Steps Of RNA Replication Research Articles

Stimulator of interferon genes (STING) is an essential proviral host factor for human rhinovirus species A and C

Human rhinoviruses (RVs) are positive-strand RNA viruses that cause respiratory tract disease in children and adults. Here we show that the innate immune signaling protein STING is required for efficient replication of members of two distinct RV species, RV-A and RV-C. The host factor activity of STING was identified in a genome-wide RNA interference (RNAi) screen and confirmed in primary human small airway epithelial cells. Replication of RV-A serotypes was strictly dependent on STING, whereas RV-B serotypes were notably less dependent. Subgenomic RV-A and RV-C RNA replicons failed to amplify in the absence of STING, revealing it to be required for a step in RNA replication. STING was expressed on phosphatidylinositol 4-phosphate (PI4P)-enriched membranes and was enriched in RV-A16 compared with RV-B14 replication organelles isolated in isopycnic gradients. The host factor activity of STING was species-specific, as murine STING (mSTING) did not rescue RV-A16 replication in STING-deficient cells. This species specificity mapped primarily to the cytoplasmic, ligand-binding domain of STING. Mouse-adaptive mutations in the RV-A16 2C protein allowed for robust replication in cells expressing mSTING, suggesting a role for 2C in recruiting STING to RV-A replication organelles. Palmitoylation of STING was not required for RV-A16 replication, nor was the C-terminal tail of STING that mediates IRF3 signaling. Despite co-opting STING to promote its replication, interferon signaling in response to STING agonists remained intact in RV-A16 infected cells. These data demonstrate a surprising requirement for a key host mediator of innate immunity to DNA viruses in the life cycle of a small pathogenic RNA virus.

In vitro selection of ribozyme ligases that use prebiotically plausible 2-aminoimidazole–activated substrates

The hypothesized central role of RNA in the origin of life suggests that RNA propagation predated the advent of complex protein enzymes. A critical step of RNA replication is the template-directed synthesis of a complementary strand. Two experimental approaches have been extensively explored in the pursuit of demonstrating protein-free RNA synthesis: template-directed nonenzymatic RNA polymerization using intrinsically reactive monomers and ribozyme-catalyzed polymerization using more stable substrates such as biological 5'-triphosphates. Despite significant progress in both approaches in recent years, the assembly and copying of functional RNA sequences under prebiotic conditions remains a challenge. Here, we explore an alternative approach to RNA-templated RNA copying that combines ribozyme catalysis with RNA substrates activated with a prebiotically plausible leaving group, 2-aminoimidazole (2AI). We applied in vitro selection to identify ligase ribozymes that catalyze phosphodiester bond formation between a template-bound primer and a phosphor-imidazolide-activated oligomer. Sequencing revealed the progressive enrichment of 10 abundant sequences from a random sequence pool. Ligase activity was detected in all 10 RNA sequences; all required activation of the ligator with 2AI and generated a 3'-5' phosphodiester bond. We propose that ribozyme catalysis of phosphodiester bond formation using intrinsically reactive RNA substrates, such as imidazolides, could have been an evolutionary step connecting purely nonenzymatic to ribozyme-catalyzed RNA template copying during the origin of life.

An RNA virus hijacks an incognito function of a DNA repair enzyme

A previously described mammalian cell activity, called VPg unlinkase, specifically cleaves a unique protein-RNA covalent linkage generated during the viral genomic RNA replication steps of a picornavirus infection. For over three decades, the identity of this cellular activity and its normal role in the uninfected cell had remained elusive. Here we report the purification and identification of VPg unlinkase as the DNA repair enzyme, 5'-tyrosyl-DNA phosphodiesterase-2 (TDP2). Our data show that VPg unlinkase activity in different mammalian cell lines correlates with their differential expression of TDP2. Furthermore, we show that recombinant TDP2 can cleave the protein-RNA linkage generated by different picornaviruses without impairing the integrity of viral RNA. Our results reveal a unique RNA repair-like function for TDP2 and suggest an unusual role in host-pathogen interactions for this cellular enzyme. On the basis of the identification of TDP2 as a potential antiviral target, our findings may lead to the development of universal therapeutics to treat the millions of individuals afflicted annually with diseases caused by picornaviruses, including myocarditis, aseptic meningitis, encephalitis, hepatitis, and the common cold.

