Kinetics of RNA replication: competition and selection among self-replicating RNA species

The process of Darwinian selection in the self-replication of single-stranded RNA by Q beta replicase was investigated by analytical and computer-simulation methods. For this system, the relative population change of the competing species was found to be a useful definition of selection value, calculable from measurable kinetic parameters and concentrations of each species. Critical differences in the criteria for selection were shown to pertain for replicase/RNA ratios greater than or less than 1, for the case that formation of double-stranded RNA occurs and when comparisons are made of closed with open systems. At a large excess of enzyme, RNA species grow exponentially without interfering with each other, and selection depends only on the fecundity of the species, i.e., their overall replication rates. For RNA concentrations greater than the replicase concentration, the selection of species is governed by their abilities to compete for enzyme. Under conditions where formation of double strands occurs, competition leads to a coexistence of the species; the selection values vanish, and the concentration ratios depend only on the template binding and double-strand formation rates. The approach to coexistence is rapid, because when its competitors are in a steady state, a species present in trace amount is amplified exponentially. When formation of hybrid double strands occurs at a substantial rate, coexistence of hybridizing species is essentially limited to cases where the formation rate of heterologous double strands is smaller than the geometric mean of the formation rates of the homologous double strands. At limiting cases, e.g. in the steady states, simple analytical expressions for the main aspects of the selection process were found. Experimental data support the analytical expressions and the simulations.     

Kinetics of poliovirus replication in HeLa cells infected by isolated RNA

Under conditions with the least toxicity for cells compatible with an optimal sensitizing effect for RNA infection, 47% of HeLa cells can be infected by viral RNA. Both RNA and virus infective centers produce identical amounts, i.e. 2000 PFU of progeny virus per infective center and both incorporate ³H uridine in equal quantities. After infection with an effective multiplicity of ten PFU of virus or RNA, virus maturation occurs thirty minutes earlier in RNA-infected cells as compared to virus-infected cells.     

野田村病毒RNA的复制机制

野田村病毒科Nodaviradae分为2个属,分别为主要感染昆虫的α野田村病毒属(Alphanodavirus)和主要感染鱼类的β野田村病毒属(Betanodavirus)。野田村病毒的基因组由2条单链正义RNA分子(RNA1和RNA2)所组成,RNA1编码蛋白A,即病毒负责复制病毒两条基因组的依赖RNA的RNA聚合酶催化亚基。RNA2编码衣壳前体蛋白α,此前体蛋白α先组装成原病毒粒子,再经历一次自我催化的成熟切割成2个病毒的衣壳蛋白β和γ,就成了成熟的有感染性的病毒粒子。在RNA复制过程中,从RNA1的3′末端会合成一个不被包装进病毒粒子的亚基因组RNA3。RNA1能在无RNA2的情况下自我复制,并持续地产生亚基因组RNA3,RNA3的合成采取的是提前终止机制。本文还介绍了野田村病毒复制的调节、非结构蛋白的功能和病毒复制在细胞内的定位。     

4 Towards non-enzymatic RNA replication

The direct path from prebiotic chemistry to the RNA World requires a plausible route for the synthesis of activated ribonucleotides and RNA templates, along with a means for the complete replication of potentially useful RNA sequences. However, many apparent roadblocks make non-enzymatic RNA replication look quite difficult, if not impossible. These problems include the slow rate, low accuracy and poor regioselectivity of non-enzymatic template copying, the hydrolysis of activated monomers and absence of good re-activation chemistry, the difficulty of strand separation after template copying, the rapidity of strand reannealing, the absence of primers in any realistic replication scenario, and the apparent incompatibly of RNA copying chemistry (which requires a high Mg²⁺ concentration) with fatty acid-based protocell membranes, which are destroyed by low Mg²⁺ concentrations. I will discuss recent progress from my laboratory on four of these issues. We have found that functional RNAs such as aptamers and ribozymes can tolerate moderate levels of 2′–5′ linkages without great loss of activity. It therefore appears that the presence of 10–25% of such linkages in the products of non-enzymatic copying would not prevent the evolution of functional RNAs. Furthermore, 2′–5′ linkages can be helpful, as they decrease the melting temperature of RNA duplexes enough to allow strand separation to occur under geophysically plausible conditions. Recently, we have found that small chemical changes to the nucleobases can greatly increase the fidelity of non-enzymatic template copying, and we have found conditions that render RNA copying chemistry compatible with vesicle integrity, thereby allowing RNA copying to occur inside fatty acid-based model protocell membranes. I will discuss potential approaches to solving the remaining issues that stand in the way of complete RNA replication. If all of the problems with RNA replication can be overcome, it should be possible to construct functioning protocells in the laboratory.     

Autocatalytic replication of a recombinant RNA

We demonstrate that a heterologous RNA sequence can be copied in vitro by Q beta replicase when it is inserted into a naturally occurring Q beta replicase template. A recombinant RNA was constructed by inserting decaadenylic acid between nucleotides 63 and 64 of MDV-1 (+) RNA, using phage T4 RNA ligase. The insert was located away from regions of the template known to be required for the binding of the replicase and for the initiation of product strand synthesis. To minimize the disruption of template structure, we inserted the heterologous sequence into a hairpin loop on the exterior of the molecule. Q beta replicase copied this recombinant RNA in vitro, and the complementary product strands served as templates for the synthesis of additional copies of the original recombinant RNA. The reaction was therefore autocatalytic and the amount of recombinant RNA increased exponentially. A 300-fold amplification of the recombinant RNA occurred within nine minutes. Insertion of biologically significant RNAs into the MDV-1 RNA sequence should allow them to be replicated autocatalytically.     

