Comparison of RNA synthesis initiation properties of non-segmented negative strand RNA virus polymerases

It is generally thought that the promoters of non-segmented, negative strand RNA viruses (nsNSVs) direct the polymerase to initiate RNA synthesis exclusively opposite the 3´ terminal nucleotide of the genome RNA by a de novo (primer independent) initiation mechanism. However, recent studies have revealed that there is diversity between different nsNSVs with pneumovirus promoters directing the polymerase to initiate at positions 1 and 3 of the genome, and ebolavirus polymerases being able to initiate at position 2 on the template. Studies with other RNA viruses have shown that polymerases that engage in de novo initiation opposite position 1 typically have structural features to stabilize the initiation complex and ensure efficient and accurate initiation. This raised the question of whether different nsNSV polymerases have evolved fundamentally different structural properties to facilitate initiation at different sites on their promoters. Here we examined the functional properties of polymerases of respiratory syncytial virus (RSV), a pneumovirus, human parainfluenza virus type 3 (PIV-3), a paramyxovirus, and Marburg virus (MARV), a filovirus, both on their cognate promoters and on promoters of other viruses. We found that in contrast to the RSV polymerase, which initiated at positions 1 and 3 of its promoter, the PIV-3 and MARV polymerases initiated exclusively at position 1 on their cognate promoters. However, all three polymerases could recognize and initiate from heterologous promoters, with the promoter sequence playing a key role in determining initiation site selection. In addition to examining de novo initiation, we also compared the ability of the RSV and PIV-3 polymerases to engage in back-priming, an activity in which the promoter template is folded into a secondary structure and nucleotides are added to the template 3´ end. This analysis showed that whereas the RSV polymerase was promiscuous in back-priming activity, the PIV-3 polymerase generated barely detectable levels of back-primed product, irrespective of promoter template sequence. Overall, this study shows that the polymerases from these three nsNSV families are fundamentally similar in their initiation properties, but have differences in their abilities to engage in back-priming.     

The first two nucleotides of the respiratory syncytial virus antigenome RNA replication product can be selected independently of the promoter terminus

There is limited knowledge regarding how the RNA-dependent RNA polymerases of the nonsegmented negative-strand RNA viruses initiate genome replication. In a previous study of respiratory syncytial virus (RSV) RNA replication, we found evidence that the polymerase could select the 5'-ATP residue of the genome RNA independently of the 3' nucleotide of the template. To investigate if a similar mechanism is used during antigenome synthesis, a study of initiation from the RSV leader (Le) promoter was performed using an intracellular minigenome assay in which RNA replication was restricted to a single step, so that the products examined were derived only from input mutant templates. Templates in which Le nucleotides 1U, or 1U and 2G, were deleted directed efficient replication, and in both cases, the replication products were initiated at the wild-type position, at position -1 or -2 relative to the template, respectively. Sequence analysis of the RNA products showed that they contained ATP and CTP at the -1 and -2 positions, respectively, thus restoring the mini-antigenome RNA to wild-type sequence. These data indicate that the RSV polymerase is able to select the first two nucleotides of the antigenome and initiate at the correct position, even if the 3'-terminal two nucleotides of the template are missing. Substitution of positions +1 and +2 of the template reduced RNA replication and resulted in increased initiation at positions +3 and +5. Together these data suggest a model for how the RSV polymerase initiates antigenome synthesis.     

The methyltransferase domain of dengue virus protein NS5 ensures efficient RNA synthesis initiation and elongation by the polymerase domain

Viral RNA-dependent RNA polymerases (RdRps) responsible for the replication of single-strand RNA virus genomes exert their function in the context of complex replication machineries. Within these replication complexes the polymerase activity is often highly regulated by RNA elements, proteins or other domains of multi-domain polymerases. Here, we present data of the influence of the methyltransferase domain (NS5-MTase) of dengue virus (DENV) protein NS5 on the RdRp activity of the polymerase domain (NS5-Pol). The steady-state polymerase activities of DENV-2 recombinant NS5 and NS5-Pol are compared using different biochemical assays allowing the dissection of the de novo initiation, transition and elongation steps of RNA synthesis. We show that NS5-MTase ensures efficient RdRp activity by stimulating the de novo initiation and the elongation phase. This stimulation is related to a higher affinity of NS5 toward the single-strand RNA template indicating NS5-MTase either completes a high-affinity RNA binding site and/or promotes the correct formation of the template tunnel. Furthermore, the NS5-MTase increases the affinity of the priming nucleotide ATP upon de novo initiation and causes a higher catalytic efficiency of the polymerase upon elongation. The complex stimulation pattern is discussed under the perspective that NS5 adopts several conformations during RNA synthesis.     

