scholarly journals Characterization of norovirus 3Dpol RNA-dependent RNA polymerase activity and initiation of RNA synthesis

2006 ◽  
Vol 87 (9) ◽  
pp. 2621-2630 ◽  
Author(s):  
Jacques Rohayem ◽  
Katrin Jäger ◽  
Ivonne Robel ◽  
Ulrike Scheffler ◽  
Achim Temme ◽  
...  
Keyword(s):  
Time Course ◽  
Rna Synthesis ◽  
De Novo ◽  
Polymerase Activity ◽  
Opposite Polarity ◽  
Transferase Activity ◽  
Structural Protein ◽  
Double Stranded Rna ◽  
Terminal Transferase

Norovirus (NV) 3Dpol is a non-structural protein predicted to play an essential role in the replication of the NV genome. In this study, the characteristics of NV 3Dpol activity and initiation of RNA synthesis have been examined in vitro. Recombinant NV 3Dpol, as well as a 3Dpol active-site mutant were expressed in Escherichia coli and purified. NV 3Dpol was able to synthesize RNA in vitro and displayed flexibility with respect to the use of Mg2+ or Mn2+ as a cofactor. NV 3Dpol yielded two different products when incubated with synthetic RNA in vitro: (i) a double-stranded RNA consisting of two single strands of opposite polarity or (ii) the single-stranded RNA template labelled at its 3′ terminus by terminal transferase activity. Initiation of RNA synthesis occurred de novo rather than by back-priming, as evidenced by the fact that the two strands of the double-stranded RNA product could be separated, and by dissociation in time-course analysis of terminal transferase and RNA synthesis activities. In addition, RNA synthesis was not affected by blocking of the 3′ terminus of the RNA template by a chain terminator, sustaining de novo initiation of RNA synthesis. NV 3Dpol displays in vitro properties characteristic of RNA-dependent RNA polymerases, allowing the implementation of this in vitro enzymic assay for the development and validation of antiviral drugs against NV, a so far non-cultivated virus and an important human pathogen.

scholarly journals In Vitro Double-Stranded RNA Synthesis by Rotavirus Polymerase Mutants with Lesions at Core Shell Contact Sites

2019 ◽  
Vol 93 (20) ◽  
Author(s):  
Courtney L. Steger ◽  
Mackenzie L. Brown ◽  
Owen M. Sullivan ◽  
Crystal E. Boudreaux ◽  
Courtney A. Cohen ◽  
...  
Keyword(s):  
Enzymatic Activity ◽  
Functional Significance ◽  
Rna Synthesis ◽  
Polymerase Activity ◽  
Core Shell ◽  
Double Stranded Rna ◽  
The Core ◽  
Contact Sites ◽  
Shell Protein

ABSTRACT The rotavirus polymerase VP1 mediates all stages of viral RNA synthesis within the confines of subviral particles and while associated with the core shell protein VP2. Transcription (positive-strand RNA [+RNA] synthesis) by VP1 occurs within double-layered particles (DLPs), while genome replication (double-stranded RNA [dsRNA] synthesis) by VP1 occurs within assembly intermediates. VP2 is critical for VP1 enzymatic activity; yet, the mechanism by which the core shell protein triggers polymerase function remains poorly understood. Structural analyses of transcriptionally competent DLPs show that VP1 is located beneath the VP2 core shell and sits slightly off-center from each of the icosahedral 5-fold axes. In this position, the polymerase is contacted by the core shell at 5 distinct surface-exposed sites, comprising VP1 residues 264 to 267, 547 to 550, 614 to 620, 968 to 980, and 1022 to 1025. Here, we sought to test the functional significance of these VP2 contact sites on VP1 with regard to polymerase activity. We engineered 19 recombinant VP1 (rVP1) proteins that contained single- or multipoint alanine mutations within each individual contact site and assayed them for the capacity to synthesize dsRNA in vitro in the presence of rVP2. Three rVP1 mutants (E265A/L267A, R614A, and D971A/S978A/I980A) exhibited diminished in vitro dsRNA synthesis. Despite their loss-of-function phenotypes, the mutants did not show major structural changes in silico, and they maintained their overall capacity to bind rVP2 in vitro via their nonmutated contact sites. These results move us toward a mechanistic understanding of rotavirus replication and identify precise VP2-binding sites on the polymerase surface that are critical for its enzymatic activation. IMPORTANCE Rotaviruses are important pathogens that cause severe gastroenteritis in the young of many animals. The viral polymerase VP1 mediates all stages of viral RNA synthesis, and it requires the core shell protein VP2 for its enzymatic activity. Yet, there are several gaps in knowledge about how VP2 engages and activates VP1. Here, we probed the functional significance of 5 distinct VP2 contact sites on VP1 that were revealed through previous structural studies. Specifically, we engineered alanine amino acid substitutions within each of the 5 VP1 regions and assayed the mutant polymerases for the capacity to synthesize RNA in the presence of VP2 in a test tube. Our results identified residues within 3 of the VP2 contact sites that are critical for robust polymerase activity. These results are important because they enhance the understanding of a key step of the rotavirus replication cycle.