Semi-conservative replication of double-stranded RNA by a virion-associated RNA polymerase

5-Bromo-UTP was found to replace UTP efficiently as a substrate for the virion-associated double-stranded RNA replicase of $\text{Penicillium}\text{stoloniferum}$ virus PsV-S. The double-stranded RNA product of the replication reaction with 5-bromo-UTP as a substrate gave in equilibrium caesium sulphate density gradient centrifugation a single band with a buoyant density of 1.647 g/ml, consistent with that of a hybrid double-stranded RNA consisting of one brominated and one unbrominated strand. After the reaction none of the original unbrominated double-stranded RNA (buoyant density 1.606 g/ml) could be detected. It is concluded that replication of double-stranded RNA in virions of PsV-S takes place by a semi-conservative mechanism.     

Dipyridamole reversibly inhibits mengovirus RNA replication

Dipyridamole is an effective inhibitor of cardiovirus growth in cell culture. The effects of dipyridamole on mengovirus replication in vivo and in vitro were examined in the hope the drug could be used as an experimental analog of the poliovirus inhibitor guanidine. Guanidine selectively inhibits poliovirus RNA synthesis but not RNA translation, and as such, has been a valuable research tool. Although guanidine does not inhibit cardiovirus infection, a compound with similar discriminatory characteristics would be experimentally useful for parallel work with these viruses. We found that mengovirus plaque formation in HeLa or L cells was inhibited nearly 100% by the presence of 80 muM dipyridamole. The inhibitory effect was reversible and targeted an early step in the replication cycle. Studies with luciferase-expressing mengovirus replicons showed that viral protein synthesis was unaffected by dipyridamole, and rather, RNA synthesis was the step targeted by the drug. This assessment was confirmed by direct analyses of viral translation and RNA synthesis activities in a Krebs-2-derived in vitro system that supported complete, infectious cardiovirus replication. In Krebs extracts, dipyridamole specifically inhibited viral RNA synthesis to more than 95%, with no concomitant effect on viral protein translation or polyprotein processing. The observed inhibition reversibly affected an early step in both minus-strand and plus-strand RNA synthesis, although inhibition of plus-strand synthesis was more profound than that of minus-strand synthesis. We conclude that dipyridamole is a potent experimental tool that readily distinguishes between cardiovirus translation and RNA replication functions.     

Inhibition of gammaherpesvirus replication by RNA interference

RNA interference (RNAi) is a conserved mechanism in which double-stranded, small interfering RNAs (siRNAs) trigger a sequence-specific gene-silencing process. Here we describe the inhibition of murine herpesvirus 68 replication by siRNAs targeted to sequences encoding Rta, an immediate-early protein known as an initiator of the lytic viral gene expression program, and open reading frame 45 (ORF 45), a conserved viral protein. Our results suggest that RNAi can block gammaherpesvirus replication and ORF 45 is required for efficient viral production.     

Inhibition of virus replication by RNA interference

RNA interference (RNAi) is a sequence-specific gene-silencing mechanism in eukaryotes, which is believed to function as a defence against viruses and transposons. Since its discovery, RNAi has been developed into a widely used technique for generating genetic knock-outs and for studying gene function by reverse genetics. Additionally, inhibition of virus replication by means of induced RNAi has now been reported for numerous viruses, including several important human pathogens such as human immunodeficiency virus type 1, hepatitis C virus, hepatitis B virus, dengue virus, poliovirus and influenza virus A. In this review, we will summarize the current data on RNAi-mediated inhibition of virus replication and discuss the possibilities for the development of RNAi-based antiviral therapeutics.     

Nodavirus RNA replication protein a induces membrane association of genomic RNA

Positive-strand RNA virus genome replication occurs in membrane-associated RNA replication complexes, whose assembly remains poorly understood. Here we show that prior to RNA replication, the multifunctional, transmembrane RNA replication protein A of the nodavirus flock house virus (FHV) recruits FHV genomic RNA1 to a membrane-associated state in both Drosophila melanogaster and Saccharomyces cerevisiae cells. Protein A has mitochondrial membrane-targeting, self-interaction, RNA-dependent RNA polymerase (RdRp), and RNA capping domains. In the absence of RdRp activity due to an active site mutation (A(D692E)), protein A stimulated RNA1 accumulation by increasing RNA1 stability. Protein A(D692E) stimulated RNA1 accumulation in wild-type cells and in xrn1(-) yeast defective in decapped RNA decay, showing that increased RNA1 stability was not due to protein A-mediated RNA1 recapping. Increased RNA1 stability was closely linked with protein A-induced membrane association of the stabilized RNA and was highly selective for RNA1. Substantial N- and C-proximal regions of protein A were dispensable for these activities. However, increased RNA1 accumulation was eliminated by deleting protein A amino acids (aa) 1 to 370 but was restored completely by adding back the transmembrane domain (aa 1 to 35) and partially by adding back peripheral membrane association sequences in aa 36 to 370. Moreover, although RNA polymerase activity was not required, even small deletions in or around the RdRp domain abolished increased RNA1 accumulation. These and other results show that prior to negative-strand RNA synthesis, multiple domains of mitochondrially targeted protein A cooperate to selectively recruit FHV genomic RNA to membranes where RNA replication complexes form.