scholarly journals Biochemical and genetic analysis of the role of the viral polymerase in enterovirus recombination

2016 ◽  
Vol 44 (14) ◽  
pp. 6883-6895 ◽  
Author(s):  
Andrew Woodman ◽  
Jamie J. Arnold ◽  
Craig E. Cameron ◽  
David J. Evans
Keyword(s):  
Genetic Recombination ◽  
Evolutionary Process ◽  
Virus Genome ◽  
Biochemical Assay ◽  
Strand Transfer ◽  
Turnover Rates ◽  
Coding Region ◽  
Cis Acting ◽  
Polymerase Fidelity

Abstract Genetic recombination in single-strand, positive-sense RNA viruses is a poorly understand mechanism responsible for generating extensive genetic change and novel phenotypes. By moving a critical cis-acting replication element (CRE) from the polyprotein coding region to the 3′ non-coding region we have further developed a cell-based assay (the 3′CRE-REP assay) to yield recombinants throughout the non-structural coding region of poliovirus from dually transfected cells. We have additionally developed a defined biochemical assay in which the only protein present is the poliovirus RNA dependent RNA polymerase (RdRp), which recapitulates the strand transfer events of the recombination process. We have used both assays to investigate the role of the polymerase fidelity and nucleotide turnover rates in recombination. Our results, of both poliovirus intertypic and intratypic recombination in the CRE-REP assay and using a range of polymerase variants in the biochemical assay, demonstrate that RdRp fidelity is a fundamental determinant of recombination frequency. High fidelity polymerases exhibit reduced recombination and low fidelity polymerases exhibit increased recombination in both assays. These studies provide the basis for the analysis of poliovirus recombination throughout the non-structural region of the virus genome and provide a defined biochemical assay to further dissect this important evolutionary process.

scholarly journals A61 Large RNA genomes: Is RNA polymerase fidelity enough?

2019 ◽  
Vol 5 (Supplement_1) ◽  
Author(s):  
F Ferron ◽  
B Canard
Keyword(s):  
Rna Polymerase ◽  
Rna Synthesis ◽  
Rna Virus ◽  
Virus Genome ◽  
Gene Products ◽  
Dna Viruses ◽  
Viral Rdrp ◽  
Polymerase Fidelity ◽  
Mechanistic Basis

Abstract Large-genome Nidoviruses and Nidovirus-like viruses reside at the current boundary of largest RNA genome sizes. They code for an unusually large number of gene products matching that of small DNA viruses (e.g. DNA bacteriophages). The order of appearance and distribution of enzyme genes along various virus families (e.g. helicase and ExoN) may be seen as an evolutionary marker in these large RNA genomes lying at the genome size boundary. A positive correlation exists between (+)RNA virus genome sizes and the presence of the RNA helicase and the ExoN domains. Although the mechanistic basis of the presence of the helicase is still unclear, the role of the ExoN activity has been linked to the existence of an RNA synthesis proofreading system. In large Nidovirales, ExoN is bound to a processive replicative RNA-dependent RNA polymerase (RdRp) and corrects mismatched bases during viral RNA synthesis. Over the last decade, a view of the overall process has been refined in Coronaviruses, and in particular in our lab (Ferron et al., PNAS, 2018). We have identified genetic markers of large RNA genomes that we wish to use to data-mine currently existing metagenomic datasets. We have also initiated a collaboration to sequence and explore new viromes that will be searched according to these criteria. Likewise, we have a collection of purified viral RdRps that are currently being used to generate RNA synthesis products that will be compared to existing NGS datasets of cognate viruses. We will be able to have an idea about how much genetic diversity is possibly achievable by viral RdRp (‘tunable fidelity’) versus the detectable diversity (i.e. after selection in the infected cell) that is actually produced.

scholarly journals cis-Acting RNA Signals in the NS5B C-Terminal Coding Sequence of the Hepatitis C Virus Genome

2004 ◽  
Vol 78 (20) ◽  
pp. 10865-10877 ◽  
Author(s):  
Haekyung Lee ◽  
Hyukwoo Shin ◽  
Eckard Wimmer ◽  
Aniko V. Paul
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Virus Genome ◽  
Coding Region ◽  
Subgenomic Replicon ◽  
Stem Loop ◽  
Coding Sequence ◽  
Cis Acting ◽  
Rna Elements

