Flock House Virus RNA Polymerase Initiates RNA Synthesis De Novo and Possesses a Terminal Nucleotidyl Transferase Activity

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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A Recombinant Hepatitis C Virus RNA-Dependent RNA Polymerase Capable of Copying the Full-Length Viral RNA

Journal of Virology ◽

10.1128/jvi.73.9.7694-7702.1999 ◽

1999 ◽

Vol 73 (9) ◽

pp. 7694-7702 ◽

Cited By ~ 156

Author(s):

Jong-Won Oh ◽

Takayoshi Ito ◽

Michael M. C. Lai

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Rna Polymerase ◽

Rna Synthesis ◽

De Novo ◽

Full Length ◽

Viral Rna ◽

Hcv Rna ◽

Independent Manner ◽

Virus Rna

ABSTRACT All of the previously reported recombinant RNA-dependent RNA polymerases (RdRp), the NS5B enzymes, of hepatitis C virus (HCV) could function only in a primer-dependent and template-nonspecific manner, which is different from the expected properties of the functional viral enzymes in the cells. We have now expressed a recombinant NS5B that is able to synthesize a full-length HCV genome in a template-dependent and primer-independent manner. The kinetics of RNA synthesis showed that this RdRp can initiate RNA synthesis de novo and yield a full-length RNA product of genomic size (9.5 kb), indicating that it did not use the copy-back RNA as a primer. This RdRp was also able to accept heterologous viral RNA templates, including poly(A)- and non-poly(A)-tailed RNA, in a primer-independent manner, but the products in these cases were heterogeneous. The RdRp used some homopolymeric RNA templates only in the presence of a primer. By using the 3′-end 98 nucleotides (nt) of HCV RNA, which is conserved in all genotypes of HCV, as a template, a distinct RNA product was generated. Truncation of 21 nt from the 5′ end or 45 nt from the 3′ end of the 98-nt RNA abolished almost completely its ability to serve as a template. Inclusion of the 3′-end variable sequence region and the U-rich tract upstream of the X region in the template significantly enhanced RNA synthesis. The 3′ end of minus-strand RNA of HCV genome also served as a template, and it required a minimum of 239 nt from the 3′ end. These data defined the cis-acting sequences for HCV RNA synthesis at the 3′ end of HCV RNA in both the plus and minus senses. This is the first recombinant HCV RdRp capable of copying the full-length HCV RNA in the primer-independent manner expected of the functional HCV RNA polymerase.

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Structural and Functional Characterization of Sapovirus RNA-Dependent RNA Polymerase

Journal of Virology ◽

10.1128/jvi.01462-06 ◽

2006 ◽

Vol 81 (4) ◽

pp. 1858-1871 ◽

Cited By ~ 51

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.

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De novo RNA synthesis catalyzed by the Zika Virus RNA polymerase domain

Scientific Reports ◽

10.1038/s41598-017-03038-8 ◽

2017 ◽

Vol 7 (1) ◽

Cited By ~ 2

Author(s):

Christina Calmels ◽

Michel Ventura ◽

Cindy Aknin ◽

Mathieu Métifiot ◽

Marie-Line Andreola

Keyword(s):

Rna Polymerase ◽

Zika Virus ◽

Rna Synthesis ◽

De Novo ◽

Polymerase Domain ◽

Virus Rna

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De novo RNA synthesis by a recombinant classical swine fever virus RNA-dependent RNA polymerase

European Journal of Biochemistry ◽

10.1046/j.1432-1033.2003.03897.x ◽

2003 ◽

Vol 270 (24) ◽

pp. 4952-4961 ◽

Cited By ~ 16

Author(s):

Guang-Hui Yi ◽

Chu-Yu Zhang ◽

Sheng Cao ◽

Hai-Xiang Wu ◽

Yi Wang

Keyword(s):

Rna Polymerase ◽

Classical Swine Fever Virus ◽

Rna Synthesis ◽

De Novo ◽

Classical Swine Fever ◽

Fever Virus ◽

Rna Dependent Rna Polymerase ◽

Virus Rna

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De novo RNA synthesis and homology modeling of the classical swine fever virus RNA polymerase

