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 Structural Variants in SARS-CoV-2 Occur at Template-Switching Hotspots

2020 ◽  
Author(s):  
Brianna Chrisman ◽  
Kelley Paskov ◽  
Nate Stockham ◽  
Kevin Tabatabaei ◽  
Jae-Yoon Jung ◽  
...  
Keyword(s):  
Secondary Structure ◽  
Evolutionary Dynamics ◽  
De Novo ◽  
Template Switching ◽  
Nucleotide Polymorphisms ◽  
Structural Variants ◽  
Consensus Sequences ◽  
The Relationship ◽  
Folded Rna

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 structural variants (SVs) 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 imperfect homologous recombination between SARS-CoV-2 replicates, and provide four independent pieces of evidence. (1) The SVs 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 structural variant hotspots have cases of both high intra-host heterogeneity and intra-host homogeneity, suggesting that these structural variants are both consequences of de novo recombination events within a host and artifacts of previous recombination. (4) Within the RNA secondary structure, the indels occur in “arms” 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 Back-priming mode of ϕ6 RNA-dependent RNA polymerase

2005 ◽  
Vol 86 (2) ◽  
pp. 521-526 ◽  
Author(s):  
Minni R. L. Laurila ◽  
Paula S. Salgado ◽  
David I. Stuart ◽  
Jonathan M. Grimes ◽  
Dennis H. Bamford
Keyword(s):  
Rna Polymerase ◽  
De Novo ◽  
Hepatitis C Virus Rna ◽  
Rna Dependent Rna Polymerase ◽  
Initiation Mechanism ◽  
Double Stranded Rna ◽  
Virus Rna ◽  
Structural Methods ◽  
Similar Element

The RNA-dependent RNA polymerase of the double-stranded RNA bacteriophage ϕ6 is capable of primer-independent initiation, as are many RNA polymerases. The structure of this polymerase revealed an initiation platform, composed of a loop in the C-terminal domain (QYKW, aa 629–632), that was essential for de novo initiation. A similar element has been identified in hepatitis C virus RNA-dependent RNA polymerase. Biochemical studies have addressed the role of this platform, revealing that a mutant version can utilize a back-priming initiation mechanism, where the 3′ terminus of the template adopts a hairpin-like conformation. Here, the mechanism of back-primed initiation is studied further by biochemical and structural methods.

scholarly journals Structural basis for VPg-induced formation of RNA-dependent RNA polymerase multimeric complexes

2014 ◽  
Vol 70 (a1) ◽  
pp. C1601-C1601
Author(s):  
Ji-Hye Lee ◽  
Yeon Bin Chung ◽  
Jong Hyeon Seok ◽  
Kang Rok Han ◽  
Sella Kim ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
De Novo ◽  
Helical Structure ◽  
Science Research ◽  
Binding Mode ◽  
Structural Basis ◽  
Murine Norovirus ◽  
Virion Protein ◽  
Electron Microscopic ◽  
Rna Dependent Rna Polymerase

Norovirus is the leading cause of epidemic acute, nonbacterial gastroenteritis, and adopts de novo and VPg (Virion protein genome linked)-primed RNA synthesis by RNA-dependent RNA polymerase (RdRp). To understand the interaction between RdRp and VPg in replication of murine norovirus-1 (MNV-1), we determined the crystal structure of MNV-1 RdRp-VPg(1-73)-RNA complex. VPg was bound to the base of the palm domain and the tip of the fingers domain of RdRp simultaneously, but RNA template could not be modeled. The binding affinity constants (Kd) for RdRp-VPg was 3.7411.57 nM and VPg(1-73) showed approximately 90-fold less affinity than that of full-length VPg. In addition to this multiple binding mode, VPg enhanced the interactions of RdRp hexamers, leading to the formation of high-order multimers or tubular fibrils with significantly increased polymerase activity, confirmed by electron microscopic and biochemical studies. Our data indicated that MNV-1 VPg with helical structure was bound to RdRp at multiple sites and induces RdRp multimerization in viral replication. The multimers of RdRp-VPg-RNA can provide a mechanistic understanding of viral polymerase multimeric arrays and a new tool for development of antivirals to control norovirus outbreaks. This work was supported by a grant of the Korea Healthcare Technology R&D Project, Ministry of Health, Welfare and Family Affairs (A085119 K.H.K), Basic Science Research Program through the National Research Foundation (NRF-2013R1A1A2064940, L.J-H), Korea University Grant (L.J-H), and the BK21 plus program of the Ministry of Education, Korea.

