Genetic recombination of poliovirus facilitates subversion of host barriers to infection

Acevedo, Ashley; Woodman, Andrew; Jj, Arnold; Yeh, Malcolm; Evans, David J.; Ce, Cameron; Andino, Raul

doi:10.1101/273060

Cited by 6 publications

(15 citation statements)

References 53 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Our interpretation that the backtracked state and copy-back synthesis contributed to recombination and were of biological relevance was supported by evaluation of RdRp derivatives known to be defective for recombination in cells (Fig. 2) 19 . PV Y275H RdRp is one of the most robust, recombination-defective mutants described 19 , but the molecular basis for the defect is not known for this mutant or the others that have been reported in the PV system or other viral systems 20,43,44 .…”

Section: Discussionmentioning

confidence: 61%

“…2) 19 . PV Y275H RdRp is one of the most robust, recombination-defective mutants described 19 , but the molecular basis for the defect is not known for this mutant or the others that have been reported in the PV system or other viral systems 20,43,44 . Here, we show that EV-A71 Y276H RdRp is equally robust and recombination defective in cells (Fig.…”

Section: Discussionmentioning

confidence: 99%

“…The Andino and Barton laboratories identified Y275H in PV RdRp as a substitution that impaired recombination substantially, among several others 19,20 . The molecular basis for the reduced recombination efficiency is not known 19,20 . The orthologous substitution is Y276H in EV-A71 RdRp.…”

Section: A Recombination-deficient Ev-a71 Rdrp Variant Attenuates Virmentioning

confidence: 99%

See 2 more Smart Citations

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

Janissen¹,

Woodman

Lee

et al. 2020

Preprint

Self Cite

View full text Add to dashboard Cite

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 pyrazinecarboxamide 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.

show abstract

Section: Discussionmentioning

confidence: 61%

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

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

Janissen¹,

Woodman

Lee

et al. 2020

Preprint

Self Cite

View full text Add to dashboard Cite

show abstract

“…The polymerase mutations identified during serial passage in escalating concentrations of ribavirin were reverse engineered into infectious cDNA clones of poliovirus to derive genetically defined virus populations (Table 2, Ribavirin Selected Mutations). In addition, several engineered variants originating from our work, and that of others, were similarly generated (Table 2, Engineered Variants) (18, 19, 53, 60). Many of these mutations lie within an extended primer grip region of the polymerase, consisting of L420, M392 and primer grip residues (K375 & R376).…”

Section: Resultsmentioning

confidence: 99%

An Extended Primer Grip of Picornavirus Polymerase Facilitates Sexual RNA Replication Mechanisms

Kempf

Watkins

Peersen

et al. 2019

Preprint

View full text Add to dashboard Cite

Picornaviruses have both asexual and sexual RNA replication mechanisms. Asexual RNA replication mechanisms involve one parental template whereas sexual RNA replication mechanisms involve two or more parental templates. Because sexual RNA replication mechanisms counteract ribavirin-induced error catastrophe, we selected for ribavirin-resistant poliovirus to identify polymerase residues that facilitate sexual RNA replication mechanisms. We used serial passage in ribavirin, beginning with a variety of ribavirin-sensitive and ribavirin-resistant parental viruses. Ribavirin-sensitive virus contained an L420A polymerase mutation while ribavirin-resistant virus contained a G64S polymerase mutation. A G64 codon mutation (G64Fix) was used to inhibit emergence of G64S-mediated ribavirin resistance. Revertants (L420) or pseudo-revertants (L420V, L420I) were selected from all independent lineages of L420A, G64Fix L420A and G64S L420A parental viruses. Ribavirin-resistant G64S mutations were selected in two independent lineages and novel ribavirin-resistance mutations were selected in the polymerase in other lineages (M299I, M323I, M392V, T353I). The structural orientation of M392, immediately adjacent to L420 and the polymerase primer grip region, led us to engineer additional polymerase mutations into poliovirus (M392A, M392L & M392V and K375R & R376K). L420A revertants and pseudorevertants (L420V, L420I) restored efficient sexual RNA replication mechanisms, confirming that ribavirin-induced error catastrophe coincides with defects in sexual RNA replication mechanisms. Viruses containing M392 mutations (M392A, M392L & M392V) and primer grip mutations (K375R & R376K) exhibited divergent RNA recombination, ribavirin sensitivity and biochemical phenotypes, consistent with changes in the fidelity of RNA synthesis. We conclude that an extended primer grip of the polymerase, including L420, M392, K375 & R376, contributes to the fidelity of RNA synthesis and to efficient sexual RNA replication mechanisms.IMPORTANCEPicornaviruses have both asexual and sexual RNA replication mechanisms. Sexual RNA replication shapes picornavirus species groups, contributes to the emergence of vaccine-derived polioviruses and counteracts error catastrophe. Can viruses distinguish between homologous and non-homologous partners during sexual RNA replication? We implicate an extended primer grip of the viral polymerase in sexual RNA replication mechanisms. By sensing RNA sequence complementarity near the active site, the extended primer grip of the polymerase has the potential to distinguish between homologous and non-homologous RNA templates during sexual RNA replication.

