Biology subjects

Arnold, J. J.

Publications and source records attributed to Arnold, J. J..

26 records · Page 2Linked to original sources

bioRxiv2022

Interfering with nucleotide excision by the coronavirus 3'-to-5' exoribonuclease

Some of the most efficacious antiviral therapeutics are ribonucleos(t)ide analogs. The presence of a 3-to-5 proofreading exoribonuclease (ExoN) in coronaviruses diminishes the potency of many ribonucleotide analogs. The ability to interfere with ExoN activity will create new possibilities for control of SARS-CoV-2 infection. ExoN is formed by a 1:1 complex of nsp14 and nsp10 proteins. We have purified and characterized ExoN using a robust, quantitative system that reveals determinants of specificity and efficiency of hydrolysis. Double-stranded RNA is preferred over single-stranded RNA. Nucleotide excision is distributive, with only one or two nucleotides hydrolyzed in a single binding event. The composition of the terminal basepair modulates excision. A stalled SARS-CoV-2 replicase in complex with either correctly or incorrectly terminated products prevents excision, suggesting that a mispaired end is insufficient to displace the replicase. Finally, we have discovered several modifications to the 3-RNA terminus that interfere with or block ExoN-catalyzed excision. While a 3-OH facilitates hydrolysis of a nucleotide with a normal ribose configuration, this substituent is not required for a nucleotide with a planar ribose configuration such as that present in the antiviral nucleotide produced by viperin. Design of ExoN-resistant, antiviral ribonucleotides should be feasible.

biochemistry↗

bioRxiv2022

IRES-independent, eIF2A/eIF2D-dependent translation of the enterovirus genome

RNA recombination in positive-strand RNA viruses is a molecular-genetic process, which permits the greatest evolution of the genome and may be essential to stabilizing the genome from the deleterious consequences of accumulated mutations. Enteroviruses represent a useful system to elucidate the details of this process. On the biochemical level, it is known that RNA recombination is catalyzed by the viral RNA-dependent RNA polymerase using a template-switching mechanism. For this mechanism to function in cells, the recombining genomes must be located in the same subcellular compartment. How a viral genome is trafficked to the site of genome replication and recombination, which is membrane associated and isolated from the cytoplasm, is not known. We hypothesized that genome translation was essential for co-localization of genomes for recombination. We show that complete inactivation of internal ribosome entry site (IRES)-mediated translation of a donor enteroviral genome enhanced recombination instead of impairing it. Recombination did not occur by a non-replicative mechanism. Rather, sufficient translation of the non-structural region of the genome occurred to support subsequent steps required for recombination. The non-canonical translation initiation factors, eIF2A and eIF2D, were required for IRES-independent translation. Our results support an eIF2A/eIF2D-dependent mechanism under conditions in which the eIF2-dependent mechanism is inactive. Detection of an IRES-independent mechanism for translation of the enterovirus genome provides an explanation for a variety of debated observations, including non-replicative recombination and persistence of enteroviral RNA lacking an IRES. The existence of an eIF2A/eIF2D-dependent mechanism in enteroviruses predicts the existence of similar mechanisms in other viruses.

microbiology↗

bioRxiv2021

The nucleotide addition cycle of the SARS-CoV-2 polymerase

Coronaviruses have evolved elaborate multisubunit machines to replicate and transcribe their genomes. Central to these machines are the RNA-dependent RNA polymerase subunit (nsp12) and its intimately associated cofactors (nsp7 and nsp8). We have used a high-throughput magnetic-tweezers approach to develop a mechanochemical description of this core polymerase. The core polymerase exists in at least three catalytically distinct conformations, one being kinetically consistent with incorporation of incorrect nucleotides. We provide the first evidence that an RdRp uses a thermal ratchet instead of a power stroke to transition from the pre- to post-translocated state. Ultra-stable magnetic tweezers enables the direct observation of coronavirus polymerase deep and long-lived backtrack that are strongly stimulated by secondary structure in the template. The framework presented here elucidates one of the most important structure-dynamics-function relationships in human health today, and will form the grounds for understanding the regulation of this complex.