(−)-Epigallocatechin-3-gallate inhibits the replication cycle of hepatitis C virus

(-)-Epigallocatechin-3-gallate (EGCG) is the most abundant catechin in green tea. In this study, we found that hepatitis C virus (HCV) infection was significantly suppressed by EGCG in an HCV cell culture (HCVcc) system using a JFH1-GFP chimeric virus, with a 50 % effective concentration (EC(50)) of 17.9 μM. The inhibitory activity of EGCG was confirmed by monitoring HCV RNA and protein expression levels in Huh7.5.1 cells infected with the JFH1 virus. Moreover, we demonstrated that the inhibitory mechanisms of EGCG were attributable to the suppression of both the HCV entry and RNA replication steps, although EGCG had little effect on translation directed by the viral internal ribosome entry site (IRES). Furthermore, HCV could be rapidly eliminated from cell cultures after two and five passages in the presence of 50 and 25 μM EGCG, respectively. These results indicate that EGCG is a potential candidate as a preventive and antiviral drug for HCV infection.

Adenosine residues in the template do not block spontaneous replication steps of RNA

Sub-freezing temperatures, azabenzotriazolide activation, multiple monomer addition, and helper displacement help to overcome what seemed like an intrinsic block of adenine-templated RNA replication steps in the absence of enzymes.

In Vivo Self-Interaction of Nodavirus RNA Replicase Protein A Revealed by Fluorescence Resonance Energy Transfer

Flock house virus (FHV) is the best-characterized member of the Nodaviridae, a family of small, positive-strand RNA viruses. Unlike most RNA viruses, FHV encodes only a single polypeptide, protein A, that is required for RNA replication. Protein A contains a C-proximal RNA-dependent RNA polymerase domain and localizes via an N-terminal transmembrane domain to the outer mitochondrial membrane, where FHV RNA replication takes place in association with invaginations referred to as spherules. We demonstrate here that protein A self-interacts in vivo by using flow cytometric analysis of fluorescence resonance energy transfer (FRET), spectrofluorometric analysis of bioluminescence resonance energy transfer, and coimmunoprecipitation. Several nonoverlapping protein A sequences were able to independently direct protein-protein interaction, including an N-terminal region previously shown to be sufficient for localization to the outer mitochondrial membrane (D. J. Miller and P. Ahlquist, J. Virol. 76:9856-9867, 2000). Mutations in protein A that diminished FRET also diminished FHV RNA replication, a finding consistent with an important role for protein A self-interaction in FHV RNA synthesis. Thus, the results imply that FHV protein A functions as a multimer rather than as a monomer at one or more steps in RNA replication.

Mutation of host delta9 fatty acid desaturase inhibits brome mosaic virus RNA replication between template recognition and RNA synthesis.

All positive-strand RNA viruses assemble their RNA replication complexes on intracellular membranes. Brome mosaic virus (BMV) replicates its RNA in endoplasmic reticulum (ER)-associated complexes in plant cells and the yeast Saccharomyces cerevisiae. BMV encodes RNA replication factors 1a, with domains implicated in RNA capping and helicase functions, and 2a, with a central polymerase-like domain. Factor 1a interacts independently with the ER membrane, viral RNA templates, and factor 2a to form RNA replication complexes on the perinuclear ER. We show that BMV RNA replication is severely inhibited by a mutation in OLE1, an essential yeast chromosomal gene encoding delta9 fatty acid desaturase, an integral ER membrane protein and the first enzyme in unsaturated fatty acid synthesis. OLE1 deletion and medium supplementation show that BMV RNA replication requires unsaturated fatty acids, not the Ole1 protein, and that viral RNA replication is much more sensitive than yeast growth to reduced unsaturated fatty acid levels. In ole1 mutant yeast, 1a still becomes membrane associated, recruits 2a to the membrane, and recognizes and stabilizes viral RNA templates normally. However, RNA replication is blocked prior to initiation of negative-strand RNA synthesis. The results show that viral RNA synthesis is highly sensitive to lipid composition and suggest that proper membrane fluidity or plasticity is essential for an early step in RNA replication. The strong unsaturated fatty acid dependence also demonstrates that modulating fatty acid balance can be an effective antiviral strategy.