In vitro synthesis of minus-strand RNA by an isolated cereal yellow dwarf virus RNA-dependent RNA polymerase requires VPg and a stem-loop structure at the 3' end of the virus RNA

Cereal yellow dwarf virus (CYDV) RNA has a 5'-terminal genome-linked protein (VPg). We have expressed the VPg region of the CYDV genome in bacteria and used the purified protein (bVPg) to raise an antiserum which was able to detect free VPg in extracts of CYDV-infected oat plants. A template-dependent RNA-dependent RNA polymerase (RdRp) has been produced from a CYDV membrane-bound RNA polymerase by treatment with BAL 31 nuclease. The RdRp was template specific, being able to utilize templates from CYDV plus- and minus-strand RNAs but not those of three unrelated viruses, Red clover necrotic mosaic virus, Cucumber mosaic virus, and Tobacco mosaic virus. RNA synthesis catalyzed by the RdRp required a 3'-terminal GU sequence and the presence of bVPg. Additionally, synthesis of minus-strand RNA on a plus-strand RNA template required the presence of a putative stem-loop structure near the 3' terminus of CYDV RNA. The base-paired stem, a single-nucleotide (A) bulge in the stem, and the sequence of a tetraloop were all required for the template activity. Evidence was produced showing that minus-strand synthesis in vitro was initiated by priming by bVPg at the 3' end of the template. The data are consistent with a model in which the RdRp binds to the stem-loop structure which positions the active site to recognize the 3'-terminal GU sequence for initiation of RNA synthesis by the addition of an A residue to VPg.     

A highly conserved lysine residue in phi29 DNA polymerase is important for correct binding of the templating nucleotide during initiation of phi29 DNA replication

DNA polymerases that initiate replication by protein-priming are able to catalyze terminal protein (TP)-primed initiation, the following transition steps and finally DNA-primed elongation. Therefore, their structures must be able to position sequentially both primers, TP and DNA, at a common binding site. For DNA-templated initiation, these DNA polymerases have to bind the origin of replication as template and TP as primer. It is likely that very precise interactions are required to position both TP and templating nucleotide at the polymerization active site. Such a specificity during TP-priming must rely on specific amino acids that must be evolutionarily conserved in this subfamily of DNA polymerases. By site-directed mutagenesis, we have analyzed the functional significance of Lys392 of phi29 DNA polymerase, immediately adjacent to the Kx3NSxYG motif, and specifically conserved among protein-primed DNA polymerases. During TP-primed initiation, mutations in this residue did not affect untemplated TP-dAMP formation, indicating that the interaction with the initiating nucleotide and TP were not affected, whereas the template-directed initiation activity was severely inhibited. Both mutant DNA polymerases had a wild-type-like (overall) DNA binding activity. We thus infer that residue Lys392 of phi29 DNA polymerase is important for the correct positioning of the templating nucleotide at the polymerization active site, a critical requirement during template-directed TP-priming at phi29 DNA origins. Consequently, mutation of this residue compromised the fidelity of the initiation reaction, not controlled by the 3'-5' exonuclease activity. During DNA-primed polymerization, the mutant polymerases showed a defect in translocation of the template strand. This translocation problem could be the consequence of a more general defect in the stabilization and positioning of a next templating nucleotide at the polymerization active site, during DNA-primed DNA synthesis.     

