Heterologous RNA replication enhancer stimulates in vitro RNA synthesis and template-switching by the carmovirus, but not by the tombusvirus, RNA-dependent RNA polymerase: Implication for modular evolution of RNA viruses

ABSTRACT Initiation of RNA synthesis by the hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp) NS5B has been extensively studied in vitro and in cellulo. Intracellular replication is thought to rely exclusively on terminal de novo initiation, as it conserves all genetic information of the genome. In vitro, however, additional modes of initiation have been observed. In this study, we aimed to clarify whether the intracellular environment allows for internal initiation of RNA replication by the HCV replicase. We used a dual luciferase replicon harboring a terminal and an internal copy of the viral genomic 5′ untranslated region, which was anticipated to support noncanonical initiation. Indeed, a shorter RNA species was detected by Northern blotting with low frequency, depending on the length and sequence composition upstream of the internal initiation site. By introducing mutations at either site, we furthermore established that internal and terminal initiation shared identical sequence requirements. Importantly, lethal point mutations at the terminal site resulted exclusively in truncated replicons. In contrast, the same mutations at the internal site abrogated internal initiation, suggesting a competitive selection of initiation sites, rather than recombination or template-switching events. In conclusion, our data indicate that the HCV replicase is capable of internal initiation in its natural environment, although functional replication likely requires only terminal initiation. Since many other positive-strand RNA viruses generate subgenomic messenger RNAs during their replication cycle, we surmise that their capability for internal initiation is a common and conserved feature of viral RdRps. IMPORTANCE Many aspects of viral RNA replication of hepatitis C virus (HCV) are still poorly understood. The process of RNA synthesis is driven by the RNA-dependent RNA polymerase (RdRp) NS5B. Most mechanistic studies on NS5B so far were performed with in vitro systems using isolated recombinant polymerase. In this study, we present a replicon model, which allows the intracellular assessment of noncanonical modes of initiation by the full HCV replicase. Our results add to the understanding of the biochemical processes underlying initiation of RNA synthesis by NS5B by the discovery of internal initiation in cellulo. Moreover, they validate observations made in vitro, showing that the viral polymerase acts very similarly in isolation and in complex with other viral and host proteins. Finally, these observations provide clues about the evolution of RdRps of positive-strand RNA viruses, which might contain the intrinsic ability to initiate internally.

Download Full-text

Strand-Specific RNA Synthesis Determinants in the RNA-Dependent RNA Polymerase of Poliovirus

Journal of Virology ◽

10.1128/jvi.78.9.4397-4407.2004 ◽

2004 ◽

Vol 78 (9) ◽

pp. 4397-4407 ◽

Cited By ~ 14

Author(s):

Christopher T. Cornell ◽

Rushika Perera ◽

Jo Ellen Brunner ◽

Bert L. Semler

Keyword(s):

Cell Culture ◽

Rna Polymerase ◽

Rna Synthesis ◽

Rna Replication ◽

Amino Acid Sequences ◽

Rna Dependent Rna Polymerase ◽

Amino Terminal ◽

Carboxy Terminal

ABSTRACT The viral RNA-dependent RNA polymerase (3Dpol) is highly conserved between the closely related enteroviruses poliovirus type 1 (PV1) and coxsackievirus B3 (CVB3). In this study, we generated PV1/CVB3 chimeric polymerase sequences in the context of full-length poliovirus transcripts to determine the role of different subdomains within the RNA-dependent RNA polymerase of PV1 that are required for functions critical for RNA replication in vitro and in cell culture. The substitution of CVB3 sequences in the carboxy-terminal portion (thumb subdomain) of the polymerase resulted in transcripts incapable of RNA replication. In contrast, three of the seven chimeras were capable of synthesizing RNA, albeit to reduced levels compared to that of wild-type PV1 RNA. Interestingly, one of the replication-competent chimeras (CPP) displayed an inability to generate positive strands, indicating the presence of amino-terminal sequences within the 3D polymerase and/or the 3D domain of the 3CD precursor polypeptide that are necessary for the assembly of strand-specific RNA synthesis complexes. In some constructs, the partial reestablishment of PV1 amino acid sequences in this region was capable of rescuing RNA replication in vitro and in cell culture.

