Expression, purification and characterization of recombinant RNA-dependent RNA polymerase from Heterobasidion RNA virus 6

RNA-dependent RNA polymerases (RdRps) are enzymes that catalyze nucleotide polymerization on RNA template in the presence of divalent metal ions. These enzymes are central for the life cycle of viruses with RNA genomes. A double-stranded RNA (dsRNA) mycovirus Heterobasidion RNA virus 6 (HetRV6) commonly infect forest pathogenic fungi from the species complex Heterobasidion annosum sensu lato. In general, the infection caused by HetRV6 is cryptic, but sometimes have negative or mutualistic impact on its host. The understanding of the HetRV6 life cycle, including RNA synthesis, can shed light on the biology of HetRV6 virus and explain its divergent impacts on the host. Furthermore, a recombinant HetRV6 RdRp can potentially be harnessed to produce dsRNA molecules applicable for silencing of the target genes in plants, fungi, insects and mammals via RNA interference (RNAi). The aim of the current study was to express, purify and characterize the in vitro enzymatic activities of RdRp from the HetRV6 virus. To this end, a complementary DNA sequence of the RdRp gene was cloned into Escherichia coli expression vector, from which the recombinant polymerase was expressed and purified using affinity, ion-exchange and size-exclusion chromatography. The purified HetRV6 RdRp was a soluble and active in vitro enzyme, which possesses polymerase and terminal nucleotidyl transferase (TNTase) activities in the absence of any primers or accessory viral or host proteins. The impact of the buffer composition, pH, temperature, divalent cations and nucleoside triphosphate concentrations was studied and the optimal reaction conditions were identified. The polymerase is fully dependent on Mn2+ ions and does not produce dsRNA in its absence, while Mg2+ ions at concentration 1‒5 mM enhance the polymerization activity. HetRV6 polymerase is active on heterologous templates. However, the requirements for successful initiation and elongation should be still studied in more detail to fully understand how the enzyme can be used for efficient dsRNA synthesis for biotechnological applications. The discovered TNTase activity can potentially be used for in vitro RNA labeling. The completed work is a useful starting point to further explore the properties of the HetRV6 RdRp in terms of possible RNA modifying tools, its structural organization and role in infection and host-microbe interactions

Joint multicast routing and channel assignment in multiradio multichannel wireless mesh networks using simulated annealing

This is the post-print version of the article - Copyright @ 2008 Springer-VerlagThis paper proposes a simulated annealing (SA) algorithm based optimization approach to search a minimum-interference multicast tree which satisfies the end-to-end delay constraint and optimizes the usage of the scarce radio network resource in wireless mesh networks. In the proposed SA multicast algorithm, the path-oriented encoding method is adopted and each candidate solution is represented by a tree data structure (i.e., a set of paths). Since we anticipate the multicast trees on which the minimum-interference channel assignment can be produced, a fitness function that returns the total channel conflict is devised. The techniques for controlling the annealing process are well developed. A simple yet effective channel assignment algorithm is proposed to reduce the channel conflict. Simulation results show that the proposed SA based multicast algorithm can produce the multicast trees which have better performance in terms of both the total channel conflict and the tree cost than that of a well known multicast algorithm in wireless mesh networks.This work was supported by the Engineering and Physical Sciences Research Council (EPSRC) of UK under Grant EP/E060722/1

Spiral attractors as the root of a new type of "bursting activity" in the Rosenzweig-MacArthur model

We study the peculiarities of spiral attractors in the Rosenzweig-MacArthur model, that describes dynamics in a food chain "prey-predator-superpredator". It is well-known that spiral attractors having a "teacup" geometry are typical for this model at certain values of parameters for which the system can be considered as slow-fast system. We show that these attractors appear due to the Shilnikov scenario, the first step in which is associated with a supercritical Andronov-Hopf bifurcation and the last step leads to the appearance of a homoclinic attractor containing a homoclinic loop to a saddle-focus equilibrium with two-dimension unstable manifold. It is shown that the homoclinic spiral attractors together with the slow-fast behavior give rise to a new type of bursting activity in this system. Intervals of fast oscillations for such type of bursting alternate with slow motions of two types: small amplitude oscillations near a saddle-focus equilibrium and motions near a stable slow manifold of a fast subsystem. We demonstrate that such type of bursting activity can be either chaotic or regular

