of Reoviridae recombination and the functions and effects of recombined RNA products are poorly understood. Here, we provide evidence that mammalian orthoreovirus regularly synthesizes RNA recombination products that retain terminal sequences but contain internal deletions, while rotavirus rarely synthesizes such products. Recombination occurs more frequently at specific sites in the mammalian orthoreovirus genome, and short regions of identical sequence are often detected at junction sites. These findings suggest that mammalian orthoreovirus recombination events are directed in part by RNA sequences. An improved understanding of recombined viral RNA synthesis may enhance our capacity to engineer improved vaccines and virotherapies in the future.

scholarly journals Reovirus RNA recombination is sequence directed and generates internally deleted defective genome segments during passage

2020 ◽  
Author(s):  
Sydni Caet Smith ◽  
Jennifer Gribble ◽  
Julia R. Diller ◽  
Michelle A. Wiebe ◽  
Timothy W. Thoner ◽  
...  
Keyword(s):  
Cultured Cells ◽  
Rna Virus ◽  
Serial Passage ◽  
Viral Population ◽  
Viral Gene ◽  
Rna Recombination ◽  
Chronic Infections ◽  
Rna Sequences ◽  
Mammalian Orthoreovirus

ABSTRACTFor viruses with segmented genomes, genetic diversity is generated by genetic drift, reassortment, and recombination. Recombination produces RNA populations distinct from full-length gene segments and can influence viral population dynamics, persistence, and host immune responses. Viruses in the Reoviridae family, including rotavirus and mammalian orthoreovirus (reovirus), have been reported to package segments containing rearrangements or internal deletions. Rotaviruses with RNA segments containing rearrangements have been isolated from immunocompromised and immunocompetent children and in vitro following serial passage at high multiplicity. Reoviruses that package small, defective RNA segments have established chronic infections in cells and in mice. However, the mechanism and extent of Reoviridae RNA recombination are undefined. Towards filling this gap in knowledge, we determined the titers and RNA segment profiles for reovirus and rotavirus following serial passage in cultured cells. The viruses exhibited occasional titer reductions characteristic of interference. Reovirus strains frequently accumulated segments that retained 5′ and 3′ terminal sequences and featured large internal deletions, while similar segments were rarely detected in rotavirus populations. Using next-generation RNA-sequencing to analyze RNA molecules packaged in purified reovirus particles, we identified distinct recombination sites within individual viral gene segments. Recombination junction sites were frequently associated with short regions of identical sequence. Taken together, these findings suggest that reovirus accumulates defective gene segments featuring internal deletions during passage and undergoes sequence-directed recombination at distinct sites.IMPORTANCEViruses in the Reoviridae family include important pathogens of humans and other animals and have segmented RNA genomes. Recombination in RNA virus populations can facilitate novel host exploration and increased disease severity. The extent, patterns, and mechanisms of Reoviridae recombination and the functions and effects of recombined RNA products are poorly understood. Here, we provide evidence that mammalian orthoreovirus regularly synthesizes RNA recombination products that retain terminal sequences but contain internal deletions, while rotavirus rarely synthesizes such products. Recombination occurs more frequently at specific sites in the mammalian orthoreovirus genome, and short regions of identical sequence are often detected at junction sites. These findings suggest that mammalian orthoreovirus recombination events are directed in part by RNA sequences. An improved understanding of recombined viral RNA synthesis may enhance our capacity to engineer improved vaccines and virotherapies in the future.

scholarly journals The RNA origin of transfer RNA aminoacylation and beyond

2011 ◽  
Vol 366 (1580) ◽  
pp. 2959-2964 ◽  
Author(s):  
Hiroaki Suga ◽  
Gosuke Hayashi ◽  
Naohiro Terasaka
Keyword(s):  
Amino Acids ◽  
Evolutionary History ◽  
Transfer Rna ◽  
Translation System ◽  
Rna Sequences ◽  
Rna Molecules ◽  
Rna Catalysts ◽  
Modern System ◽  
History Of

Aminoacylation of tRNA is an essential event in the translation system. Although in the modern system protein enzymes play the sole role in tRNA aminoacylation, in the primitive translation system RNA molecules could have catalysed aminoacylation onto tRNA or tRNA-like molecules. Even though such RNA enzymes so far are not identified from known organisms, in vitro selection has generated such RNA catalysts from a pool of random RNA sequences. Among them, a set of RNA sequences, referred to as flexizymes (Fxs), discovered in our laboratory are able to charge amino acids onto tRNAs. Significantly, Fxs allow us to charge a wide variety of amino acids, including those that are non-proteinogenic, onto tRNAs bearing any desired anticodons, and thus enable us to reprogramme the genetic code at our will. This article summarizes the evolutionary history of Fxs and also the most recent advances in manipulating a translation system by integration with Fxs.