Download Full-text

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

Journal of Virology ◽

10.1128/jvi.01137-15 ◽

2015 ◽

Vol 89 (15) ◽

pp. 7722-7734 ◽

Cited By ~ 21

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.

Download Full-text

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.

Download Full-text

Spatial Perturbations within an RNA Promoter Specifically Recognized by a Viral RNA-Dependent RNA Polymerase (RdRp) Reveal That RdRp Can Adjust Its Promoter Binding Sites

Journal of Virology ◽

10.1128/jvi.73.1.198-204.1999 ◽

1999 ◽

Vol 73 (1) ◽

pp. 198-204 ◽

Cited By ~ 16

Author(s):

Scott Stevenson Stawicki ◽

C. Cheng Kao

Keyword(s):

Rna Polymerase ◽

Rna Synthesis ◽

Core Promoter ◽

Brome Mosaic Virus ◽

Viral Rna ◽

Rna Dependent Rna Polymerase ◽

Dna And Rna ◽

In The Wild ◽

Nucleotide Insertions

ABSTRACT RNA synthesis during viral replication requires specific recognition of RNA promoters by the viral RNA-dependent RNA polymerase (RdRp). Four nucleotides (−17, −14, −13, and −11) within the brome mosaic virus (BMV) subgenomic core promoter are required for RNA synthesis by the BMV RdRp (R. W. Siegel et al., Proc. Natl. Acad. Sci. USA 94:11238–11243, 1997). The spatial requirements for these four nucleotides and the initiation (+1) cytidylate were examined in RNAs containing nucleotide insertions and deletions within the BMV subgenomic core promoter. Spatial perturbations between nucleotides −17 and −11 resulted in decreased RNA synthesis in vitro. However, synthesis was still dependent on the key nucleotides identified in the wild-type core promoter and the initiation cytidylate. In contrast, changes between nucleotides −11 and +1 had a less severe effect on RNA synthesis but resulted in RNA products initiated at alternative locations in addition to the +1 cytidylate. The results suggest a degree of flexibility in the recognition of the subgenomic promoter by the BMV RdRp and are compared with functional regions in other DNA and RNA promoters.

Download Full-text

Differential Rescue of Poliovirus RNA Replication Functions by Genetically Modified RNA Polymerase Precursors

Journal of Virology ◽

10.1128/jvi.78.23.13007-13018.2004 ◽

2004 ◽

Vol 78 (23) ◽

pp. 13007-13018 ◽

Cited By ~ 7

Author(s):

Christopher T. Cornell ◽

Jo Ellen Brunner ◽

Bert L. Semler

Keyword(s):

Rna Polymerase ◽

Rna Synthesis ◽

Genetically Modified ◽

Rna Replication ◽

Amino Acid Sequences ◽

In Vitro Translation ◽

Wild Type ◽

Positive Strand ◽

Positive Strand Rna

ABSTRACT We have previously described the RNA replication properties of poliovirus transcripts harboring chimeric RNA polymerase sequences representing suballelic exchanges between poliovirus type 1 (PV1) and coxsackievirus B3 (CVB3) utilizing an in vitro translation and RNA replication assay (C. Cornell, R. Perera, J. E. Brunner, and B. L. Semler, J. Virol. 78:4397-4407, 2004). We showed that three of the seven chimeras were capable of RNA replication in vitro, although replication levels were greatly reduced compared to that of wild-type transcripts. Interestingly, one of the replication-competent transcripts displayed a strand-specific RNA synthesis defect suggesting (i) a differential replication complex assembly mechanism involving 3D and/or precursor molecules (i.e., 3CD) required for negative- versus positive-strand RNA synthesis or (ii) effect(s) on the ability of the 3D polymerase to form higher-ordered structures required for positive-strand RNA synthesis. In this study, we have attempted to rescue defective RNA replication in vitro by cotranslating nonstructural proteins from a transcript encoding a large precursor polyprotein (P3) to complement 3D polymerase and/or precursor polypeptide functions altered in each of the chimeric constructs. Utilization of a wild-type P3 construct revealed that all transcripts containing chimeric PV1/CVB3 polymerase sequences can be complemented in trans for both negative- and positive-strand RNA synthesis. Furthermore, data from experiments utilizing genetically modified forms of the P3 polyprotein, containing mutations within 3C or 3D sequences, strongly suggest the existence of different protein-protein and protein-RNA interactions required for positive- versus negative-strand RNA synthesis. These results, combined with data from in vitro RNA elongation assays, indicate that the delivery of active 3D RNA polymerase to replication complexes requires a series of macromolecular interactions that rely on the presence of specific 3D amino acid sequences.