Characterization of an autonomous subgenomic pestivirus RNA replicon.

As an initial approach to define the requirements for the replication of bovine viral diarrhea virus (BVDV), a member of the Flaviviridae family with a positive-strand RNA genome, full-length genomic and subgenomic RNAs were originated by in vitro transcription of diverse BVDV cDNA constructs and transfected into eucaryotic host cells. RNA replication was measured either directly by an RNase protection method or by monitoring the synthesis of viral protein. When full-length BVDV cRNA was initially applied, the synthesis of negative-strand RNA intermediates as well as progeny positive-strand RNA was detected posttransfection in the cytoplasm of the host cells. Compared to the negative-strand RNA intermediate, an excess of positive-strand RNA was synthesized. Surprisingly, a subgenomic RNA molecule, DI9c, corresponding to a previously characterized defective interfering particle, was found to support both steps of RNA replication in the absence of a helper virus as well, thus functioning as an autonomous replicon. DI9c comprises the 5' and 3' untranslated regions of the BVDV genome and the coding regions of the autoprotease Npro and the nonstructural proteins NS3, NS4A, NS4B, NS5A, and NS5B. Most interestingly, the NS2 polypeptide was thus determined to be nonessential for RNA replication. As expected, deletion of the genomic 3' end as well as abolition of the catalytic function of the virus-encoded serine protease resulted in DI9c molecules that were unable to replicate. Deletion of the entire Npro gene also destroyed the ability of DI9c molecules to replicate. On the other hand, DI9c derivatives in which the 5' third of the Npro gene was fused to a ubiquitin gene, allowing the proteolytic release of NS3 in trans, turned out to be replication competent. These results suggest that the RNA sequence located at the 5' end of the open reading frame exerts an essential role during BVDV replication. Replication of DI9c and DI9c derivatives was found not to be limited to host cells of bovine origin, indicating that cellular factors functioning as potential parts of the viral replication machinery are well conserved between different mammalian cells. Our data provide an important step toward the ready identification and characterization of viral factors and genomic elements involved in the life cycle of pestiviruses. The implications for other Flaviviridae and, in particular, the BVDV-related human hepatitis C virus are discussed.

The NS1 protein of human respiratory syncytial virus is a potent inhibitor of minigenome transcription and RNA replication.

The NS1 protein (139 amino acids) is one of the two nonstructural proteins of human respiratory syncytial virus (RSV) and is encoded by a very abundant mRNA transcribed from the promoter-proximal RSV gene. The function of NS1 was unknown and was investigated here by using a reconstituted transcription and RNA replication system that involves a minireplicon and viral proteins (N, P, L and M2-1) expressed from separate cotransfected plasmids. Coexpression of the NS1 cDNA strongly inhibited transcription and RNA replication mediated by the RSV polymerase, even when the level of expressed NS1 protein was substantially below that observed in RSV-infected cells. The effect depended on synthesis of NS1 protein rather than NS1 RNA alone. Transcription and both steps of RNA replication, namely, synthesis of the antigenome and the genome, appeared to be equally sensitive to inhibition. The efficiency of encapsidation of the plasmid-derived minigenome was not altered by coexpression of NS1, indicating that the inhibition occurs at a later step. In two different dicistronic minigenomes, transcription of each gene was equally sensitive to inhibition by NS1. This suggested that the gradient of transcriptional polarity was unaffected and that the effect of NS1 instead probably involves an early event such as polymerase entry on the genome. NS1-mediated inhibition of transcription and RNA replication was not affected by coexpression of the M2 mRNA, which has two open reading frames encoding the transcriptional elongation factor M2-1 and the putative negative regulatory factor M2-2. The potent nature of the NS1-mediated inhibition suggests that negative regulation is an authentic function of the NS1 protein, albeit not necessarily the only one.

Yeast mutations in multiple complementation groups inhibit brome mosaic virus RNA replication and transcription and perturb regulated expression of the viral polymerase-like gene.

