scholarly journals The structure of a dimeric form of SARS-CoV-2 polymerase

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Florian A. Jochheim ◽  
Dimitry Tegunov ◽  
Hauke S. Hillen ◽  
Jana Schmitzová ◽  
Goran Kokic ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Catalytic Subunit ◽  
Template Switching ◽  
Rna Dependent Rna Polymerase ◽  
Genomic Rnas ◽  
Dimeric Form

AbstractThe coronavirus SARS-CoV-2 uses an RNA-dependent RNA polymerase (RdRp) to replicate and transcribe its genome. Previous structures of the RdRp revealed a monomeric enzyme composed of the catalytic subunit nsp12, two copies of subunit nsp8, and one copy of subunit nsp7. Here we report an alternative, dimeric form of the enzyme and resolve its structure at 5.5 Å resolution. In this structure, the two RdRps contain only one copy of nsp8 each and dimerize via their nsp7 subunits to adopt an antiparallel arrangement. We speculate that the RdRp dimer facilitates template switching during production of sub-genomic RNAs.

scholarly journals Dimeric form of SARS-CoV-2 polymerase

2021 ◽  
Author(s):  
Florian Alexander Jochheim ◽  
Dimitry Tegunov ◽  
Hauke S Hillen ◽  
Jana Schmitzova ◽  
Goran Kokic ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Catalytic Subunit ◽  
Template Switching ◽  
Rna Dependent Rna Polymerase ◽  
Genomic Rnas ◽  
Dimeric Form

The coronavirus SARS-CoV-2 uses an RNA-dependent RNA polymerase (RdRp) to replicate and transcribe its genome. Structures of the RdRp revealed a monomeric enzyme composed of the catalytic subunit nsp12, two copies of subunit nsp8, and one copy of subunit nsp7. Here we report an alternative, dimeric form of the coronavirus polymerase and resolve its structure at 4.8 Å resolution. In this structure, the two RdRps contain only one copy of nsp8 and dimerize via their nsp7 subunits to adopt an antiparallel arrangement. We speculate that the RdRp dimer facilitates template switching during production of sub-genomic RNAs for transcription.

scholarly journals Indels in SARS-CoV-2 occur at template-switching hotspots

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Brianna Sierra Chrisman ◽  
Kelley Paskov ◽  
Nate. Stockham ◽  
Kevin Tabatabaei ◽  
Jae-Yoon Jung ◽  
...  
Keyword(s):  
Secondary Structure ◽  
Rna Polymerase ◽  
Evolutionary Dynamics ◽  
De Novo ◽  
Template Switching ◽  
Nucleotide Polymorphisms ◽  
Rna Dependent Rna Polymerase ◽  
Consensus Sequences ◽  
Insertions And Deletions

AbstractThe evolutionary dynamics of SARS-CoV-2 have been carefully monitored since the COVID-19 pandemic began in December 2019. However, analysis has focused primarily on single nucleotide polymorphisms and largely ignored the role of insertions and deletions (indels) as well as recombination in SARS-CoV-2 evolution. Using sequences from the GISAID database, we catalogue over 100 insertions and deletions in the SARS-CoV-2 consensus sequences. We hypothesize that these indels are artifacts of recombination events between SARS-CoV-2 replicates whereby RNA-dependent RNA polymerase (RdRp) re-associates with a homologous template at a different loci (“imperfect homologous recombination”). We provide several independent pieces of evidence that suggest this. (1) The indels from the GISAID consensus sequences are clustered at specific regions of the genome. (2) These regions are also enriched for 5’ and 3’ breakpoints in the transcription regulatory site (TRS) independent transcriptome, presumably sites of RNA-dependent RNA polymerase (RdRp) template-switching. (3) Within raw reads, these indel hotspots have cases of both high intra-host heterogeneity and intra-host homogeneity, suggesting that these indels are both consequences of de novo recombination events within a host and artifacts of previous recombination. We briefly analyze the indels in the context of RNA secondary structure, noting that indels preferentially occur in “arms” and loop structures of the predicted folded RNA, suggesting that secondary structure may be a mechanism for TRS-independent template-switching in SARS-CoV-2 or other coronaviruses. These insights into the relationship between structural variation and recombination in SARS-CoV-2 can improve our reconstructions of the SARS-CoV-2 evolutionary history as well as our understanding of the process of RdRp template-switching in RNA viruses.