Download Full-text

Mechanism of Stimulation of Plus-Strand Synthesis by an RNA Replication Enhancer in a Tombusvirus

Journal of Virology ◽

10.1128/jvi.79.15.9777-9785.2005 ◽

2005 ◽

Vol 79 (15) ◽

pp. 9777-9785 ◽

Cited By ~ 25

Author(s):

Tadas Panavas ◽

Peter D. Nagy

Keyword(s):

Rna Synthesis ◽

Rna Viruses ◽

Rna Replication ◽

Dual Function ◽

Long Distance ◽

Replication Assay ◽

Rna Interaction ◽

Stimulation Of

ABSTRACT Replication of RNA viruses is regulated by cis-acting RNA elements, including promoters, replication silencers, and replication enhancers (REN). To dissect the function of an REN element involved in plus-strand RNA synthesis, we developed an in vitro trans-replication assay for tombusviruses, which are small plus-strand RNA viruses. In this assay, two RNA strands were tethered together via short complementary regions with the REN present in the nontemplate RNA, whereas the promoter was located in the template RNA. We found that the template activity of the tombusvirus replicase preparation was stimulated in trans by the REN, suggesting that the REN is a functional enhancer when located in the vicinity of the promoter. In addition, this study revealed that the REN has dual function during RNA synthesis. (i) It binds to the viral replicase. (ii) It interacts with the core plus-strand initiation promoter via a long-distance RNA-RNA interaction, which leads to stimulation of initiation of plus-strand RNA synthesis by the replicase in vitro. We also observed that this RNA-RNA interaction increased the in vivo accumulation and competitiveness of defective interfering RNA, a model template. We propose that REN is important for asymmetrical viral RNA replication that leads to more abundant plus-strand RNA progeny than the minus-strand intermediate, a hallmark of replication of plus-strand RNA viruses.

Download Full-text

169 The inhibition of flavivirus RNA synthesis by the heteropolyanion HPA-23 and related compounds in the in vitro RNA-Dependent RNA polymerase assay

Antiviral Research ◽

10.1016/0166-3542(94)90292-5 ◽

1994 ◽

Vol 23 ◽

pp. 126

Keyword(s):

Rna Polymerase ◽

Rna Synthesis ◽

Rna Dependent Rna Polymerase ◽

Related Compounds

Download Full-text

Template-Dependent Initiation of Sindbis Virus RNA Replication In Vitro

Journal of Virology ◽

10.1128/jvi.72.8.6546-6553.1998 ◽

1998 ◽

Vol 72 (8) ◽

pp. 6546-6553 ◽

Cited By ~ 64

Author(s):

Julie A. Lemm ◽

Anders Bergqvist ◽

Carol M. Read ◽

Charles M. Rice

Keyword(s):