ABSTRACT The cis-replicating RNA elements in the 5′ and 3′ nontranslated regions (NTRs) of the hepatitis C virus (HCV) genome have been thoroughly studied before. However, no cis-replicating elements have been identified in the coding sequences of the HCV polyprotein until very recently. The existence of highly conserved and stable stem-loop structures in the RNA polymerase NS5B coding sequence, however, has been previously predicted (A. Tuplin, J. Wood, D. J. Evans, A. H. Patel, and P. Simmonds, RNA 8:824-841, 2002). We have selected for our studies a 249-nt-long RNA segment in the C-terminal NS5B coding region (NS5BCR), which is predicted to form four stable stem-loop structures (SL-IV to SL-VII). By deletion and mutational analyses of the RNA structures, we have determined that two of the stem-loops (SL-V and SL-VI) are essential for replication of the HCV subgenomic replicon in Huh-7 cells. Mutations in the loop and the top of the stem of these RNA elements abolished replicon RNA synthesis but had no effect on translation. In vitro gel shift and filter-binding assays revealed that purified NS5B specifically binds to SL-V. The NS5B-RNA complexes were specifically competed away by unlabeled homologous RNA, to a small extent by 3′ NTR RNA, and only poorly by 5′ NTR RNA. The other two stem-loops (SL-IV and SL-VII) of the NS5BCR domain were found to be important but not essential for colony formation by the subgenomic replicon. The precise function(s) of these cis-acting RNA elements is not known.

scholarly journals cis-Acting Packaging Signals in the Influenza Virus PB1, PB2, and PA Genomic RNA Segments

2005 ◽  
Vol 79 (16) ◽  
pp. 10348-10355 ◽  
Author(s):  
Yuying Liang ◽  
Ying Hong ◽  
Tristram G. Parslow
Keyword(s):  
Influenza Virus ◽  
Influenza A ◽  
Virus Assembly ◽  
Virus Genome ◽  
Coding Region ◽  
Coding Sequences ◽  
Viral Packaging ◽  
Cis Acting ◽  
Virus Particles ◽  
Coding Regions

ABSTRACT The influenza A virus genome consists of eight negative-sense RNA segments. The cis-acting signals that allow these viral RNA segments (vRNAs) to be packaged into influenza virus particles have not been fully elucidated, although the 5′ and 3′ untranslated regions (UTRs) of each vRNA are known to be required. Efficient packaging of the NA, HA, and NS segments also requires coding sequences immediately adjacent to the UTRs, but it is not yet known whether the same is true of other vRNAs. By assaying packaging of genetically tagged vRNA reporters during plasmid-directed influenza virus assembly in cells, we have now mapped cis-acting sequences that are sufficient for packaging of the PA, PB1, and PB2 segments. We find that each involves portions of the distal coding regions. Efficient packaging of the PA or PB1 vRNAs requires at least 40 bases of 5′ and 66 bases of 3′ coding sequences, whereas packaging of the PB2 segment requires at least 80 bases of 5′ coding region but is independent of coding sequences at the 3′ end. Interestingly, artificial reporter vRNAs carrying mismatched ends (i.e., whose 5′ and 3′ ends are derived from different vRNA segments) were poorly packaged, implying that the two ends of any given vRNA may collaborate in forming specific structures to be recognized by the viral packaging machinery.

scholarly journals A Novel cis-Acting Element Facilitates Minus-Strand DNA Synthesis during Reverse Transcription of the Hepatitis B Virus Genome

2004 ◽  
Vol 78 (12) ◽  
pp. 6252-6262 ◽  
Author(s):  
Myeong-Kyun Shin ◽  
Jehan Lee ◽  
Wang-Shick Ryu
Keyword(s):  
Dna Synthesis ◽  
Reverse Transcription ◽  
Donor Site ◽  
Virus Genome ◽  
Acceptor Site ◽  
Strand Transfer ◽  
Minus Strand ◽  
Stem Loop ◽  
Cis Acting ◽  
Donor And Acceptor