scholarly journals Structural and Functional Characterization of Sapovirus RNA-Dependent RNA Polymerase

2006 ◽  
Vol 81 (4) ◽  
pp. 1858-1871 ◽  
Author(s):  
Stephen W. B. Fullerton ◽  
Martina Blaschke ◽  
Bruno Coutard ◽  
Julia Gebhardt ◽  
Alexander Gorbalenya ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Synthesis ◽  
De Novo ◽  
Functional Characterization ◽  
Transferase Activity ◽  
Dependent Manner ◽  
Rna Dependent Rna Polymerase ◽  
Key Enzyme

ABSTRACT Sapoviruses are one of the major agents of acute gastroenteritis in childhood. They form a tight genetic cluster (genus) in the Caliciviridae family that regroups both animal and human pathogenic strains. No permissive tissue culture has been developed for human sapovirus, limiting its characterization to surrogate systems. We report here on the first extensive characterization of the key enzyme of replication, the RNA-dependent RNA polymerase (RdRp) associated with the 3Dpol-like protein. Enzymatically active sapovirus 3Dpol and its defective mutant were expressed in Escherichia coli and purified. The overall structure of the sapovirus 3Dpol was determined by X-ray crystallography to 2.32-Å resolution. It revealed a right hand fold typical for template-dependent polynucleotide polymerases. The carboxyl terminus is located within the active site cleft, as observed in the RdRp of some (norovirus) but not other (lagovirus) caliciviruses. Sapovirus 3Dpol prefers Mn2+ over Mg2+ but may utilize either as a cofactor in vitro. In a synthetic RNA template-dependent reaction, sapovirus 3Dpol synthesizes a double-stranded RNA or labels the template 3′ terminus by terminal transferase activity. Initiation of RNA synthesis occurs de novo on heteropolymeric templates or in a primer-dependent manner on polyadenylated templates. Strikingly, this mode of initiation of RNA synthesis was also described for norovirus, but not for lagovirus, suggesting structural and functional homologies in the RNA-dependent RNA polymerase of human pathogenic caliciviruses. This first experimental evidence makes sapovirus 3Dpol an attractive target for developing drugs to control calicivirus infection in humans.

scholarly journals A conserved arginine in NS5 binds genomic 3’stem-loop RNA for primer-independent initiation of flavivirus RNA-replication

RNA ◽  
2021 ◽  
pp. rna.078949.121
Author(s):  
Sai Wang ◽  
Kitt Wing Ki Chan ◽  
Min Jie Alvin Tan ◽  
Charlotte Flory ◽  
Dahai Luo ◽  
...  
Keyword(s):  
Protein Interactions ◽  
De Novo ◽  
Rna Replication ◽  
Polymerase Activity ◽  
Structural Protein ◽  
Binding Studies ◽  
Stem Loop ◽  
Cis Acting ◽  
Rna Polymerase Activity

Replication of the RNA genome of flaviviruses without a primer involves RNA-protein interactions that have been shown to include the recognition of the stem-loop A (SLA) in the 5’ untranslated region (UTR) by the non-structural protein 5 (NS5). We show that DENV2 NS5 arginine 888, located within the C-terminal 18 residues, is completely conserved in all flaviviruses and interacts specifically with the top-loop of 3’SL in the 3’UTR which contains the pentanucleotide 5’-CACAG-3’ previously shown to be critical for flavivirus RNA replication. We present virological and biochemical data showing the importance of this Arg 888 in virus viability and de novo initiation of RNA polymerase activity in vitro. Based on our binding studies, we hypothesize that ternary complex formation of NS5 with 3’SL, followed by dimerization, leads to the formation of the de novo initiation complex that could be regulated by the reversible zipping and unzipping of cis-acting RNA elements.