show abstract

“…We hypothesized that EQI is avirulent without the acquisition of the VP1-K244E mutation in vivo . To reduce mutation rates and restrict generation of viral quasispecies, we engineered the viral RNA-dependent RNA polymerase (RdRp) of EQI to harbor previously identified high-fidelity mutations G64R and L123F (abbreviated as HF in Fig 5A) [39–41] and recombination deficient mutation Y276H (labelled as Rec - ) [42]. We employed a luciferase-based replicon system to assess the impact of these mutations on genome replication.…”

Section: Resultsmentioning

confidence: 99%

Electrostatic interactions at the five-fold axis alter heparin-binding phenotype and drive EV-A71 virulence in mice

Tee

Tan

Yogarajah

et al. 2019

Preprint

View full text Add to dashboard Cite

19Enterovirus A71 (EV-A71) causes hand, foot and mouth disease epidemics with neurological 20 complications and fatalities. However, the neuropathogenesis of EV-A71 remains poorly 21 understood. In mice, adaptation and virulence determinants have been mapped to mutations at 22 VP1-145, VP1-244 and VP2-149. We hypothesized that heparin-binding phenotype shapes 23 EV-A71 virulence in mice. We constructed six viruses with varying residues at VP1-98, VP1-24 145 (which are both heparin-binding determinants) and VP2-149 (based on the wild type 25 98E/145Q/149K, termed EQK) to generate KQK, KEK, EEK, EEI and EQI variants. We 26 demonstrated that the weak heparin-binder EEI was highly lethal in mice. The initially strong 27 heparin-binding EQI variant acquired an additional mutation VP1-K244E, which confers weak 28 heparin-binding phenotype resulting in elevated viremia and increased brain inflammation and 29 virus antigens in mice, with subsequent high virulence. EEI and EQI-K244E variants 30 inoculated into mice disseminated efficiently and displayed high viremia. Increasing 31 polymerase fidelity and impairing recombination of EQI attenuated virulence, suggesting the 32 importance of population diversity in EV-A71 pathogenesis in vivo. Combining in silico 33 docking and deep sequencing approaches, we inferred that virus population diversity is shaped 34 by electrostatic interactions at the five-fold axis of the virus surface. Electrostatic surface 35 charges facilitate virus adaptation by generating poor heparin-binding variants for better in vivo 36 dissemination in mice, likely due to reduced adsorption to heparin-rich peripheral tissues, 37 which ultimately results in increased neurovirulence. The dynamic switching from heparin-38 binding to weak heparin-binding phenotype in vivo explained the neurovirulence of EV-A71. 39 40 Author summary 41 Enterovirus A71 (EV-A71) is the primary cause of hand, foot and mouth disease, and it can 42 also infect the central nervous system and cause fatal outbreaks in young children. EV-A71 43 pathogenesis remains elusive. In this study, we demonstrated that EV-A71 variants with strong 44 affinity to heparan sulfate (heparin) have a growth advantage in tissue culture, but are 45 disadvantageous in vivo. When inoculated into mice, strong heparin-binding virus variants are 46 more likely to be adsorbed to peripheral tissues, resulting in impaired ability to disseminate 47 and being cleared from the bloodstream rapidly. The lower viremia level resulted in no 48 neuroinvasion. In contrast, weak heparin-binding variants show greater levels of viremia, 49 dissemination and subsequent neurovirulence in mice. We also provide evidence that the ability 50 of EV-A71 to bind heparin is mediated by electrostatic surface charges due to amino acids on 51 the virus capsid surface. In mice, EV-A71 undergoes adaptive mutation to acquire greater 52 negative surface charges, thus generating new virulent variants with weak heparin-binding 53 which allows greater viral spread. Our study underlines the...

show abstract

Genetic recombination of poliovirus facilitates subversion of host barriers to infection

Cited by 6 publications

References 53 publications

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

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

An Extended Primer Grip of Picornavirus Polymerase Facilitates Sexual RNA Replication Mechanisms

Electrostatic interactions at the five-fold axis alter heparin-binding phenotype and drive EV-A71 virulence in mice

Contact Info

Product

Resources

About