biophysics↗

bioRxiv2020

Signatures and mechanisms of efficacious therapeutic ribonucleotides against SARS-CoV-2 revealed by analysis of its replicase using magnetic tweezers

The nucleotide analog Remdesivir (RDV) is the only FDA-approved antiviral therapy to treat infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The physical basis for efficient utilization of RDV by SARS-CoV-2 polymerase is unknown. Here, we characterize the impact of RDV and other nucleotide analogs on RNA synthesis by the polymerase using a high-throughput, single-molecule, magnetic-tweezers platform. The location of the modification in the ribose or in the base dictates the catalytic pathway(s) used for its incorporation. We reveal that RDV incorporation does not terminate viral RNA synthesis, but leads the polymerase into deep backtrack, which may appear as termination in traditional ensemble assays. SARS-CoV-2 is able to evade the endogenously synthesized product of the viperin antiviral protein, ddhCTP, though the polymerase incorporates this nucleotide analog well. This experimental paradigm is essential to the discovery and development of therapeutics targeting viral polymerases. TeaserWe revise Remdesivirs mechanism of action and reveal SARS-CoV-2 ability to evade interferon-induced antiviral ddhCTP

biophysics↗

bioRxiv2020

Induced copy-back RNA synthesis as a novel therapeutic mechanism against RNA viruses

The viral RNA-dependent RNA polymerase (RdRp) is a well-established target for development of broad-spectrum antiviral therapeutics. Incorporation of ribonucleotide analogues by the RdRp will either cause termination of RNA synthesis or mutagenesis of the RNA product. We demonstrated recently that incorporation of a pyrazine-carboxamide ribonucleotide into nascent RNA leads to pausing and backtracking of the elongating RdRp. Here, we provide evidence for the single-stranded RNA product of backtracking serving as an intermediate in RdRp-catalyzed, template-switching reactions. This intermediate is used for both intramolecular template-switching (copy-back RNA synthesis) and intermolecular template-switching (homologous RNA recombination). The use of a magnetic-tweezers platform to monitor RdRp elongation dynamics permitted direct observation of copy-back synthesis and illuminated properties of the RdRp that promote copy-back synthesis, including stability of the RdRp-nascent-RNA complex and the dimensions of the RdRp nucleic-acid-binding channel. In cells, recombination was stimulated by the presence of a pyrazine-carboxamide ribonucleotide. The effect of the drug on recombination was diminished for a recombination-defective virus, but this virus was not resistant to the drug. The discovery that a ribonucleotide analogue can induce copy-back RNA synthesis suggests that this third mechanistic class of compounds may function by promoting formation of defective viral genomes. This study identifies RdRp-catalyzed intra- and intermolecular template switching as a viable new mechanistic target with potentially broad-spectrum appeal.

microbiology↗

bioRxiv2020

Temperature controlled high-throughput magnetic tweezers show striking difference in activation energies of replicating viral RNA-dependent RNA polymerases.

RNA virus survival depends on efficient viral genome replication, which is performed by the viral RNA dependent RNA polymerase (RdRp). The recent development of high throughput magnetic tweezers has enabled the simultaneous observation of dozens of viral RdRp elongation traces on kilobases long templates, and this has shown that RdRp nucleotide addition kinetics is stochastically interrupted by rare pauses of 1-1000 s duration, of which the short-lived ones (1-10 s) are the temporal signature of a low fidelity catalytic pathway. We present a simple and precise temperature controlled system for magnetic tweezers to characterize the replication kinetics temperature dependence between 25{degrees}C and 45{degrees}C of RdRps from three RNA viruses, i.e. the double-stranded RNA bacteriophage {Phi}6, and the positive-sense single-stranded RNA poliovirus (PV) and human rhinovirus C (HRV-C). We found that {Phi}6 RdRp is largely temperature insensitive, while PV and HRV-C RdRps replication kinetics are activated by temperature. Furthermore, the activation energies we measured for PV RdRp catalytic state corroborate previous estimations from ensemble pre-steady state kinetic studies, further confirming the catalytic origin of the short pauses and their link to temperature independent RdRp fidelity. This work will enable future temperature controlled study of biomolecular complex at the single molecule level.