The Respiratory Syncytial Virus Polymerase Has Multiple RNA Synthesis Activities at the Promoter

Respiratory syncytial virus (RSV) is an RNA virus in the Family Paramyxoviridae. Here, the activities performed by the RSV polymerase when it encounters the viral antigenomic promoter were examined. RSV RNA synthesis was reconstituted in vitro using recombinant, isolated polymerase and an RNA oligonucleotide template representing nucleotides 1–25 of the trailer complement (TrC) promoter. The RSV polymerase was found to have two RNA synthesis activities, initiating RNA synthesis from the +3 site on the promoter, and adding a specific sequence of nucleotides to the 3′ end of the TrC RNA using a back-priming mechanism. Examination of viral RNA isolated from RSV infected cells identified RNAs initiated at the +3 site on the TrC promoter, in addition to the expected +1 site, and showed that a significant proportion of antigenome RNAs contained specific nucleotide additions at the 3′ end, demonstrating that the observations made in vitro reflected events that occur during RSV infection. Analysis of the impact of the 3′ terminal extension on promoter activity indicated that it can inhibit RNA synthesis initiation. These findings indicate that RSV polymerase-promoter interactions are more complex than previously thought and suggest that there might be sophisticated mechanisms for regulating promoter activity during infection.     

Modification of the 5' terminus of Sindbis virus genomic RNA allows nsP4 RNA polymerases with nonaromatic amino acids at the N terminus to function in RNA replication

We have previously shown that Sindbis virus RNA polymerase requires an N-terminal aromatic amino acid or histidine for wild-type or pseudo-wild-type function; mutant viruses with a nonaromatic amino acid at the N terminus of the polymerase, but which are otherwise wild type, are unable to produce progeny viruses and will not form a plaque at any temperature tested. We now show that such mutant polymerases can function to produce progeny virus sufficient to form plaques at both 30 and 40 degrees C upon addition of AU, AUA, or AUU to the 5' terminus of the genomic RNA or upon substitution of A for U as the third nucleotide of the genome. These results are consistent with the hypothesis that (i) 3'-UA-5' is required at the 3' terminus of the minus-strand RNA for initiation of plus-strand genomic RNA synthesis; (ii) in the wild-type virus this sequence is present in a secondary structure that can be opened by the wild-type polymerase but not by the mutant polymerase; (iii) the addition of AU, AUA, or AUU to the 5' end of the genomic RNA provides unpaired 3'-UA-5' at the 3' end of the minus strand that can be utilized by the mutant polymerase, and similarly, the effect of the U3A mutation is to destabilize the secondary structure, freeing 3'-terminal UA; and (iv) the N terminus of nsP4 may directly interact with the 3' terminus of the minus-strand RNA for the initiation of the plus-strand genomic RNA synthesis. This hypothesis is discussed in light of our present results as well as of previous studies of alphavirus RNAs, including defective interfering RNAs.     

Nucleotide sequence analysis of two simian virus 40 mutants with deletions in the late region of the genome.

Two mutants of simian virus 40, dl-1261 and dl-1262, have deletions that map between coordinated 0.90 and 0.95 (Cole et al., J. Virol 24:277--294, 1977). Both affect the structure of the two minor proteins VP2 and VP3. The precise location and size of the deletions have now been determined by nucleotide sequence analysis. Mutant dl-1261 is deleted of 54 base pairs, is temperature sensitive for the protein defined by the D complementation group, and promotes the synthesis of shorter VP2 and VP3 polypeptides. Mutant dl-1262 is viable irrespective of temperature and has a deletion of 36 base pairs, 23 of which overlap the deletion in dl-1261. Since these mutants produce normal VP1, the deleted regions probably have no function in the splicing of precursor RNA to the VP1 mRNA.     

Nucleolar targeting of hepatitis delta antigen abolishes its ability to initiate viral antigenomic RNA replication

Hepatitis delta virus (HDV) is a small RNA virus that contains one 1.7-kb single-stranded circular RNA of negative polarity. The HDV particle also contains two isoforms of hepatitis delta antigen (HDAg), small (SHDAg) and large HDAg. SHDAg is required for the replication of HDV, which is presumably carried out by host RNA-dependent RNA polymerases. The localization and the HDAg and host RNA polymerase responsible for HDV replication remain important issues to be addressed. In this study, using recombinant SHDAg fused with a heterologous nucleolar localization sequence (NoLS) to confine its subcellular localization in nucleoli, we aimed to study the effect of SHDAg subcellular localization on HDV RNA replication. The initiation of genomic RNA synthesis from antigenomic template was hardly detectable when SHDAg was fused with the NoLS motif and localized mainly in nucleoli. In contrast, the initiation of antigenomic RNA synthesis was not affected. Drug treatment to release a SHDAg-NoLS mutant from nucleoli could partially restore the replication of HDV genomic RNA from antigenomic RNA. This also recovered the cointeraction between SHDAg and RNA polymerase II. These data strongly suggest that nuclear polymerase (RNA polymerase II) is involved in the synthesis of genomic RNA and that the synthesis of antigenomic RNA can occur in nucleoli. Our results support the idea that the replication of HDV genomic RNA or antigenomic RNA is likely to be carried out by different machineries in different subcellular localizations.     