Effects of memristor-based coupling in the ensemble of FitzHugh-Nagumo elements

In this paper, we study the impact of electrical and memristor-based couplings on some neuron-like spiking regimes, previously observed in the ensemble of two identical FitzHugh-Nagumo elements with chemical excitatory coupling. We demonstrate how increasing strength of these couplings affects on such stable periodic regimes as spiking in-phase, anti-phase and sequential activity. We show that the presence of electrical and memristor-based coupling does not essentially affect regimes of in-phase activity. Such regimes do not changes remaining stable ones. However, it is not the case for regimes of anti-phase and sequential activity. All such regimes can transform into periodic or chaotic ones which are very similar to the regimes of in-phase activity. Concerning the regimes of sequential activity, this transformation depends continuously on the coupling parameters, whereas some anti-phase regimes can disappear via a saddle-node bifurcation and nearby orbits tend to regimes of in-phase activity. Also, we show that new interesting neuron-like phenomena can appear from the regimes of sequential activity when increasing the strength of electrical and/or memristor-based coupling. The corresponding regimes can be associated with the appearance of spiral attractors containing a saddle-focus equilibrium with homoclinic orbit and, thus, they correspond to chaotic motions near the invariant manifold of synchronization, which contains all in-phase limit cycles. Such new regimes can lead to the emergence of extreme events in the system of coupled neurons. In particular, the interspike intervals can become arbitrarily large when orbit pass very close to the saddle-focus. Finally, we show that the further increase in the strength of electrical coupling and/or memristor-based coupling leads to decreasing interspike intervals and, thus, it helps to avoid such extreme behavior

RNA-Dependent RNA Polymerase from Heterobasidion RNA Virus 6 Is an Active Replicase In Vitro

Heterobasidion RNA virus 6 (HetRV6) is a double-stranded (ds)RNA mycovirus and a member of the recently established genus Orthocurvulavirus within the family Orthocurvulaviridae. The purpose of the study was to determine the biochemical requirements for RNA synthesis catalyzed by HetRV6 RNA-dependent RNA polymerase (RdRp). HetRV6 RdRp was expressed in Escherichia coli and isolated to near homogeneity using liquid chromatography. The enzyme activities were studied in vitro using radiolabeled UTP. The HetRV6 RdRp was able to initiate RNA synthesis in a primer-independent manner using both virus-related and heterologous single-stranded (ss)RNA templates, with a polymerization rate of about 46 nt/min under optimal NTP concentration and temperature. NTPs with 2′-fluoro modifications were also accepted as substrates in the HetRV6 RdRp-catalyzed RNA polymerization reaction. HetRV6 RdRp transcribed viral RNA genome via semi-conservative mechanism. Furthermore, the enzyme demonstrated terminal nucleotidyl transferase (TNTase) activity. Presence of Mn2+ was required for the HetRV6 RdRp catalyzed enzymatic activities. In summary, our study shows that HetRV6 RdRp is an active replicase in vitro that can be potentially used in biotechnological applications, molecular biology, and biomedicine

RNA-Dependent RNA Polymerase from Heterobasidion RNA Virus 6 Is an Active Replicase In Vitro

Heterobasidion RNA virus 6 (HetRV6) is a double-stranded (ds)RNA mycovirus and a member of the recently established genus Orthocurvulavirus within the family Orthocurvulaviridae. The purpose of the study was to determine the biochemical requirements for RNA synthesis catalyzed by HetRV6 RNA-dependent RNA polymerase (RdRp). HetRV6 RdRp was expressed in Escherichia coli and isolated to near homogeneity using liquid chromatography. The enzyme activities were studied in vitro using radiolabeled UTP. The HetRV6 RdRp was able to initiate RNA synthesis in a primer-independent manner using both virus-related and heterologous single-stranded (ss)RNA templates, with a polymerization rate of about 46 nt/min under optimal NTP concentration and temperature. NTPs with 2′-fluoro modifications were also accepted as substrates in the HetRV6 RdRp-catalyzed RNA polymerization reaction. HetRV6 RdRp transcribed viral RNA genome via semi-conservative mechanism. Furthermore, the enzyme demonstrated terminal nucleotidyl transferase (TNTase) activity. Presence of Mn2+ was required for the HetRV6 RdRp catalyzed enzymatic activities. In summary, our study shows that HetRV6 RdRp is an active replicase in vitro that can be potentially used in biotechnological applications, molecular biology, and biomedicine

Native RNA purification method for small RNA molecules based on asymmetrical flow field-flow fractionation

RNA molecules provide promising new possibilities for the prevention and treatment of viral infections and diseases. The rapid development of RNA biology and medicine requires advanced methods for the purification of RNA molecules, which allow fast and efficient RNA processing, preferably under non-denaturing conditions. Asymmetrical flow field-flow fractionation (AF4) enables gentle separation and purification of macromolecules based on their diffusion coefficients. The aim of the study was to develop an AF4 method for efficient purification of enzymatically produced antiviral small interfering (si)RNA molecules and to evaluate the overall potential of AF4 in the separation of short single-stranded (ss) and double-stranded (ds) RNA molecules. We show that AF4 separates monomeric ssRNA from dsRNA molecules of the same size and monomeric ssRNA from multimeric forms of the same ssRNA. The developed AF4 method enabled the separation of enzymatically produced 27-nt siRNAs from partially digested substrate dsRNA, which is potentially toxic for mammalian cells. The recovery of AF4-purified enzymatically produced siRNA molecules was about 70%, which is about 20% higher than obtained using anion-exchange chromatography. The AF4-purified siRNAs were not toxic for mammalian cells and fully retained their biological activity as confirmed by efficient inhibition of herpes simplex virus 1 replication in cell culture. Our work is the first to develop AF4 methods for the separation of short RNA molecules.Peer reviewe