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 Role of an Internal and Two 3′-Terminal RNA Elements in Assembly of Tombusvirus Replicase

2005 ◽  
Vol 79 (16) ◽  
pp. 10608-10618 ◽  
Author(s):  
Zivile Panaviene ◽  
Tadas Panavas ◽  
Peter D. Nagy
Keyword(s):  
Rna Virus ◽  
Viral Rna ◽  
Host Cells ◽  
Replication Proteins ◽  
Rna Sequences ◽  
Alternative Structures ◽  
Recognition Element ◽  
Rna Elements

ABSTRACT Plus-strand RNA virus replication requires the assembly of the viral replicase complexes on intracellular membranes in the host cells. The replicase of Cucumber necrosis virus (CNV), a tombusvirus, contains the viral p33 and p92 replication proteins and possible host factors. In addition, the assembly of CNV replicase is stimulated in the presence of plus-stranded viral RNA (Z. Panaviene et al., J. Virol. 78:8254-8263, 2004). To define cis-acting viral RNA sequences that stimulate replicase assembly, we performed a systematic deletion approach with a model tombusvirus replicon RNA in Saccharomyces cerevisiae, which also coexpressed p33 and p92 replication proteins. In vitro replicase assays performed with purified CNV replicase preparations from yeast revealed critical roles for three RNA elements in CNV replicase assembly: the internal p33 recognition element (p33RE), the replication silencer element (RSE), and the 3′-terminal minus-strand initiation promoter (gPR). Deletion or mutagenesis of these elements reduced the activity of the CNV replicase to a minimal level. In addition to the primary sequences of gPR, RSE, and p33RE, formation of two alternative structures among these elements may also play a role in replicase assembly. Altogether, the role of multiple RNA elements in tombusvirus replicase assembly could be an important factor to ensure fidelity of template selection during replication.

scholarly journals Inhibition of Nipah Virus by Defective Interfering Particles

2020 ◽  
Vol 221 (Supplement_4) ◽  
pp. S460-S470 ◽  
Author(s):  
Stephen R Welch ◽  
Natasha L Tilston ◽  
Michael K Lo ◽  
Shannon L M Whitmer ◽  
Jessica R Harmon ◽  
...  
Keyword(s):  
Rna Virus ◽  
Nipah Virus ◽  
Vero Cells ◽  
Viral Population ◽  
Alternative Source ◽  
Highly Pathogenic ◽  
Defective Interfering Particles ◽  
Viral Genomes ◽  
Antiviral Control

Abstract The error-prone nature of RNA-dependent RNA polymerases drives the diversity of RNA virus populations. Arising within this diversity is a subset of defective viral genomes that retain replication competency, termed defective interfering (DI) genomes. These defects are caused by aberrant viral polymerase reinitiation on the same viral RNA template (deletion DI species) or the nascent RNA strand (copyback DI species). DI genomes have previously been shown to alter the dynamics of a viral population by interfering with normal virus replication and/or by stimulating the innate immune response. In this study, we investigated the ability of artificially produced DI genomes to inhibit Nipah virus (NiV), a highly pathogenic biosafety level 4 paramyxovirus. High multiplicity of infection passaging of both NiV clinical isolates and recombinant NiV in Vero cells generated an extensive DI population from which individual DIs were identified using next-generation sequencing techniques. Assays were established to generate and purify both naturally occurring and in silico-designed DIs as fully encapsidated, infectious virus-like particles termed defective interfering particles (DIPs). We demonstrate that several of these NiV DIP candidates reduced NiV titers by up to 4 logs in vitro. These data represent a proof-of-principle that a therapeutic application of DIPs to combat NiV infections may be an alternative source of antiviral control for this disease.

scholarly journals The Proteasomal Rpn11 Metalloprotease Suppresses Tombusvirus RNA Recombination and Promotes Viral Replication via Facilitating Assembly of the Viral Replicase Complex

2014 ◽  
Vol 89 (5) ◽  
pp. 2750-2763 ◽  
Author(s):  
K. Reddisiva Prasanth ◽  
Daniel Barajas ◽  
Peter D. Nagy
Keyword(s):  
Viral Replication ◽  
Genetic Recombination ◽  
Rna Virus ◽  
Viral Rna ◽  
Rna Recombination ◽  
Viral Recombination ◽  
Antiviral Responses ◽  
New Hosts