Brome mosaic virus (BMV), a member of the alphavirus-like superfamily of positive-strand RNA viruses, encodes two proteins, 1a and 2a, that interact with each other, with unidentified host proteins, and with host membranes to form the viral RNA replication complex. Yeast expressing 1a and 2a support replication and subgenomic mRNA synthesis by BMV RNA3 derivatives. Using a multistep selection and screening process, we have isolated yeast mutants in multiple complementation groups that inhibit BMV-directed gene expression. Three complementation groups, represented by mutants mab1-1, mab2-1, and mab3-1 (for maintenance of BMV functions), were selected for initial study. Each of these mutants has a single, recessive, chromosomal mutation that inhibits accumulation of positive- and negative-strand RNA3 and subgenomic mRNA. BMV-directed gene expression was inhibited when the RNA replication template was introduced by in vivo transcription from DNA or by transfection of yeast with in vitro transcripts, confirming that cytoplasmic RNA replication steps were defective. mab1-1, mab2-1, and mab3-1 slowed yeast growth to varying degrees and were temperature-sensitive, showing that the affected genes contribute to normal cell growth. In wild-type yeast, expression of the helicase-like 1a protein increased the accumulation of 2a mRNA and the polymerase-like 2a protein, revealing a new level of viral regulation. In association with their other effects, mab1-1 and mab2-1 blocked the ability of 1a to stimulate 2a mRNA and protein accumulation, whereas mab3-1 had elevated 2a protein accumulation. Together, these results show that BMV RNA replication in yeast depends on multiple host genes, some of which directly or indirectly affect the regulated expression and accumulation of 2a.

Poliovirus RNA Polymerase Mutation 3D-M394T Results in a Temperature-Sensitive Defect in RNA Synthesis

Mutant ts10 is an RNA-negative temperature-sensitive mutant of Mahoney type 1 poliovirus. Mutant ts10 3Dpolwas purified from infected cells and was shown to be rapidly heat-inactivated at 45° when compared to wild-type polymerase. Sequencing of mutant ts10 genomic RNA revealed a U to C transition at nt 7167 resulting in an amino acid change of methionine 394 of 3Dpolto threonine. The 3D-M394T mutation was engineered into a wild-type infectious clone of poliovirus type 1. The resultant mutant virus, 3D-105, had a temperature-sensitive phenotype in plaque assays. The translation and replication of wild-type, ts10, and 3D-105 virion RNAs were all characterized in HeLa S10 translation-RNA replication reactionsin vitro.The optimum temperatures for the replication of the wild-type and mutant viral RNAs in the HeLa S10 translation-replication reactions were 37 and 34°, respectively. To characterize the temperature-sensitive defect in the replication of the mutant RNA, we used preinitiation RNA replication complexes which were formed in HeLa S10in vitroreactions containing guanidine HCl. Negative-strand RNA synthesis in 3D-M394T mutant preinitiation replication complexes was normal at 34° but was rapidly and irreversibly inhibited at 39.5°. To differentiate between the initiation and elongation steps in RNA replication, we compared the elongation rates in mutant and wild-type replication complexes at 39.5°. The results showed that the elongation rates for nascent negative strands in both the mutant and wild-type replication complexes were identical. Therefore, the results indicate that the heat-sensitive step in negative-strand synthesis exhibited by the 3D-M394T replication complexes is in the initiation of RNA synthesis and not in the elongation of nascent chains.

A single nucleotide substitution in the internal ribosome entry site of foot-and-mouth disease virus leads to enhanced cap-independent translation in vivo