Discovery of a potent respiratory syncytial virus RNA polymerase inhibitor

Targeting viral polymerases has been a proven and attractive strategy for antiviral drug discovery. Herein we describe our effort in improving the antiviral activity and physical properties of a series of benzothienoazepine compounds as respiratory syncytial virus (RSV) RNA polymerase inhibitors. The antiviral activity and spectrum of this class was significantly improved by exploring the amino substitution of the pyridine ring, resulting in the discovery of the most potent RSV A polymerase inhibitors reported to date.     

Template nucleotide moieties required for de novo initiation of RNA synthesis by a recombinant viral RNA-dependent RNA polymerase

The recombinant RNA-dependent RNA polymerase of the bovine viral diarrhea virus specifically requires a cytidylate at the 3' end for the de novo initiation of RNA synthesis (C. C. Kao, A. M. Del Vecchio, and W. Zhong, Virology 253:1-7, 1999). Using RNAs containing nucleotide analogs, we found that the N3 and C4-amino group at the initiation cytidine were required for RNA synthesis. However, the ribose C2'-hydroxyl of the initiating cytidylate can accept several modifications and retain the ability to direct synthesis. The only unacceptable modification is a protonated C2'-amino group. Quite strikingly, the recognition of the functional groups for the initiation cytidylate and other template nucleotides are different. For example, a C5-methyl group in cytidine can direct RNA synthesis at all template positions except at the initiation cytidylate and C2'-amino modifications are tolerated better after the +11 position. When a 4-thiouracil (4sU) base analog that allows only imperfect base pairing with the nascent RNA is placed at different positions in the template, the efficiency of synthesis is correlated with the calculated stability of the template-nascent RNA duplex adjacent to the position of the 4sU. These results define the requirements for the specific interactions required for the initiation of RNA synthesis and will be compared to the mechanisms of initiation by other RNA-dependent and DNA-dependent RNA polymerases.     

Functional analysis of two cavities in flavivirus NS5 polymerase

Flavivirus NS5 protein encodes methyltransferase and RNA-dependent RNA polymerase (RdRp) activities. Structural analysis of flavivirus RdRp domains uncovered two conserved cavities (A and B). Both cavities are located in the thumb subdomains and represent potential targets for development of allosteric inhibitors. In this study, we used dengue virus as a model to analyze the function of the two RdRp cavities. Amino acids from both cavities were subjected to mutagenesis analysis in the context of genome-length RNA and recombinant NS5 protein; residues critical for viral replication were subjected to revertant analysis. For cavity A, we found that only one (Lys-756) of the seven selected amino acids is critical for viral replication. Alanine substitution of Lys-756 did not affect the RdRp activity, suggesting that this residue functions through a nonenzymatic mechanism. For cavity B, all four selected amino acids (Leu-328, Lys-330, Trp-859, and Ile-863) are critical for viral replication. Biochemical and revertant analyses showed that three of the four mutated residues (Leu-328, Trp-859, and Ile-863) function at the step of initiation of RNA synthesis, whereas the fourth residue (Lys-330) functions by interacting with the viral NS3 helicase domain. Collectively, our results have provided direct evidence for the hypothesis that cavity B, but not cavity A, from dengue virus NS5 polymerase could be a target for rational drug design.     