ABSTRACTRNA viruses co-opt a large number of cellular proteins that affect virus replication and, in some cases, viral genetic recombination. RNA recombination helps viruses in an evolutionary arms race with the host's antiviral responses and adaptation of viruses to new hosts. Tombusviruses and a yeast model host are used to identify cellular factors affecting RNA virus replication and RNA recombination. In this study, we have examined the role of the conserved Rpn11p metalloprotease subunit of the proteasome, which couples deubiquitination and degradation of proteasome substrates, in tombusvirus replication and recombination inSaccharomyces cerevisiaeand plants. Depletion or mutations of Rpn11p lead to the rapid formation of viral RNA recombinants in combination with reduced levels of viral RNA replication in yeast orin vitrobased on cell extracts. Rpn11p interacts with the viral replication proteins and is recruited to the viral replicase complex (VRC). Analysis of the multifunctional Rpn11p has revealed that the primary role of Rpn11p is to act as a “matchmaker” that brings the viral p92polreplication protein and the DDX3-like Ded1p/RH20 DEAD box helicases into VRCs. Overexpression of Ded1p can complement the defect observed inrpn11mutant yeast by reducing TBSV recombination. This suggests that Rpn11p can suppress tombusvirus recombination via facilitating the recruitment of the cellular Ded1p helicase, which is a strong suppressor of viral recombination, into VRCs. Overall, this work demonstrates that the co-opted Rpn11p, which is involved in the assembly of the functional proteasome, also functions in the proper assembly of the tombusvirus VRCs.IMPORTANCERNA viruses evolve rapidly due to genetic changes based on mutations and RNA recombination. Viral genetic recombination helps viruses in an evolutionary arms race with the host's antiviral responses and facilitates adaptation of viruses to new hosts. Cellular factors affect viral RNA recombination, although the role of the host in virus evolution is still understudied. In this study, we used a plant RNA virus, tombusvirus, to examine the role of a cellular proteasomal protein, called Rpn11, in tombusvirus recombination in a yeast model host, in plants, andin vitro. We found that the cellular Rpn11 is subverted for tombusvirus replication and Rpn11 has a proteasome-independent function in facilitating viral replication. When the Rpn11 level is knocked down or a mutated Rpn11 is expressed, then tombusvirus RNA goes through rapid viral recombination and evolution. Taken together, the results show that the co-opted cellular Rpn11 is a critical host factor for tombusviruses by regulating viral replication and genetic recombination.

scholarly journals Insertions of codons encoding basic amino acids in H7 hemagglutinins of influenza A viruses occur by recombination with RNA at hotspots near snoRNA binding sites

2020 ◽  
Author(s):  
Alexander P. Gultyaev ◽  
Monique I. Spronken ◽  
Mathis Funk ◽  
Ron A.M. Fouchier ◽  
Mathilde Richard
Keyword(s):  
Amino Acids ◽  
Cleavage Site ◽  
Influenza A ◽  
Rna Recombination ◽  
Influenza A Viruses ◽  
Rna Sequences ◽  
Rna Molecules ◽  
Basic Amino Acids ◽  
Molecular Determinant ◽  
Ha Protein

ABSTRACTThe presence of multiple basic amino acids in the protease cleavage site of the hemagglutinin (HA) protein is the main molecular determinant of virulence of highly pathogenic avian influenza (HPAI) viruses. Recombination of HA RNA with other RNA molecules of host or virus origin is a dominant mechanism of multi basic cleavage site (MBCS) acquisition for H7 subtype HA. Using alignments of HA RNA sequences from documented cases of MBCS insertion due to recombination, we show that such recombination with host RNAs is most likely to occur at particular hotspots in ribosomal RNAs (rRNAs), transfer RNAs (tRNAs) and viral RNAs. The locations of these hotspots in highly abundant RNAs indicate that RNA recombination is facilitated by the binding of small nucleolar RNA (snoRNA) near the recombination points.

scholarly journals In VitroPassage Selects for Chlamydia muridarum with Enhanced Infectivity in Cultured Cells but Attenuated Pathogenicity in Mouse Upper Genital Tract

2015 ◽  
Vol 83 (5) ◽  
pp. 1881-1892 ◽  
Author(s):  
Chaoqun Chen ◽  
Zhou Zhou ◽  
Turner Conrad ◽  
Zhangsheng Yang ◽  
Jin Dai ◽  
...  
Keyword(s):  
Genital Tract ◽  
Selection Pressure ◽  
Cultured Cells ◽  
Serial Passage ◽  
Host Cells ◽  
Content Type ◽  
Chlamydia Muridarum ◽  
Low Incidence

