A specific sequence in the genome of respiratory syncytial virus regulates the generation of copy-back defective viral genomes

Sun, Yan; Kim, Young Ho; Speranza, Emily; Taylor, Louis J.; Agarwal, Divyansh; Gerhardt, Dawn M.; Bennett, Richard S.; Connor, John H.; Grant, Gregory R.; López, Carolina B.

doi:10.1101/349001

Cited by 12 publications

(29 citation statements)

References 57 publications

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“…However, different from deletion and point mutation DVGs, copy-back DVGs are frequently found in discrete dominant populations in an infected cell or tissue, and the same copy-back DVG seems to arise in independent infections with the same parental virus 54,55 or during infections with different virus strains 56 . The demonstration of hotspots for the generation of copyback DVGs from respiratory syncytial virus (RSV) and the identification of specific nucleotides that determine where copy-back DVGs rejoin further demonstrate that the generation of copy-back DVGs is not completely random, but instead that specific sequences encoded in the viral genome direct or facilitate their formation 50 in some infections, DVG generation is not a completely stochastic process and, instead, virus-encoded sequences favour the production and/or amplification of predominant DVGs. It remains to be determined whether conservation is a property of certain DVG types and which specific sequences and/or RNA structures lead to DVG generation in these conditions.…”

Section: Mechanisms Of Dvg Generationmentioning

confidence: 99%

“…While recent NGS data reveal that hundreds of DVGs can arise within a single viral infection, the fact that a smaller subset of dominant DVGs are repeatedly detected in different samples 50 indicates that complex dynamics are at play within the viral population. These complexities include competition (and possibly compensation or cooperation) between different DVGs and positive selection of the best competitors that implicates their relative fitness in relation to the wild-type parental virus and other DVGs.…”

Section: Impact On Viral Evolution and Dynamicsmentioning

confidence: 99%

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Defective viral genomes are key drivers of the virus–host interaction

2019

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Viruses survive often harsh host environments, yet we know little about the strategies they utilize to adapt and subsist given their limited genomic resources. We are beginning to appreciate the surprising versatility of viral genomes and how replicationcompetent and -defective virus variants can provide means for adaptation, immune escape and virus perpetuation. This Review summarizes current knowledge of the types of defective viral genomes generated during the replication of RNA viruses and the functions that they carry out. We highlight the universality and diversity of defective viral genomes during infections and discuss their predicted role in maintaining a fit virus population, their impact on human and animal health, and their potential to be harnessed as antiviral tools. Review ARticleNATuRe MicRoBiology extinction interfere with replication of a wild-type viral genome 29,30 . Hypermutations and mutations leading to frame shifts also result in defective viruses 31 (Fig. 1a). One example is mutations introduced by the cellular protein APOBEC3G into pro-retroviruses 32 . APOBEC3G deaminates deoxycytidine nucleotides in the viral DNA, leading to hypermutations that interfere with the ability of the viral genome to integrate into the host genome and replicate. Interestingly, in opposition to an interfering activity for these mutated pro-viruses, it has been proposed that defective proviruses enhance fitness and that complementation among them drives viral persistence and pathogenesis 33,34 .Deletions. The genomic viral species most commonly referred to as DVGs are truncated viral genomes resulting from large internal deletions occurring during viral replication. Deletion DVGs tend to bear single large truncations that remove several or all essential genes required for self-propagation while retaining 5′ and 3′ ends as well as other RNA structural elements required for polymerase binding, replication and/or packaging [35][36][37] . Multiple variants also exist, including DVGs that can be described as 'mosaic' in that they appear to be the result of multiple recombination and rearrangement events, including deletions, insertions, duplications and even inversions of parts of the genome [38][39][40] (Fig. 1b). Copy-backs.Copy-back and snap-back DVGs are rearranged genomes in which a sequence is duplicated in reverse complement to create theoretical stem-like structures (panhandle structures for copy-back DVGs or hairpin structures for snap-back DVGs) 41-43 . Copy-back DVGs have been reported in many negative-sense (ns) RNA viruses and are generated from the 5′ end of the genome in a process that produces DVGs with complementary 3′ and 5′ termini 44,45 . If the complementary region of the DVG comprises almost the entire sequence, the DVG can be further characterized as a snap-back DVG 43 (Fig. 1c). Copy-back DVGs are predicted to be generated when the viral RNA-dependent RNA polymerase (RdRP) detaches from the template and reattaches to the nascent strand, copying back the end of the genome 42,46 .

