Mechanism for de novo initiation at two sites in the respiratory syncytial virus promoter

Cressey, Tessa N.; Noton, Sarah L.; Nagendra, Kartikeya; Braun, Molly R.; Fearns, Rachel

doi:10.1093/nar/gky480

Cited by 26 publications

(39 citation statements)

References 46 publications

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“…1b, c). Using an established RdRp assay 13,41 , we demonstrated the characteristic RNA products of both de novo initiation (≤25 nt) and back primer (>25 nt) activities by the wt polymerase L:P (lane 6), but not the catalytically inactive polymerase L(D811A):P (lane 3) using a short trailer complementary 25 (TrC25) RNA template. The Le or TrC sequences of the genome serve as the promoters for the RSV polymerase.…”

Section: Resultsmentioning

confidence: 99%

Cryo-EM structure of the respiratory syncytial virus RNA polymerase

et al. 2020

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The respiratory syncytial virus (RSV) RNA polymerase, constituted of a 250 kDa large (L) protein and tetrameric phosphoprotein (P), catalyzes three distinct enzymatic activitiesnucleotide polymerization, cap addition, and cap methylation. How RSV L and P coordinate these activities is poorly understood. Here, we present a 3.67 Å cryo-EM structure of the RSV polymerase (L:P) complex. The structure reveals that the RNA dependent RNA polymerase (RdRp) and capping (Cap) domains of L interact with the oligomerization domain (P OD) and C-terminal domain (P CTD) of a tetramer of P. The density of the methyltransferase (MT) domain of L and the N-terminal domain of P (P NTD) is missing. Further analysis and comparison with other RNA polymerases at different stages suggest the structure we obtained is likely to be at an elongation-compatible stage. Together, these data provide enriched insights into the interrelationship, the inhibitors, and the evolutionary implications of the RSV polymerase.

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Section: Resultsmentioning

confidence: 99%

Cryo-EM structure of the respiratory syncytial virus RNA polymerase

et al. 2020

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“…Both processes are initiated by the L-P complex from a common promoter at the 3´end of the genome [17][18][19]. The polymerase initiates transcription and antigenome synthesis from two distinct sites within the same promoter, and the relative concentrations of the respective initiating NTPs can determine which initiation site the polymerase selects [20]. This, coupled with the fact that the polymerase can only engage in antigenome synthesis following accumulation of sufficient soluble N protein to encapsidate the antigenome, provides an explanation for how a polymerase molecule engaging with the genome template can become committed to either mRNA transcription versus antigenome production [21], but how transcription and replication are temporally controlled remains unclear.…”

Section: Introductionmentioning

confidence: 99%

Polymerase-tagged respiratory syncytial virus reveals a dynamic rearrangement of the ribonucleocapsid complex during infection

et al. 2020

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The ribonucleocapsid complex of respiratory syncytial virus (RSV) is responsible for both viral mRNA transcription and viral replication during infection, though little is known about how this dual function is achieved. Here, we report the use of a recombinant RSV virus with a FLAG-tagged large polymerase protein, L, to characterize and localize RSV ribonucleocapsid structures during the early and late stages of viral infection. Through proximity ligation assays and super-resolution microscopy, viral RNA and proteins in the ribonucleocapsid complex were revealed to dynamically rearrange over time, particularly between 6 and 8 hours post infection, suggesting a connection between the ribonucleocapsid structure and its function. The timing of ribonucleocapsid rearrangement corresponded with an increase in RSV genome RNA accumulation, indicating that this rearrangement is likely involved with the onset of RNA replication and secondary transcription. Additionally, early overexpression of RSV M2-2 from in vitro transcribed mRNA was shown to inhibit virus infection by rearranging the ribonucleocapsid complex. Collectively, these results detail a critical understanding into the localization and activity of RSV L and the ribonucleocapsid complex during RSV infection.

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“…Given that the ebolavirus gene start signals begin with a C residue (27,28), this could reflect a preference of the polymerase for initiating with GTP. A recent study of RSV initiation showed that its polymerase has a template-independent affinity for the initiating NTPs and that this plays a role in initiation site selection (29). Thus, it is possible that ebolavirus polymerases preferentially bind GTP due to template-independent affinity, and that this affinity combined with positioning by the promoter helps guide initiation at the correct site.…”

Section: Discussionmentioning

confidence: 99%

Ebolavirus polymerase uses an unconventional genome replication mechanism

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Cressey

Hume

et al. 2019

Proc. Natl. Acad. Sci. U.S.A.

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Most nonsegmented negative strand (NNS) RNA virus genomes have complementary 3′ and 5′ terminal nucleotides because the promoters at the 3′ ends of the genomes and antigenomes are almost identical to each other. However, according to published sequences, both ends of ebolavirus genomes show a high degree of variability, and the 3′ and 5′ terminal nucleotides are not complementary. If correct, this would distinguish the ebolaviruses from other NNS RNA viruses. Therefore, we investigated the terminal genomic and antigenomic nucleotides of three different ebolavirus species, Ebola (EBOV), Sudan, and Reston viruses. Whereas the 5′ ends of ebolavirus RNAs are highly conserved with the sequence ACAGG-5′, the 3′ termini are variable and are typically 3′-GCCUGU, ACCUGU, or CCUGU. A small fraction of analyzed RNAs had extended 3′ ends. The majority of 3′ terminal sequences are consistent with a mechanism of nucleotide addition by hairpin formation and back-priming. Using single-round replicating EBOV minigenomes, we investigated the effect of the 3′ terminal nucleotide on viral replication and found that the EBOV polymerase initiates replication opposite the 3′-CCUGU motif regardless of the identity of the 3′ terminal nucleotide(s) and of the position of this motif relative to the 3′ end. Deletion or mutation of the first residue of the 3′-CCUGU motif completely abolished replication initiation, suggesting a crucial role of this nucleotide in directing initiation. Together, our data show that ebolaviruses have evolved a unique replication strategy among NNS RNA viruses resulting in 3′ overhangs. This could be a mechanism to avoid antiviral recognition.Ebola virus replication | Ebola virus genome ends | variable 3′ genome ends | ebolavirus replication initiation | nonsegmented negative strand RNA virus replication

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Mechanism for de novo initiation at two sites in the respiratory syncytial virus promoter

Cited by 26 publications

References 46 publications

Cryo-EM structure of the respiratory syncytial virus RNA polymerase

Cryo-EM structure of the respiratory syncytial virus RNA polymerase

Polymerase-tagged respiratory syncytial virus reveals a dynamic rearrangement of the ribonucleocapsid complex during infection

Ebolavirus polymerase uses an unconventional genome replication mechanism

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