ABSTRACT Hepadnaviruses replicate through reverse transcription of an RNA pregenome, resulting in a relaxed circular DNA genome. The first 3 or 4 nucleotides (nt) of minus-strand DNA are synthesized by the use of a bulge in a stem-loop structure near the 5′ end of the pregenome as a template. This primer is then transferred to a complementary UUCA motif, termed an acceptor, within DR1* near the 3′ end of the viral pregenome via 4-nt homology, and it resumes minus-strand DNA synthesis: this process is termed minus-strand transfer or primer translocation. Aside from the sequence identity of the donor and acceptor, little is known about the sequence elements contributing to minus-strand transfer. Here we report a novel cis-acting element, termed the β5 region (28 nt in length), located 20 nt upstream of DR1*, that facilitates minus-strand DNA synthesis. The deletion or inversion of the sequence including the β5 region diminished minus-strand DNA synthesis initiated at DR1*. Furthermore, the insertion of the β5 region into its own position in a mutant in which the sequences including the β5 region were replaced restored minus-strand DNA synthesis at DR1*. We speculate that the β5 region facilitates minus-strand transfer, possibly by bringing the acceptor site in proximity to the donor site via base pairing or by interacting with protein factors involved in this process.

scholarly journals Genetic recombination variation in wild Robertsonian mice: on the role of chromosomal fusions and Prdm9 allelic background

2014 ◽  
Vol 281 (1786) ◽  
pp. 20140297 ◽  
Author(s):  
Laia Capilla ◽  
Nuria Medarde ◽  
Alexandra Alemany-Schmidt ◽  
Maria Oliver-Bonet ◽  
Jacint Ventura ◽  
...  
Keyword(s):  
House Mouse ◽  
Genetic Recombination ◽  
Chromosomal Rearrangements ◽  
Evolutionary Process ◽  
Genome Shuffling ◽  
Mus Musculus Domesticus ◽  
Recombination Rates ◽  
Chromosomal Distribution ◽  
Epigenetic Signatures

Despite the existence of formal models to explain how chromosomal rearrangements can be fixed in a population in the presence of gene flow, few empirical data are available regarding the mechanisms by which genome shuffling contributes to speciation, especially in mammals. In order to shed light on this intriguing evolutionary process, here we present a detailed empirical study that shows how Robertsonian (Rb) fusions alter the chromosomal distribution of recombination events during the formation of the germline in a Rb system of the western house mouse ( Mus musculus domesticus ). Our results indicate that both the total number of meiotic crossovers and the chromosomal distribution of recombination events are reduced in mice with Rb fusions and that this can be related to alterations in epigenetic signatures for heterochromatinization. Furthermore, we detected novel house mouse Prdm9 allelic variants in the Rb system. Remarkably, mean recombination rates were positively correlated with a decrease in the number of ZnF domains in the Prdm9 gene. The suggestion that recombination can be modulated by both chromosomal reorganizations and genetic determinants that control the formation of double-stranded breaks during meiosis opens new avenues for understanding the role of recombination in chromosomal speciation.

On the role of conflict in the evolutionary process

2016 ◽  
pp. 87-91
Author(s):  
V.I. Bol’shakov ◽  
◽  
Yu.I. Dubrov ◽  
Keyword(s):  
Evolutionary Process

scholarly journals Intra-Colonial Viral Infections in Western Honey Bees (Apis mellifera)

2021 ◽  
Vol 9 (5) ◽  
pp. 1087
Author(s):  
Loreley Castelli ◽  
María Laura Genchi García ◽  
Anne Dalmon ◽  
Daniela Arredondo ◽  
Karina Antúnez ◽  
...  
Keyword(s):  
Apis Mellifera ◽  
Honey Bee ◽  
Viral Infections ◽  
Sampling Period ◽  
Virus Genome ◽  
The Individual ◽  
Bee Virus ◽  
Insight Into ◽  
Bee Viruses