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Section: Mechanisms Of Dvg Generationmentioning

confidence: 99%

Section: Impact On Viral Evolution and Dynamicsmentioning

confidence: 99%

Defective viral genomes are key drivers of the virus–host interaction

2019

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“…Sequence-and factordependent pausing is well represented in the DNA-dependent RNA polymerase literature 45 . In case of the respiratory syncytial virus (RSV), sequences in the genome rich in G:C nucleotides were recently discovered that promote copy-back RNA synthesis 46 . In this system, these sequences alter polymerase elongation capacity 46 .…”

Section: Discussionmentioning

confidence: 99%

“…In case of the respiratory syncytial virus (RSV), sequences in the genome rich in G:C nucleotides were recently discovered that promote copy-back RNA synthesis 46 . In this system, these sequences alter polymerase elongation capacity 46 . It is intriguing to speculate that these sequences promote backtracking.…”

Section: Discussionmentioning

confidence: 99%

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

Janissen¹,

Woodman

Lee

et al. 2020

Preprint

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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.

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“…However, the process of DVGs generation may not be as stochastic as initially proposed; previous studies have shown that specific sequences in the genome of vesicular stomatitis virus (VSV) favour the generation of defective RNAs (22). Additionally, a recent study has shown that the generation of DVGs in respiratory syncytial virus (RSV) infections may favour specific regions of the on September 2, 2020 by guest http://jvi.asm.org/ Downloaded from genome suggesting the existence of hotspots that act as rejoin points for the viral polymerase during the formation of copyback DVGs (23).…”

Section: Downloaded Frommentioning

confidence: 99%

Innate Intracellular Antiviral Responses Restrict the Amplification of Defective Virus Genomes of Parainfluenza Virus 5

Wignall-Fleming

Vasou

Young

et al. 2020

J Virol

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During the replication of parainfluenza virus 5 (PIV5), copyback defective virus genomes (DVGs) are erroneously produced and are packaged into “infectious” virus particles. Copyback DVGs are the primary inducers of innate intracellular responses, including the interferon (IFN) response. While DVGs can interfere with the replication of nondefective (ND) virus genomes and activate the IFN-induction cascade before ND PIV5 can block the production of IFN, we demonstrate that the converse is also true, i.e., high levels of ND virus can block the ability of DVGs to activate the IFN-induction cascade. By following the replication and amplification of DVGs in A549 cells that are deficient in a variety of innate intracellular antiviral responses, we show that DVGs induce an uncharacterized IFN-independent innate response(s) that limits their replication. High-throughput sequencing was used to characterize the molecular structure of copyback DVGs. While there appears to be no sequence-specific break or rejoining points for the generation of copyback DVGs, our findings suggest there are region, size, and/or structural preferences selected for during for their amplification. IMPORTANCE Copyback defective virus genomes (DVGs) are powerful inducers of innate immune responses both in vitro and in vivo. They impact the outcome of natural infections, may help drive virus‐host coevolution, and promote virus persistence. Due to their potent interfering and immunostimulatory properties, DVGs may also be used therapeutically as antivirals and vaccine adjuvants. However, little is known of the host cell restrictions which limit their amplification. We show here that the generation of copyback DVGs readily occurs during parainfluenza virus 5 (PIV5) replication, but that their subsequent amplification is restricted by the induction of innate intracellular responses. Molecular characterization of PIV5 copyback DVGs suggests that while there are no genome sequence-specific breaks or rejoin points for the generation of copyback DVGs, genome region, size, and structural preferences are selected for during their evolution and amplification.

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A specific sequence in the genome of respiratory syncytial virus regulates the generation of copy-back defective viral genomes

Cited by 12 publications

References 57 publications

Defective viral genomes are key drivers of the virus–host interaction

Defective viral genomes are key drivers of the virus–host interaction

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

Innate Intracellular Antiviral Responses Restrict the Amplification of Defective Virus Genomes of Parainfluenza Virus 5

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