scholarly journals An in vitro Evolutionary Journey of an Artificial RNA Replication System Towards Biological Complexity

2021 ◽  
Vol 61 (4) ◽  
pp. 240-244
Author(s):  
Taro FURUBAYASHI ◽  
Norikazu ICHIHASHI
Keyword(s):  
Rna Replication ◽  
Biological Complexity ◽  
Replication System

scholarly journals Automated in vitro evolution of a translation-coupled RNA replication system in a droplet flow reactor

2018 ◽  
Vol 8 (1) ◽  
Author(s):  
Tomoaki Yoshiyama ◽  
Tetsuo Ichii ◽  
Tetsuya Yomo ◽  
Norikazu Ichihashi
Keyword(s):  
Flow Reactor ◽  
Rna Replication ◽  
In Vitro Evolution ◽  
Droplet Flow ◽  
Replication System

scholarly journals Stimulation of Poliovirus Synthesis in a HeLa Cell-Free In Vitro Translation-RNA Replication System by Viral Protein 3CDpro

2005 ◽  
Vol 79 (10) ◽  
pp. 6358-6367 ◽  
Author(s):  
David Franco ◽  
Harsh B. Pathak ◽  
Craig E. Cameron ◽  
Bart Rombaut ◽  
Eckard Wimmer ◽  
...  
Keyword(s):  
Hela Cell ◽  
Rna Binding ◽  
Virus Production ◽  
Rna Replication ◽  
Cellular Protein ◽  
In Vitro Translation ◽  
Replication System ◽  
Encoding Protein ◽  
Stimulation Of

ABSTRACT The plus-strand RNA genome of poliovirus serves three distinct functions in the life cycle of the virus. The RNA is translated and then replicated, and finally the progeny RNAs are encapsidated. These processes can be faithfully reproduced in a HeLa cell-free in vitro translation-RNA replication system that produces viable poliovirus. We have previously observed a stimulation of virus synthesis when an mRNA, encoding protein 3CDpro, is added to the translation-RNA replication reactions of poliovirus RNA. Our aim in these experiments was to further define the factors that affect the stimulatory activity of 3CDpro in virus synthesis. We observed that purified 3CDpro protein also enhances virus synthesis by about 100-fold but has no effect on the translation of the polyprotein. Optimal stimulation is observed only when 3CDpro is present early in the incubation period. The stimulation, however, is abolished by a mutation either in the RNA binding domain of 3CDpro, 3CproR84S/I86A, or by each of two groups of complementary mutations R455A/R456A and D339A/S341A/D349A at interface I in the 3Dpol domain of 3CDpro. Surprisingly, virus synthesis is strongly inhibited by the addition of both 3Cpro and 3CDpro at the beginning of incubation. We also examined the effect of other viral or cellular proteins on virus synthesis in the in vitro system. No enhancement of virus synthesis occurred with viral proteins 3BC, 3ABC, 3BCD, 3Dpol, and 3Cpro or with cellular protein PCBP2. These results suggest that 3CDpro has to be present in the reaction at the time the replication complexes are assembled and that both the 3Cpro and 3Dpol domains of the protein are required for its activity that stimulates virus production.

scholarly journals Development of an in vitro bacteriophage N4 DNA replication system.

1986 ◽  
Vol 261 (23) ◽  
pp. 10506-10510
Author(s):  
J K Rist ◽  
M Pearle ◽  
A Sugino ◽  
L B Rothman-Denes
Keyword(s):  
Dna Replication ◽  
Replication System ◽  
Bacteriophage N4

scholarly journals Hijacking Components of the Cellular Secretory Pathway for Replication of Poliovirus RNA

2006 ◽  
Vol 81 (2) ◽  
pp. 558-567 ◽  
Author(s):  
George A. Belov ◽  
Nihal Altan-Bonnet ◽  
Gennadiy Kovtunovych ◽  
Catherine L. Jackson ◽  
Jennifer Lippincott-Schwartz ◽  
...  
Keyword(s):  
Secretory Pathway ◽  
Brefeldin A ◽  
Rna Replication ◽  
Bound State ◽  
Small Gtpases ◽  
Nucleotide Exchange ◽  
Virus Growth ◽  
Guanine Nucleotide Exchange ◽  
Membrane Reorganization