Virus Research ◽

10.1016/j.virusres.2005.03.003 ◽

2005 ◽

Vol 112 (1-2) ◽

pp. 9-23 ◽

Cited By ~ 7

Author(s):

Pengwei Zhang ◽

Jian Xie ◽

Guanghui Yi ◽

Chuyu Zhang ◽

Rong Zhou

Keyword(s):

Rna Polymerase ◽

Homology Modeling ◽

Classical Swine Fever Virus ◽

Rna Synthesis ◽

De Novo ◽

Classical Swine Fever ◽

Fever Virus ◽

Virus Rna

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Mechanism for De Novo RNA Synthesis and Initiating Nucleotide Specificity by T7 RNA Polymerase

Journal of Molecular Biology ◽

10.1016/j.jmb.2007.03.041 ◽

2007 ◽

Vol 370 (2) ◽

pp. 256-268 ◽

Cited By ~ 47

Author(s):

William P. Kennedy ◽

Jamila R. Momand ◽

Y. Whitney Yin

Keyword(s):

Rna Polymerase ◽

Rna Synthesis ◽

De Novo ◽

T7 Rna Polymerase ◽

Nucleotide Specificity

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Mutations in Conserved Domain II of the Large (L) Subunit of the Sendai Virus RNA Polymerase Abolish RNA Synthesis

Virology ◽

10.1006/viro.1999.9933 ◽

1999 ◽

Vol 262 (2) ◽

pp. 375-383 ◽

Cited By ~ 39

Author(s):

Sherin Smallwood ◽

Colin D. Easson ◽

Joyce A. Feller ◽

Sandra M. Horikami ◽

Sue A. Moyer

Keyword(s):

Rna Polymerase ◽

Rna Synthesis ◽

Sendai Virus ◽

Conserved Domain ◽

Virus Rna

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RNA-Dependent RNA Polymerase from Heterobasidion RNA Virus 6 Is an Active Replicase In Vitro

Viruses ◽

10.3390/v13091738 ◽

2021 ◽

Vol 13 (9) ◽

pp. 1738

Author(s):

Alesia A. Levanova ◽

Eeva J. Vainio ◽

Jarkko Hantula ◽

Minna M. Poranen

Keyword(s):

Rna Polymerase ◽

Rna Synthesis ◽

Rna Virus ◽

Polymerization Reaction ◽

Rna Dependent Rna Polymerase ◽

Independent Manner ◽

Nucleotidyl Transferase ◽

Rna Polymerization ◽

The Family

Heterobasidion RNA virus 6 (HetRV6) is a double-stranded (ds)RNA mycovirus and a member of the recently established genus Orthocurvulavirus within the family Orthocurvulaviridae. The purpose of the study was to determine the biochemical requirements for RNA synthesis catalyzed by HetRV6 RNA-dependent RNA polymerase (RdRp). HetRV6 RdRp was expressed in Escherichia coli and isolated to near homogeneity using liquid chromatography. The enzyme activities were studied in vitro using radiolabeled UTP. The HetRV6 RdRp was able to initiate RNA synthesis in a primer-independent manner using both virus-related and heterologous single-stranded (ss)RNA templates, with a polymerization rate of about 46 nt/min under optimal NTP concentration and temperature. NTPs with 2′-fluoro modifications were also accepted as substrates in the HetRV6 RdRp-catalyzed RNA polymerization reaction. HetRV6 RdRp transcribed viral RNA genome via semi-conservative mechanism. Furthermore, the enzyme demonstrated terminal nucleotidyl transferase (TNTase) activity. Presence of Mn2+ was required for the HetRV6 RdRp catalyzed enzymatic activities. In summary, our study shows that HetRV6 RdRp is an active replicase in vitro that can be potentially used in biotechnological applications, molecular biology, and biomedicine.

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

eLife ◽

10.7554/elife.09591 ◽

2015 ◽

Vol 4 ◽

Cited By ~ 120

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.

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