scholarly journals De Novo Initiation of RNA Synthesis by the RNA-Dependent RNA Polymerase (NS5B) of Hepatitis C Virus

2000 ◽  
Vol 74 (2) ◽  
pp. 851-863 ◽  
Author(s):  
Guangxiang Luo ◽  
Robert K. Hamatake ◽  
Danielle M. Mathis ◽  
Jason Racela ◽  
Karen L. Rigat ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Rna Polymerase ◽  
Rna Synthesis ◽  
De Novo ◽  
Oligonucleotide Primer ◽  
Transferase Activity ◽  
Rdrp Activity ◽  
Rna Dependent Rna Polymerase ◽  
Hcv Ns5b

ABSTRACT Hepatitis C virus (HCV) NS5B protein possesses an RNA-dependent RNA polymerase (RdRp) activity, a major function responsible for replication of the viral RNA genome. To further characterize the RdRp activity, NS5B proteins were expressed from recombinant baculoviruses, purified to near homogeneity, and examined for their ability to synthesize RNA in vitro. As a result, a highly active NS5B RdRp (1b-42), which contains an 18-amino acid C-terminal truncation resulting from a newly created stop codon, was identified among a number of independent isolates. The RdRp activity of the truncated NS5B is comparable to the activity of the full-length protein and is 20 times higher in the presence of Mn2+ than in the presence of Mg2+. When a 384-nucleotide RNA was used as the template, two major RNA products were synthesized by 1b-42. One is a complementary RNA identical in size to the input RNA template (monomer), while the other is a hairpin dimer RNA synthesized by a “copy-back” mechanism. Substantial evidence derived from several experiments demonstrated that the RNA monomer was synthesized through de novo initiation by NS5B rather than by a terminal transferase activity. Synthesis of the RNA monomer requires all four ribonucleotides. The RNA monomer product was verified to be the result of de novo RNA synthesis, as two expected RNA products were generated from monomer RNA by RNase H digestion. In addition, modification of the RNA template by the addition of the chain terminator cordycepin at the 3′ end did not affect synthesis of the RNA monomer but eliminated synthesis of the self-priming hairpin dimer RNA. Moreover, synthesis of RNA on poly(C) and poly(U) homopolymer templates by 1b-42 NS5B did not require the oligonucleotide primer at high concentrations (≥50 μM) of GTP and ATP, further supporting a de novo initiation mechanism. These findings suggest that HCV NS5B is able to initiate RNA synthesis de novo.

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.

The role of RNA-polymerase II transcription in embryonic nucleologenesis by bovine embryos

Biologia ◽  
2010 ◽  
Vol 65 (3) ◽  
Author(s):  
Mária Kovalská ◽  
Ida Petrovičová ◽  
František Strejček ◽  
Marian Adamkov ◽  
Erika Halašová ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
De Novo ◽  
Control Group ◽  
Bovine Embryos ◽  
Embryonic Genome Activation ◽  
Embryonic Genome ◽  
Autoradiographic Labeling ◽  
Genome Activation

AbstractThe early stages of embryonic development are maternally driven. As development proceeds, maternally inherited informational molecules decay, and embryogenesis becomes dependent on de novo synthesized RNAs of embryonic genome. The aim of the present study is to investigate the role of de novo transcription in the development of embryos during embryonic genome activation. Autoradiography for detection of transcriptional activity and transmission electron microscopy were applied in in vitro produced bovine embryos cultured to the late 8-cell stage with or without (control group) α-amanitin, specific inhibitor of RNA-polymerases II and III transcription. The α-amanitin (AA) groups presented three sets of embryos cultivated with AA in different time intervals (6, 9 and 12 h). In control group, nucleoplasm and nucleolar structures displayed strong autoradiographic labeling and showed initial development of fibrillo-granular nucleoli. In α-amanitin groups, lack of autoradiographic labeling and disintegrated nucleolus precursor bodies (NPBs) stage were observed. Inhibition of RNA polymerase II (RNA pol II) already in the early phases of embryonic genome activation has detrimental effect on nucleolar formation and embryo survival, what was shown for the first time.

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