scholarly journals Predicting Intraserotypic Recombination in Enterovirus 71

2018 ◽  
Vol 93 (4) ◽  
Author(s):  
Andrew Woodman ◽  
Kuo-Ming Lee ◽  
Richard Janissen ◽  
Yu-Nong Gong ◽  
Nynke H. Dekker ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Iii ◽  
Enterovirus 71 ◽  
Template Switching ◽  
Rna Dependent Rna Polymerase ◽  
A Cell ◽  
Strain Type ◽  
Species Groups ◽  
Recombination Junction ◽  
Recombination Assay

ABSTRACTEnteroviruses are well known for their ability to cause neurological damage and paralysis. The model enterovirus is poliovirus (PV), the causative agent of poliomyelitis, a condition characterized by acute flaccid paralysis. A related virus, enterovirus 71 (EV-A71), causes similar clinical outcomes in recurrent outbreaks throughout Asia. Retrospective phylogenetic analysis has shown that recombination between circulating strains of EV-A71 produces the outbreak-associated strains which exhibit increased virulence and/or transmissibility. While studies on the mechanism(s) of recombination in PV are ongoing in several laboratories, little is known about factors that influence recombination in EV-A71. We have developed a cell-based assay to study recombination of EV-A71 based upon previously reported assays for poliovirus recombination. Our results show that (i) EV-A71 strain type and RNA sequence diversity impacts recombination frequency in a predictable manner that mimics the observations found in nature; (ii) recombination is primarily a replicative process mediated by the RNA-dependent RNA polymerase; (iii) a mutation shown to reduce recombination in PV (L420A) similarly reduces EV-A71 recombination, suggesting conservation in mechanism(s); and (iv) sequencing of intraserotypic recombinant genomes indicates that template switching occurs by a mechanism that may require some sequence homology at the recombination junction and that the triggers for template switching may be sequence independent. The development of this recombination assay will permit further investigation on the interplay between replication, recombination and disease.IMPORTANCERecombination is a mechanism that contributes to genetic diversity. We describe the first assay to study EV-A71 recombination. Results from this assay mimic what is observed in nature and can be used by others to predict future recombination events within the enterovirus species A group. In addition, our results highlight the central role played by the viral RNA-dependent RNA polymerase (RdRp) in the recombination process. Further, our results show that changes to a conserved residue in the RdRp from different species groups have a similar impact on viable recombinant virus yields, which is indicative of conservation in mechanism.

scholarly journals Mechanism of RNA Recombination in Carmo- and Tombusviruses: Evidence for Template Switching by the RNA-Dependent RNA Polymerase In Vitro

2003 ◽  
Vol 77 (22) ◽  
pp. 12033-12047 ◽  
Author(s):  
Chi-Ping Cheng ◽  
Peter D. Nagy
Keyword(s):  
Rna Polymerase ◽  
Template Switching ◽  
Rna Recombination ◽  
Cloning And Sequencing ◽  
Rna Dependent Rna Polymerase ◽  
Replication Process ◽  
Satellite Rnas ◽  
Rna Ligation ◽  
Rna Formation