RNA viruses play a significant role in the current high losses of pollinators. Although many studies have focused on the epidemiology of western honey bee (Apis mellifera) viruses at the colony level, the dynamics of virus infection within colonies remains poorly explored. In this study, the two main variants of the ubiquitous honey bee virus DWV as well as three major honey bee viruses (SBV, ABPV and BQCV) were analyzed from Varroa-destructor-parasitized pupae. More precisely, RT-qPCR was used to quantify and compare virus genome copies across honey bee pupae at the individual and subfamily levels (i.e., patrilines, sharing the same mother queen but with different drones as fathers). Additionally, virus genome copies were compared in cells parasitized by reproducing and non-reproducing mite foundresses to assess the role of this vector. Only DWV was detected in the samples, and the two variants of this virus significantly differed when comparing the sampling period, colonies and patrilines. Moreover, DWV-A and DWV-B exhibited different infection patterns, reflecting contrasting dynamics. Altogether, these results provide new insight into honey bee diseases and stress the need for more studies about the mechanisms of intra-colonial disease variation in social insects.

Inflammatory Myositis Secondary to Anti-Retroviral Therapy in a Child; Case Report and Review of the Literature

2021 ◽  
pp. 1-7
Author(s):  
Marie Monaghan ◽  
Charlotte Loh ◽  
Stephen Jones ◽  
Agyepong Oware ◽  
Kathryn Urankar ◽  
...  
Keyword(s):  
Drug Regimen ◽  
Dramatic Improvement ◽  
Strand Transfer ◽  
Inflammatory Myositis ◽  
Once Daily ◽  
Immune Mediated ◽  
Show Evidence ◽  
Transfer Inhibitor ◽  
Integrase Strand Transfer Inhibitor

Here, we describe a five year old girl with congenital HIV who had a six-week onset of rapidly deteriorating mobility and progressive proximal muscle weakness, associated with a raised Creatine Kinase (CK) level of 4330 U/L [25–200 U/L], subsequently diagnosed with an inflammatory myositis. Potential causes were investigated by paediatric neurology and immunology teams. Her viral load had been undetectable over the preceding two years, excluding a primary HIV myositis. While MRI scanning did not show evidence of definite myositis, a muscle biopsy showed evidence of an inflammatory process, comprising a moderate endomysial, perimysial and perivascular mononuclear (CD8 + T cell) infiltrate with increased MHC expression. No particular features of dermatomyositis or immune-mediated necrotising myopathy were identified and there were no features of an inclusion body myositis. Given the absence of active HIV infection, the role of anti-retroviral medications was considered. She had had a recent switch in medication, from twice daily Raltegravir (an Integrase Strand Transfer Inhibitor, INSTI) to once daily Dolutegravir (an INSTI) while continuing on an established daily protocol of Abacavir and Lamivudine (Nucleoside Reverse Transcriptase Inhibitors). Changing the Dolutegravir back to Raltegravir, in combination with continuing Lamivudine and Abacavir for two months made no difference to her weakness or CK levels. Moreover, this drug regimen had been well-tolerated over the preceding 19 month period. Changing the anti-retroviral regime completely to a single drug class (Protease Inhibitors) of Ritonavir and Darunavir, resulted in a dramatic improvement in her symptomatology. Within ten days she regained the ability to stand and walk, with a reduction in her CK from 1700 U/L at time of switch to 403 U/L [25–200]. This case highlights the potential risk of developing inflammatory myositis from anti-retrovirals even 19 months into treatment.

scholarly journals The role of diversity and tolerance in economic development

2021 ◽  
Author(s):  
Witold Kwasnicki
Keyword(s):  
Economic Growth ◽  
Economic Development ◽  
Industrial Development ◽  
Evolutionary Process ◽  
Evolutionary Model ◽  
Industry Development ◽  
Radical Innovations ◽  
Long Run

AbstractThis paper presents an evolutionary model of industry development, and uses simulations to investigation the role of diversity and heterogeneity in firms’ behaviour, and hence industrial development. The simulations suggest that economic growth is increased with greater variety, in the sense of the evolutionary process approaching the equilibrium faster and also, in the long run, moving faster from one equilibrium to a new, more advanced, equilibrium. This occurs due to higher variety caused by a more tolerant environment, and due to the higher probability of emergence of radical innovations.

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