biophysics↗

bioRxiv2018

A speed-fidelity trade-off determines the mutation rate and virulence of an RNA virus

Mutation rates can evolve through genetic drift, indirect selection due to genetic hitchhiking, or direct selection on the physicochemical cost of high fidelity. However, for many systems, it has been difficult to disentangle the relative impact of these forces empirically. In RNA viruses, an observed correlation between mutation rate and virulence has led many to argue that their extremely high mutation rates are advantageous, because they may allow for increased adaptability. This argument has profound implications, as it suggests that pathogenesis in many viral infections depends on rare or de novo mutations. Here we present data for an alternative model whereby RNA viruses evolve high mutation rates as a byproduct of selection for increased replicative speed. We find that a poliovirus antimutator, 3DG64S, has a significant replication defect and that wild type and 3DG64S populations have similar adaptability in two distinct cellular environments. Experimental evolution of 3DG64S under r-selection led to reversion and compensation of the fidelity phenotype. Mice infected with 3DG64S exhibited delayed morbidity at doses well above the LD50, consistent with attenuation by slower growth as opposed to reduced mutational supply. Furthermore, compensation of the 3DG64S growth defect restored virulence, while compensation of the fidelity phenotype did not. Our data are consistent with the kinetic proofreading model for biosynthetic reactions and suggest that speed is more important than accuracy. In contrast to what has been suggested for many RNA viruses, we find that within host spread is associated with viral replicative speed and not standing genetic diversity.\n\nAuthor SummaryMutation rate evolution has long been a fundamental problem in evolutionary biology. The polymerases of RNA viruses generally lack proofreading activity and exhibit extremely high mutation rates. Since most mutations are deleterious and mutation rates are tuned by natural selection, we asked why hasnt the virus evolved to have a lower mutation rate? We used experimental evolution and a murine infection model to show that RNA virus mutation rates may actually be too high and are not necessarily adaptive. Rather, our data indicate that viral mutation rates are driven higher as a result of selection for viruses with faster replication kinetics. We suggest that viruses have high mutation rates, not because they facilitate adaption, but because it is hard to be both fast and accurate.

microbiology↗

bioRxiv2018

Genetic recombination of poliovirus facilitates subversion of host barriers to infection

The contribution of RNA recombination to viral fitness and pathogenesis is poorly defined. Here, we isolate a recombination-deficient, poliovirus variant and find that, while recombination is detrimental to virus replication in tissue culture, recombination is important for pathogenesis in infected animals. Notably, recombination-defective virus exhibits severe attenuation following intravenous inoculation that is associated with a significant reduction in population size during intra-host spread. Because the impact of high mutational loads manifests most strongly at small population sizes, our data suggest that the repair of mutagenized genomes is an essential function of recombination and that this function may drive the long-term maintenance of recombination in viral species despite its associated fitness costs.\n\nSignificance StatementRNA recombination is a widespread but poorly understood feature of RNA virus replication. For poliovirus, recombination is involved in the emergence of neurovirulent circulating vaccine-derived poliovirus, which has hampered global poliovirus eradication efforts. This emergence illustrates the power of recombination to drive major adaptive change; however, it remains unclear if these adaptive events represent the primary role of recombination in virus survival. Here, we identify a viral mutant with a reduced rate of recombination and find that recombination also plays a central role in the spread of virus within animal hosts. These results highlight a novel approach for improving the safety of live attenuated vaccines and further our understanding of the role of recombination in virus pathogenesis and evolution.

microbiology↗