ABSTRACT Infection of cells with poliovirus induces a massive intracellular membrane reorganization to form vesicle-like structures where viral RNA replication occurs. The mechanism of membrane remodeling remains unknown, although some observations have implicated components of the cellular secretory and/or autophagy pathways. Recently, we showed that some members of the Arf family of small GTPases, which control secretory trafficking, became membrane-bound after the synthesis of poliovirus proteins in vitro and associated with newly formed membranous RNA replication complexes in infected cells. The recruitment of Arfs to specific target membranes is mediated by a group of guanine nucleotide exchange factors (GEFs) that recycle Arf from its inactive, GDP-bound state to an active GTP-bound form. Here we show that two different viral proteins independently recruit different Arf GEFs (GBF1 and BIG1/2) to the new structures that support virus replication. Intracellular Arf-GTP levels increase ∼4-fold during poliovirus infection. The requirement for these GEFs explains the sensitivity of virus growth to brefeldin A, which can be rescued by the overexpression of GBF1. The recruitment of Arf to membranes via specific GEFs by poliovirus proteins provides an important clue toward identifying cellular pathways utilized by the virus to form its membranous replication complex.

scholarly journals Relaxed Substrate Specificity in Qβ Replicase through Long-Term In Vitro Evolution

Life ◽  
2021 ◽  
Vol 12 (1) ◽  
pp. 32
Author(s):  
Kohtoh Yukawa ◽  
Ryo Mizuuchi ◽  
Norikazu Ichihashi
Keyword(s):  
Substrate Specificity ◽  
Early Life ◽  
Evolutionary History ◽  
Rna Replication ◽  
Life Forms ◽  
In Vitro Evolution ◽  
Present Evidence ◽  
Major Transition

A change from RNA- to DNA-based genetic systems is hypothesized as a major transition in the evolution of early life forms. One of the possible requirements for this transition is a change in the substrate specificity of the replication enzyme. It is largely unknown how such changes would have occurred during early evolutionary history. In this study, we present evidence that an RNA replication enzyme that has evolved in the absence of deoxyribonucleotide triphosphates (dNTPs) relaxes its substrate specificity and incorporates labeled dNTPs. This result implies that ancient replication enzymes, which probably evolved in the absence of dNTPs, could have incorporated dNTPs to synthesize DNA soon after dNTPs became available. The transition from RNA to DNA, therefore, might have been easier than previously thought.

scholarly journals The Role of Tissue Oxygen Tension in Dengue Virus Replication

Cells ◽  
2018 ◽  
Vol 7 (12) ◽  
pp. 241 ◽  
Author(s):  
Efseveia Frakolaki ◽  
Panagiota Kaimou ◽  
Maria Moraiti ◽  
Katerina Kalliampakou ◽  
Kalliopi Karampetsou ◽  
...  
Keyword(s):  
Dengue Virus ◽  
Oxygen Tension ◽  
Atmospheric Oxygen ◽  
Rna Replication ◽  
Metabolic Reprogramming ◽  
Anaerobic Glycolysis ◽  
Low Oxygen ◽  
Hypoxia Response

Low oxygen tension exerts a profound effect on the replication of several DNA and RNA viruses. In vitro propagation of Dengue virus (DENV) has been conventionally studied under atmospheric oxygen levels despite that in vivo, the tissue microenvironment is hypoxic. Here, we compared the efficiency of DENV replication in liver cells, monocytes, and epithelial cells under hypoxic and normoxic conditions, investigated the ability of DENV to induce a hypoxia response and metabolic reprogramming and determined the underlying molecular mechanism. In DENV-infected cells, hypoxia had no effect on virus entry and RNA translation, but enhanced RNA replication. Overexpression and silencing approaches as well as chemical inhibition and energy substrate exchanging experiments showed that hypoxia-mediated enhancement of DENV replication depends on the activation of the key metabolic regulators hypoxia-inducible factors 1α/2α (HIF-1α/2α) and the serine/threonine kinase AKT. Enhanced RNA replication correlates directly with an increase in anaerobic glycolysis producing elevated ATP levels. Additionally, DENV activates HIF and anaerobic glycolysis markers. Finally, reactive oxygen species were shown to contribute, at least in part through HIF, both to the hypoxia-mediated increase of DENV replication and to virus-induced hypoxic reprogramming. These suggest that DENV manipulates hypoxia response and oxygen-dependent metabolic reprogramming for efficient viral replication.