Although modernChlamydia muridarumhas been passaged for decades, there are no reports on the consequences of serial passage with strong selection pressure on its fitness. In order to explore the potential for Pasteurian selection to induce genomic and phenotypic perturbations toC. muridarum, a starter population was passaged in cultured cells for 28 generations without standard infection assistance. The resultant population, designated CMG28, displays markedly reducedin vitrodependence on centrifugation for infection and low incidence and severity of upper genital tract pathology following intravaginal inoculation into mice compared to the parentalC. muridarumpopulation, CMG0. Deep sequencing of CMG0 and CMG28 revealed novel protein variants in the hypothetical genes TC0237 (Q117E) and TC0668 (G322R).In vitroattachment assays of isogenic plaque clone pairs with mutations in either TC0237 and TC0668 or only TC0237 reveal that TC0237(Q117E) is solely responsible for enhanced adherence to host cells. Paradoxically, double mutants, but not TC0237(Q117E) single mutants, display severely attenuatedin vivopathogenicity. These findings implicate TC0237 and TC0668 as novel genetic factors involved in chlamydial attachment and pathogenicity, respectively, and show that serial passage under selection pressure remains an effective tool for studyingChlamydiapathogenicity.

scholarly journals Influenza A Virus Negative Strand RNA is Translated for CD8+ T Cell Immunosurveillance

2018 ◽  
Author(s):  
Heather D. Hickman ◽  
Jacqueline W. Mays ◽  
James Gibbs ◽  
Ivan Kosik ◽  
Javier Magadan ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Influenza A Virus ◽  
Influenza A ◽  
Cultured Cells ◽  
Rna Virus ◽  
Cd8 T Cell ◽  
Negative Strand

AbstractTo probe the limits of CD8+ T cell immunosurveillance, we inserted the model peptide SIINFEKL into influenza A virus (IAV) negative strand gene segments. Although IAV genomic RNA is widely considered as non-coding, there is a conserved, relatively long open reading frame present in the genomic strand of segment eight, encoding a potential protein termed NEG8. The biosynthesis of NEG8 from IAV has yet to be demonstrated. While we failed to detect NEG8 protein expression in IAV infected cells, cell surface Kb-SIINFEKL complexes are generated when SIINFEKL is genetically appended to the predicted COOH-terminus of NEG8, as shown by activation of OT-I T cells in vitro and in vivo. Moreover, recombinant IAV encoding SIINFEKL embedded in the negative strand of the NA-stalk coding sequence also activates OT-I T cells in vivo. Together, our findings demonstrate both the translation of sequences on the negative strand of a single stranded RNA virus and its relevance anti-viral immunosurveillance.SignificanceEvery gene encodes complementary information on the opposite strand that can potentially be used for immunosurveillance. In this study, we show that the influenza A virus “non-coding” strand translated into polypeptides during a viral infection of either cultured cells or mice that can be recognized by CD8+ T cells. Our findings raise the possibility that influenza virus uses its negative strand to generate proteins useful to the virus. More generally, it adds to a growing literature showing that immunosurveillance extends to gene sequences generally thought not to be converted into proteins. The relevance of translating this “dark” information extends from viral immunity to cancer immunotherapy and autoimmunity.

RNA design by in vitro RNA recombination and synthesis

1987 ◽  
Vol 65 (8) ◽  
pp. 677-692 ◽  
Author(s):  
Robert Cedergren ◽  
Henri Grosjean
Keyword(s):  
In Vitro Transcription ◽  
Biologically Active ◽  
Future Research ◽  
Rna Recombination ◽  
Rna Sequences ◽  
Active Length ◽  
Rna Fragments ◽  
Rna Design ◽  
Relationship Of

The techniques of in vitro RNA synthesis and recombination are presented. These include the site-specific cleavage of RNA, the manipulation of terminal phosphates, and the ligation of RNA fragments. Areas of promising future research include the establishment of RNA cloning vectors and the use of in vitro transcription of natural or designed RNA genes. The chemical synthesis approach now offers the possibility of making large amounts of biologically active length RNAs and of incorporating modified or reporter nucleotides into RNA sequences for physical studies. The new RNA techniques taken with DNA technology will permit a new approach towards understanding the complexity of RNA metabolism and the relationship of structure to function in RNAs.

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