Molecular Basis for the Selective Inhibition of Respiratory Syncytial Virus RNA Polymerase by 2'-Fluoro-4'-Chloromethyl-Cytidine Triphosphate

Respiratory syncytial virus (RSV) causes severe lower respiratory tract infections, yet no vaccines or effective therapeutics are available. ALS-8176 is a first-in-class nucleoside analog prodrug effective in RSV-infected adult volunteers, and currently under evaluation in hospitalized infants. Here, we report the mechanism of inhibition and selectivity of ALS-8176 and its parent ALS-8112. ALS-8176 inhibited RSV replication in non-human primates, while ALS-8112 inhibited all strains of RSV in vitro and was specific for paramyxoviruses and rhabdoviruses. The antiviral effect of ALS-8112 was mediated by the intracellular formation of its 5''-triphosphate metabolite (ALS-8112-TP) inhibiting the viral RNA polymerase. ALS-8112 selected for resistance-associated mutations within the region of the L gene of RSV encoding the RNA polymerase. In biochemical assays, ALS-8112-TP was efficiently recognized by the recombinant RSV polymerase complex, causing chain termination of RNA synthesis. ALS-8112-TP did not inhibit polymerases from host or viruses unrelated to RSV such as hepatitis C virus (HCV), whereas structurally related molecules displayed dual RSV/HCV inhibition. The combination of molecular modeling and enzymatic analysis showed that both the 2''F and the 4''ClCH₂ groups contributed to the selectivity of ALS-8112-TP. The lack of antiviral effect of ALS-8112-TP against HCV polymerase was caused by Asn291 that is well-conserved within positive-strand RNA viruses. This represents the first comparative study employing recombinant RSV and HCV polymerases to define the selectivity of clinically relevant nucleotide analogs. Understanding nucleotide selectivity towards distant viral RNA polymerases could not only be used to repurpose existing drugs against new viral infections, but also to design novel molecules.     

Crystal Structure of the Human Primase

DNA replication in bacteria and eukaryotes requires the activity of DNA primase, a DNA-dependent RNA polymerase that lays short RNA primers for DNA polymerases. Eukaryotic and archaeal primases are heterodimers consisting of small catalytic and large accessory subunits, both of which are necessary for RNA primer synthesis. Understanding of RNA synthesis priming in eukaryotes is currently limited due to the lack of crystal structures of the full-length primase and its complexes with substrates in initiation and elongation states. Here we report the crystal structure of the full-length human primase, revealing the precise overall organization of the enzyme, the relative positions of its functional domains, and the mode of its interaction with modeled DNA and RNA. The structure indicates that the dramatic conformational changes in primase are necessary to accomplish the initiation and then elongation of RNA synthesis. The presence of a long linker between the N- and C-terminal domains of p58 provides the structural basis for the bulk of enzyme''s conformational flexibility. Deletion of most of this linker affected the initiation and elongation steps of the primer synthesis.     

Molecular Basis for Nucleotide Conservation at the Ends of the Dengue Virus Genome

The dengue virus (DV) is an important human pathogen from the Flavivirus genus, whose genome- and antigenome RNAs start with the strictly conserved sequence pppAG. The RNA-dependent RNA polymerase (RdRp), a product of the NS5 gene, initiates RNA synthesis de novo, i.e., without the use of a pre-existing primer. Very little is known about the mechanism of this de novo initiation and how conservation of the starting adenosine is achieved. The polymerase domain NS5Pol_DV of NS5, upon initiation on viral RNA templates, synthesizes mainly dinucleotide primers that are then elongated in a processive manner. We show here that NS5Pol_DV contains a specific priming site for adenosine 5′-triphosphate as the first transcribed nucleotide. Remarkably, in the absence of any RNA template the enzyme is able to selectively synthesize the dinucleotide pppAG when Mn²⁺ is present as catalytic ion. The T794 to A799 priming loop is essential for initiation and provides at least part of the ATP-specific priming site. The H798 loop residue is of central importance for the ATP-specific initiation step. In addition to ATP selection, NS5Pol_DV ensures the conservation of the 5′-adenosine by strongly discriminating against viral templates containing an erroneous 3′-end nucleotide in the presence of Mg²⁺. In the presence of Mn²⁺, NS5Pol_DV is remarkably able to generate and elongate the correct pppAG primer on these erroneous templates. This can be regarded as a genomic/antigenomic RNA end repair mechanism. These conservational mechanisms, mediated by the polymerase alone, may extend to other RNA virus families having RdRps initiating RNA synthesis de novo.