Rna Polymerase ◽

Sindbis Virus ◽

Rna Synthesis ◽

Rna Replication ◽

Functional Assay ◽

Minus Strand ◽

Virus Rna ◽

A Genome ◽

Strand Initiation

ABSTRACT Recent insights into the early events in Sindbis virus RNA replication suggest a requirement for either the P123 or P23 polyprotein, as well as mature nsP4, the RNA-dependent RNA polymerase, for initiation of minus-strand RNA synthesis. Based on this observation, we have succeeded in reconstituting an in vitro system for template-dependent initiation of SIN RNA replication. Extracts were isolated from cells infected with vaccinia virus recombinants expressing various SIN proteins and assayed by the addition of exogenous template RNAs. Extracts from cells expressing P123C>S, a protease-defective P123 polyprotein, and nsP4 synthesized a genome-length minus-sense RNA product. Replicase activity was dependent upon addition of exogenous RNA and was specific for alphavirus plus-strand RNA templates. RNA synthesis was also obtained by coexpression of nsP1, P23C>S, and nsP4. However, extracts from cells expressing nsP4 and P123, a cleavage-competent P123 polyprotein, had much less replicase activity. In addition, a P123 polyprotein containing a mutation in the nsP2 protease which increased the efficiency of processing exhibited very little, if any, replicase activity. These results provide further evidence that processing of the polyprotein inactivates the minus-strand initiation complex. Finally, RNA synthesis was detected when soluble nsP4 was added to a membrane fraction containing P123C>S, thus providing a functional assay for purification of the nsP4 RNA polymerase.

Download Full-text

The Imidazopyrrolopyridine Analogue AG110 Is a Novel, Highly Selective Inhibitor of Pestiviruses That Targets the Viral RNA-Dependent RNA Polymerase at a Hot Spot for Inhibition of Viral Replication

Journal of Virology ◽

10.1128/jvi.00388-07 ◽

2007 ◽

Vol 81 (20) ◽

pp. 11046-11053 ◽

Cited By ~ 36

Author(s):

Jan Paeshuyse ◽

Jean-Michel Chezal ◽

Matheus Froeyen ◽

Pieter Leyssen ◽

Hélène Dutartre ◽

...

Keyword(s):

Rna Polymerase ◽

Rna Synthesis ◽

Hot Spot ◽

Resistance Mutation ◽

Drug Resistance Mutation ◽

Drug Binding ◽

Viral Rna ◽

Rna Dependent Rna Polymerase ◽

Diarrhea Virus

ABSTRACT Ethyl 2-methylimidazo[1,2-a]pyrrolo[2,3-c]pyridin-8-carboxylate (AG110) was identified as a potent inhibitor of pestivirus replication. The 50% effective concentration values for inhibition of bovine viral diarrhea virus (BVDV)-induced cytopathic effect, viral RNA synthesis, and production of infectious virus were 1.2 ± 0.5 μM, 5 ± 1 μM, and 2.3 ± 0.3 μM, respectively. AG110 proved inactive against the hepatitis C virus and a flavivirus. AG110 inhibits BVDV replication at a time point that coincides with the onset of intracellular viral RNA synthesis. Drug-resistant mutants carry the E291G mutation in the viral RNA-dependent RNA polymerase (RdRp). AG110-resistant virus is cross-resistant to the cyclic urea compound 1453 which also selects for the E291G drug resistance mutation. Moreover, BVDV that carries the F224S mutation (because of resistance to the imidazopyridine 5-[(4-bromophenyl)methyl]-2-phenyl-5H-imidazo[4,5-c]pyridine [BPIP]and VP32947) is also resistant to AG110. AG110 did not inhibit the in vitro activity of recombinant BVDV RdRp but inhibited the activity of BVDV replication complexes (RCs). Molecular modeling revealed that E291 is located in a small cavity near the tip of the finger domain of the RdRp about 7 Å away from F224. Docking of AG110 in the crystal structure of the BVDV RdRp revealed several potential contacts including with Y257. The E291G mutation might enable the free rotation of Y257, which might in turn destabilize the backbone of the loop formed by residues 223 to 226, rendering more mobility to F224 and, hence, reducing the affinity for BPIP and VP32947. It is concluded that a single drug-binding pocket exists within the finger domain region of the BVDV RdRp that consists of two separate but potentially overlapping binding sites rather than two distinct drug-binding pockets.

Download Full-text