Three different M1 RNA-containing viruslike particle types in Saccharomyces cerevisiae: in vitro M1 double-stranded RNA synthesis

1986 ◽  
Vol 6 (5) ◽  
pp. 1552-1561
Author(s):  
R Esteban ◽  
R B Wickner
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Rna Synthesis ◽  
Major Coat Protein ◽  
Double Stranded Rna ◽  
Dsrna Molecule ◽  
Protein Toxin ◽  
New Type ◽  
Viruslike Particles ◽  
M1 Rna

Killer strains of Saccharomyces cerevisiae bear at least two different double-stranded RNAs (dsRNAs) encapsidated in 39-nm viruslike particles (VLPs) of which the major coat protein is coded by the larger RNA (L-A dsRNA). The smaller dsRNA (M1 or M2) encodes an extracellular protein toxin (K1 or K2 toxin). Based on their densities on CsCl gradients, L-A- and M1-containing particles can be separated. Using this method, we detected a new type of M1 dsRNA-containing VLP (M1-H VLP, for heavy) that has a higher density than those previously reported (M1-L VLP, for light). M1-H and M1-L VLPs are present together in the same strains and in all those we tested. M1-H, M1-L, and L-A VLPs all have the same types of proteins in the same approximate proportions, but whereas L-A VLPs and M1-L VLPs have one dsRNA molecule per particle, M1-H VLPs contain two M1 dsRNA molecules per particle. Their RNA polymerase produces mainly plus single strands that are all extruded in the case of M1-H particles but are partially retained inside the M1-L particles to be used later for dsRNA synthesis. We show that M1-H VLPs are formed in vitro from the M1-L VLPs. We also show that the peak of M1 dsRNA synthesis is in fractions lighter than M1-L VLPs, presumably those carrying only a single plus M1 strand. We suggest that VLPs carrying two M1 dsRNAs (each 1.8 kilobases) can exist because the particle is designed to carry one L-A dsRNA (4.5 kilobases).

Glucocorticoid-regulated compartmentalization of cell surface-associated glycoproteins in rat hepatoma cells: evidence for an independent response that requires receptor function and de novo RNA synthesis

1987 ◽  
Vol 7 (4) ◽  
pp. 1508-1517
Author(s):  
O K Haffar ◽  
A K Vallerga ◽  
S A Marenda ◽  
H J Witchel ◽  
G L Firestone
Keyword(s):  
Cell Surface ◽  
Time Course ◽  
Rna Synthesis ◽  
De Novo ◽  
Receptor Occupancy ◽  
Receptor Function ◽  
Intracellular Fraction ◽  
Culture Cells ◽  
Viral Glycoproteins ◽  
Rat Hepatoma

The role of glucocorticoid hormones in the compartmentalization of cell surface-associated mouse mammary tumor virus (MMTV) glycoproteins was examined in M1.54, a cloned line of MMTV-infected rat hepatoma tissue culture cells. The expression of cellular [2-3H]mannose-labeled and cell surface 125I-labeled MMTV glycoproteins was examined throughout a time course of exposure to dexamethasone, a synthetic glucocorticoid. Posttranslational localization of cell surface MMTV glycoproteins required 6 h of exposure to hormone and occurred approximately 4 h after their initial production in an intracellular fraction. This regulated localization to the cell surface correlated with glucocorticoid receptor occupancy and was inhibited by exposure to RU 38486, a powerful antagonist of glucocorticoid-mediated responses. Cell surface immunoprecipitation demonstrated that actinomycin D, an inhibitor of de novo RNA synthesis, prevented regulated expression of cell surface viral glycoproteins, suggesting that newly synthesized cellular components mediate this process. The localization of cell surface MMTV glycoproteins appeared normal in a transcriptional variant (CR1) that produces basal levels of MMTV RNA and glycoprotein precursors in the presence of dexamethasone. Thus, regulated compartmentalization of viral glycoproteins is not an obligate consequence of a critical precursor concentration. Taken together, our results suggest that posttranslational trafficking of cell surface-destined MMTV glycoproteins resulted from an independent glucocorticoid hormone response that required receptor function and de novo RNA synthesis.