ABSTRACT RNA recombination occurs frequently during replication of tombusviruses and carmoviruses, which are related small plus-sense RNA viruses of plants. The most common recombinants generated by these viruses are either defective interfering (DI) RNAs or chimeric satellite RNAs, which are thought to be generated by template switching of the viral RNA-dependent RNA polymerase (RdRp) during the viral replication process. To test if RNA recombination is mediated by the viral RdRp, we used either a purified recombinant RdRp of Turnip crinkle carmovirus or a partially purified RdRp preparation of Cucumber necrosis tombusvirus. We demonstrated that these RdRp preparations generated RNA recombinants in vitro. The RdRp-driven template switching events occurred between either identical templates or two different RNA templates. The template containing a replication enhancer recombined more efficiently than templates containing artificial sequences. We also observed that AU-rich sequences promote recombination more efficiently than GC-rich sequences. Cloning and sequencing of the generated recombinants revealed that the junction sites were located frequently at the ends of the templates (end-to-end template switching). We also found several recombinants that were generated by template switching involving internal positions in the RNA templates. In contrast, RNA ligation-based RNA recombination was not detected in vitro. Demonstration of the ability of carmo- and tombusvirus RdRps to switch RNA templates in vitro supports the copy-choice models of RNA recombination and DI RNA formation for these viruses.

scholarly journals Isolation and Analysis of Rare Norovirus Recombinants from Coinfected Mice Using Drop-Based Microfluidics

2015 ◽  
Vol 89 (15) ◽  
pp. 7722-7734 ◽  
Author(s):  
Huidan Zhang ◽  
Shelley K. Cockrell ◽  
Abimbola O. Kolawole ◽  
Assaf Rotem ◽  
Adrian W. R. Serohijos ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Viruses ◽  
Phylogenetic Analyses ◽  
Low Frequency ◽  
Template Switching ◽  
Murine Norovirus ◽  
Rna Dependent Rna Polymerase ◽  
Infectious Gastroenteritis

ABSTRACTHuman noroviruses (HuNoVs) are positive-sense RNA viruses that can cause severe, highly infectious gastroenteritis. HuNoV outbreaks are frequently associated with recombination between circulating strains. Strain genotyping and phylogenetic analyses show that noroviruses often recombine in a highly conserved region near the junction of the viral polyprotein (open reading frame 1 [ORF1]) and capsid (ORF2) genes and occasionally within the RNA-dependent RNA polymerase (RdRP) gene. Although genotyping methods are useful for tracking changes in circulating viral populations, they report only the dominant recombinant strains and do not elucidate the frequency or range of recombination events. Furthermore, the relatively low frequency of recombination in RNA viruses has limited studies to cell culture orin vitrosystems, which do not reflect the complexities and selective pressures present in an infected organism. Using two murine norovirus (MNV) strains to model coinfection, we developed a microfluidic platform to amplify, detect, and recover individual recombinants followingin vitroandin vivocoinfection. One-step reverse transcriptase PCR (RT-PCR) was performed in picoliter drops with primers that identified the wild-type and recombinant progenies and scanned for recombination breakpoints at ∼1-kb intervals. We detected recombination between MNV strains at multiple loci spanning the viral protease, RdRP, and capsid ORFs and isolated individual recombinant RNA genomes that were present at a frequency of 1/300,000 or higher. This study is the first to examine norovirus recombination following coinfection of an animal and suggests that the exchange of RNA among viral genomes in an infected host occurs in multiple locations and is an important driver of genetic diversity.IMPORTANCERNA viruses increase diversity and escape host immune barriers by genomic recombination. Studies using a number of viral systems indicate that recombination occurs via template switching by the virus-encoded RNA-dependent RNA polymerase (RdRP). However, factors that govern the frequency and positions of recombination in an infected organism remain largely unknown. This work leverages advances in the applied physics of drop-based microfluidics to isolate and sequence rare recombinants arising from the coinfection of mice with two distinct strains of murine norovirus. This study is the first to detect and analyze norovirus recombination in an animal model.

scholarly journals Isolation of reconstructed functional ribonucleoprotein complexes of Machupo virus

2021 ◽  
Author(s):  
Jesse D. Pyle ◽  
Sean P. J. Whelan
Keyword(s):  
Rna Polymerase ◽  
Mammalian Cells ◽  
Severe Disease ◽  
Host Cells ◽  
Genomic Rna ◽  
Replication Machinery ◽  
Rna Dependent Rna Polymerase ◽  
Genomic Rnas ◽  
Ribonucleoprotein Complexes ◽  
Machupo Virus