Mutants of foot-and-mouth disease virus (FMDV) with altered biological properties can be selected during the course of persistent infection of BHK-21 cells with FMDV C-S8c1 (J. C. de la Torre, E. Martínez-Salas, J. Díez, A. Villaverde, F. Gebauer, E. Rocha, M. Dávila, and E. Domingo, J. Virol. 62:2050-2058, 1988). Two nucleotide substitutions, U to C at position -376 and A to G at position -15, (counting as +1 the A of the first functional AUG), were fixed within the internal ribosome entry site (IRES) of R100, the virus rescued after 100 passages of the carrier BHK-21 cells. IRES-directed cap-independent protein synthesis was quantitated by using bicistronic constructs of the form chloramphenicol acetyltransferase gene-IRES-luciferase gene. The IRES from R100 was 1.5- to 5-fold more active than that of C-S8c1 in directing cap-independent luciferase synthesis. This enhanced translational activity was observed when the RNAs were transcribed either in the nucleus or in the cytoplasm by a weak or a strong promoter, respectively. C-S8c1 and R100 IRES elements were functional in both FMDV-sensitive and FMDV-resistant cells (including persistently infected R cells), indicating that factors mediating cap-independent protein synthesis are not limited in any of the analyzed cell lines. Constructs in which each of the two mutations in the R100 IRES were analyzed separately indicate that the transition at position -376 is responsible for the enhanced activity of the R100 IRES. By estimating the effect that an increase in the initial translation efficiency may have on subsequent RNA replication steps, we suggest that the modifications in the IRES elements can account for the previously described hypervirulence of FMDV R100 for BHK-21 cells. The results show that a single point mutation in an IRES element of a picornavirus can cause an increase in translation efficiency.

An RNA sequence of hundreds of nucleotides at the 5' end of poliovirus RNA is involved in allowing viral protein synthesis.

Twenty-one mutations were engineered in the 5' noncoding region of poliovirus type 1 RNA, using an infectious cDNA copy of the viral genome. RNA was made from these constructs and used to transfect HeLa cells. Viable virus was recovered from 12 of these transfection experiments, including six strains with a recognizable phenotype, mapping in four different regions. One mutant of each site was studied in more detail. Mutant 5NC-11, having a 4-base insertion at nucleotide 70, was dramatically deficient in RNA synthesis, suggesting that the far 5' end of the genome is primarily involved in one or more steps of RNA replication. Mutants 5NC-13, 5NC-114, and 5NC-116, mapping at nucleotides 224, 270, and 392, respectively, showed a similar behavior; they made very little viral protein, they did not inhibit host cell translation, and they synthesized a significant amount of viral RNA, although with some delay compared with wild type. These three mutants were efficiently complemented by all other poliovirus mutants tested, except those with lesions in protein 2A. Our results imply that these three mutants map in a region (region P) primarily involved in viral protein synthesis and that their inability to shut off host cell translation is secondary to a quantitative defect in protein 2A. The exact function of region P is still to be determined, but our data supports the hypothesis of a single functional module allowing viral protein synthesis and extending over several hundred nucleotides.

Dependence of the activity of the poliovirus replicase on a host cell protein

Two poliovirus-specific RNA polymerase activities have been identified: a poly(U) polymerase that copies poly(A)·oligo(U) and a replicase that copies natural heteropolymers with some preference for poliovirus RNA. Both activities purified together until a step of a salt gradient elution from poly(U)-agarose, when poly(U) polymerase but no replicase was recovered. Addition of a salt wash fraction from the ribosomes of uninfected cells to this poly(U) polymerase fraction reconstituted replicase activity, and a host factor was purified 50 fold from the ribosomal salt wash. None of the available initiation factors or elongation factors for protein synthesis were able to reconstitute replicase activity. Host factor activity could be supplied by adding oligo(U), suggesting that the factor acts at the initiation step of RNA replication. With the purified replicase-host factor combination, only poly(A)-containing RNAs were copied, and a preference for poliovirus RNA was shown.

Polar effects of an amber mutation in f2 bacteriophage

Bacteriophage f2 mutant sus-3, containing an amber mutation near the N-terminus of the coat protein cistron, is polar. Bacteria infected by sus-3 synthesize little RNA polymerase or viral RNA. RNA from infecting phage is incorporated into a double strand only 15 to 25% as well as is RNA from normal virus. Suppression of the amber mutation results in a two- to fourfold increase in the amount of parental sus-3 double-stranded RNA. Two independent experiments established that the sus-3 amber mutation has no effect on the ability of double-stranded RNA to function in phage RNA replication, in contrast to our previous prediction (Lodish & Zinder, 1966 b). All of the polar effects of the sus-3 mutation can be ascribed to decreased synthesis of the enzyme required for the first step of RNA replication (production of complementary RNA). The results eliminate explanations of polarity which postulate a coupling between translation and synthesis of virus RNA.