RNA polymerase activity in virions from Ustilago maydis

1984 ◽  
Vol 4 (1) ◽  
pp. 188-194
Author(s):  
B S Ben-Tzvi ◽  
Y Koltin ◽  
M Mevarech ◽  
A Tamarkin
Keyword(s):  
Rna Polymerase ◽  
Viral Genome ◽  
Ustilago Maydis ◽  
Polymerase Activity ◽  
Reaction Products ◽  
Double Stranded Rna ◽  
Rna Molecules ◽  
Rna Transcripts ◽  
Rna Polymerase Activity

RNA polymerase activity is associated with the double-stranded RNA virions of Ustilago maydis. The reaction products of the polymerase activity are single-stranded RNA molecules. The RNA molecules synthesized are homologous to the three classes of double-stranded RNA molecules that typify the viral genome. The single-stranded RNA synthesized is released from the virions. The molecular weight of the single-stranded RNA transcripts is about half the size of the double-stranded RNA segments, and thus, it appears that in the in vitro reaction, full-length transcripts can be obtained.

scholarly journals Caspofungin and LTX-315 inhibit SARS-CoV-2 replication by targeting the nsp12 polymerase

2020 ◽  
Author(s):  
Min Wang ◽  
Fei Ye ◽  
Jiaqi Su ◽  
Jingru Zhao ◽  
Bin Yuan ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Vero Cells ◽  
Polymerase Activity ◽  
Structural Protein ◽  
Live Virus ◽  
Drug Candidates ◽  
Virtual Screen ◽  
Virus Assay ◽  
Promising Target

Abstract The ongoing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, previously designated as 2019-nCoV) outbreak has caused global concern1. Currently, there are no clinically approved specific drugs or vaccines available for this virus. The viral polymerase is a promising target for developing broad- spectrum antiviral drugs. Here, based on the highly similar structure of SARS- CoV non-structural protein 12 (nsp12) polymerase subunit2, we applied virtual screen for the available compounds, including both the FDA-approved and under- clinic drugs, to identify potential antiviral molecules against SARS-CoV-2. We found two drugs, the clinically approved anti-fungi drug Caspofungin Acetate (Cancidas) and the oncolytic peptide LTX-315, can bind SARS-CoV-2 nsp12 protein to block the polymerase activity in vitro. Further live virus assay revealed that both Caspofungin Acetate and LTX-315 can effectively inhibit SARS-CoV-2 replication in vero cells. These findings present promising drug candidates for treatment of related diseases and would also stimulate the development of pan- coronavirus antiviral agents.Authors Min Wang, Fei Ye, Jiaqi Su, Jingru Zhao, and Bin Yuan contributed equally to this work.

scholarly journals Identification of Pol IV and RDR2-dependent precursors of 24 nt siRNAs guiding de novo DNA methylation in Arabidopsis

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
Todd Blevins ◽  
Ram Podicheti ◽  
Vibhor Mishra ◽  
Michelle Marasco ◽  
Jing Wang ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Cytosine Methylation ◽  
De Novo ◽  
Transferase Activity ◽  
Small Interfering Rnas ◽  
Pol Iv ◽  
Nuclear Rna ◽  
Rna Polymerase Iv ◽  
Nuclear Rna Polymerase

In Arabidopsis thaliana, abundant 24 nucleotide small interfering RNAs (24 nt siRNA) guide the cytosine methylation and silencing of transposons and a subset of genes. 24 nt siRNA biogenesis requires nuclear RNA polymerase IV (Pol IV), RNA-dependent RNA polymerase 2 (RDR2) and DICER-like 3 (DCL3). However, siRNA precursors are mostly undefined. We identified Pol IV and RDR2-dependent RNAs (P4R2 RNAs) that accumulate in dcl3 mutants and are diced into 24 nt RNAs by DCL3 in vitro. P4R2 RNAs are mostly 26-45 nt and initiate with a purine adjacent to a pyrimidine, characteristics shared by Pol IV transcripts generated in vitro. RDR2 terminal transferase activity, also demonstrated in vitro, may account for occasional non-templated nucleotides at P4R2 RNA 3’ termini. The 24 nt siRNAs primarily correspond to the 5’ or 3’ ends of P4R2 RNAs, suggesting a model whereby siRNAs are generated from either end of P4R2 duplexes by single dicing events.

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.

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