scholarly journals Biochemical and Genetic Studies of the Initiation of Human Rhinovirus 2 RNA Replication: Identification of a cis-Replicating Element in the Coding Sequence of 2Apro

2001 ◽  
Vol 75 (22) ◽  
pp. 10979-10990 ◽  
Author(s):  
Kinga Gerber ◽  
Eckard Wimmer ◽  
Aniko V. Paul
Keyword(s):  
Rna Replication ◽  
Human Rhinovirus ◽  
Viral Sequence ◽  
Coding Region ◽  
Terminal Protein ◽  
Genetic Studies ◽  
Poliovirus Type ◽  
Coding Sequence

ABSTRACT We have previously shown that the RNA polymerase 3Dpolof human rhinovirus 2 (HRV2) catalyzes the covalent linkage of UMP to the terminal protein (VPg) using poly(A) as a template (K. Gerber, E. Wimmer, and A. V. Paul, J. Virol. 75:10969–10978, 2001). The products of this in vitro reaction are VPgpU, VPgpUpU, and VPg-poly(U), the 5′ end of minus-strand RNA. In the present study we used an assay system developed for poliovirus 3Dpol (A. V. Paul, E. Rieder, D. W. Kim, J. H. van Boom, and E. Wimmer, J. Virol. 74: 10359–10370, 2000) to search for a viral sequence or structure in HRV2 RNA that would provide specificity to this reaction. We now show that a small hairpin in HRV2 RNA [cre(2A)], located in the coding sequence of 2Apro, serves as the primary template for HRV2 3Dpol in the uridylylation of HRV2 VPg, yielding VPgpU and VPgpUpU. The in vitro reaction is strongly stimulated by the addition of purified HRV2 3CDpro. Our analyses suggest that HRV2 3Dpol uses a “slide-back” mechanism during synthesis of the VPg-linked precursors. The corresponding cis- replicating RNA elements in the 2CATPase coding region of poliovirus type 1 Mahoney (I. Goodfellow, Y. Chaudhry, A. Richardson, J. Meredith, J. W. Almond, W. Barclay, and D. J. Evans, J. Virol. 74:4590–4600, 2000) and VP1 of HRV14 (K. L. McKnight and S. M. Lemon, RNA 4:1569–1584, 1998) can be functionally exchanged in the assay with cre(2A) of HRV2. Mutations of either the first or the second A in the conserved A1A2A3CA sequence in the loop of HRV2 cre(2A) abolished both viral growth and the RNA's ability to serve as a template in the in vitro VPg uridylylation reaction.

scholarly journals Transcription polymerase–catalyzed emergence of novel RNA replicons

Science ◽  
2020 ◽  
Vol 368 (6487) ◽  
pp. eaay0688 ◽  
Author(s):  
Nimit Jain ◽  
Lucas R. Blauch ◽  
Michal R. Szymanski ◽  
Rhiju Das ◽  
Sindy K. Y. Tang ◽  
...  
Keyword(s):  
Experimental Model ◽  
Hepatitis Delta Virus ◽  
De Novo ◽  
Rna Replication ◽  
Hereditary Material ◽  
Rolling Circle ◽  
Naturally Occurring ◽  
T7 Bacteriophage ◽  
Delta Virus

Transcription polymerases can exhibit an unusual mode of regenerating certain RNA templates from RNA, yielding systems that can replicate and evolve with RNA as the information carrier. Two classes of pathogenic RNAs (hepatitis delta virus in animals and viroids in plants) are copied by host transcription polymerases. Using in vitro RNA replication by the transcription polymerase of T7 bacteriophage as an experimental model, we identify hundreds of new replicating RNAs, define three mechanistic hallmarks of replication (subterminal de novo initiation, RNA shape-shifting, and interrupted rolling-circle synthesis), and describe emergence from DNA seeds as a mechanism for the origin of novel RNA replicons. These results inform models for the origins and replication of naturally occurring RNA genetic elements and suggest a means by which diverse RNA populations could be propagated as hereditary material in cellular contexts.

Sign in / Sign up

Export Citation Format

Share Document