Arenaviruses initiate infection by delivering a transcriptionally-competent ribonucleoprotein (RNP) complex into the cytosol of host cells. The arenavirus RNP consists of the large (L) RNA-dependent RNA polymerase (RdRP) bound to a nucleoprotein (NP)-encapsidated genomic RNA (vRNA) template. During transcription and replication, L must transiently displace RNA-bound NP to allow for template access into the RdRP active site. Concomitant with RNA replication new subunits of NP must be added to the nascent complementary RNAs (cRNA) as they emerge from the product exit channel of L. Interactions between L and NP thus play a central role in arenavirus gene expression. We developed an approach to purify recombinant functional RNPs from mammalian cells in culture using a synthetic vRNA, affinity-tagged L and NP. Negative-stain electron microscopy of purified RNPs revealed they adopt diverse and flexible structures, like RNPs of other Bunyavirales members. Monodisperse L-NP and trimeric ring-like NP complexes were also obtained in excess of flexible RNPs, suggesting that these heterodimeric structures self-assemble in the absence of suitable RNA templates. This work allows for further biochemical analysis of the interaction between arenavirus L and NP proteins and provides a framework for future high-resolution structural analyses of this replication-associated complex. IMPORTANCE Arenaviruses are rodent-borne pathogens that can cause severe disease in humans. All arenaviruses begin the infection cycle with delivery of the virus replication machinery into the cytoplasm of the host cell. This machinery consists of an RNA-dependent RNA polymerase – which copies the viral genome segments and synthesizes all four viral mRNAs – bound to the two nucleoprotein-encapsidated genomic RNAs. How this complex assembles remains a mystery. Our findings provide direct evidence for the formation of diverse intracellular arenavirus replication complexes using purification strategies for the polymerase, nucleoprotein, and genomic RNA of Machupo virus, which causes Bolivian hemorrhagic fever in humans. We demonstrate that the polymerase and nucleoprotein assemble into higher-order structures within cells, providing a model for the molecular events of arenavirus RNA synthesis. These findings provide a framework for probing the architectures and functions of the arenavirus replication machinery, and thus advancing antiviral strategies targeting this essential complex.

scholarly journals Small-Scale Isolation of Viral RNA-Dependent RNA Polymerase from Protoplasts Inoculated with In Vitro Transcripts

2001 ◽  
Vol 91 (8) ◽  
pp. 747-752
Author(s):  
Scott Adkins ◽  
Dennis J. Lewandowski
Keyword(s):  
Rna Polymerase ◽  
Brome Mosaic Virus ◽  
Small Scale ◽  
Rna Dependent Rna Polymerase ◽  
Cowpea Chlorotic Mottle Virus ◽  
Genomic Rnas ◽  
Chlorotic Mottle Virus ◽  
Chlorotic Mottle ◽  
In Vitro Transcripts

Cowpea chlorotic mottle virus (CCMV) replicated in tobacco suspension cell protoplasts inoculated with in vitro transcripts of CCMV RNA1, 2, and 3. CCMV RNA-dependent RNA polymerase (RdRp) isolated from these protoplasts specifically recognized CCMV and Brome mosaic virus (BMV) subgenomic RNA promoters and directed in vitro RNA synthesis in a manner indistinguishable from CCMV RdRp more laboriously isolated from systemically infected cowpea leaves. Omission of CCMV RNA3 from the protoplast inoculum or replacement with in vitro transcripts of BMV RNA3 reduced CCMV (+)-strand RNA1 and 2 accumulation to ≈1/40 and ≈1/10, respectively, of the level attained when CCMV RNA3 was present. The absence of CCMV RNA3 did not prevent assembly and isolation of highly active, template-dependent and template-specific CCMV RdRp, which directed synthesis of products identical in size to those of RdRp isolated from protoplasts inoculated with all three CCMV genomic RNAs. These results demonstrate that CCMV RNA1 and 2 are sufficient for CCMV replication and RdRp assembly in tobacco protoplasts. This approach for isolation of functional viral RdRp will be especially useful for viruses for which large quantities of infected tissue are unavailable, such as those with specific tissue tropisms or mutants incapable of systemic movement.

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