THE RESPIRATORY SYNCYTIAL VIRUS POLYMERASE CAN PERFORM RNA SYNTHESIS WITH MODIFIED PRIMERS AND NUCLEOTIDE ANALOGS

Abstract

Respiratory syncytial virus (RSV) is significant for public health, capable of causing respiratory tract disease in infants, the elderly and the immunocompromised. The RSV polymerase is an attractive target for antiviral drug development, but as yet, there is no high throughput assay for analyzing RSV polymerase activity, specifically. In this study, using a primer elongation assay as a basis, we analyzed the tolerance of the RSV polymerase for modifications at the 5´ end of the primer, and nucleotide analogs. The RSV polymerase was found to accept primers containing 5´ biotin or digoxygenin modifications, and nucleotide analogs that are reactive or fluorescent, including 5-ethynyl UTP, 8-azido ATP, 2-aminopurine, and thieno-GTP. These findings provide a menu of options for developing non-isotopic high throughput assays for RSV polymerase RNA synthesis activity, and yield insight regarding the molecular biology of the polymerase complex.

Introduction

Respiratory syncytial virus (RSV) is a major public health concern world-wide (Nair et al., 2010). RSV infection generally causes upper respiratory infections with mild flu-like symptoms, but in some cases, infection can cause more serious lower respiratory tract disease. Infants and young children, the elderly, and immunocompromised individuals are at elevated risk for life-threatening RSV infection (Falsey et al., 2005; Nair et al., 2010). Moreover, severe RSV infection early in life has been linked to life-long respiratory problems, including asthma, wheezing and allergic sensitization (Blanken et al., 2013; Mochizuki et al., 2017; Sigurs et al., 2010; Wenzel et al., 2002). As yet, there is no vaccine to protect against RSV infection, and antiviral treatments are limited. Palivizumab is a monoclonal antibody that prevents RSV infection by inhibiting viral fusion with target cells. However, it is only effective prophylactically, and its use is restricted to high-risk infants (Fenton et al., 2004). Ribavirin is a small-molecule with activity against RSV, but it is no longer recommended because of poor efficacy and serious side effects (Cockerill et al., 2019). Although treatment options are currently limited, analysis of the kinetics of RSV infection have shown that viral load during the first three days post-infection correlates with severity of symptoms and progression to life-threatening disease in very young children (DeVincenzo et al., 2010; El Saleeby et al., 2011). This indicates that it would be possible to control RSV disease, if effective antiviral agents were available. Thus, there is a strong rationale for identifying inhibitors of RSV replication.

RSV has a single-stranded genome of negative sense RNA, which is transcribed and replicated by the viral RNA dependent RNA polymerase (Collins et al., 2013; Fearns and Plemper, 2017). The core polymerase consists of the large polymerase subunit (L) and phosphoprotein (P) (Mazumder and Barik, 1994). The 250 kDa L protein has three enzymatic domains that catalyze RNA polymerization, mRNA cap addition, and cap methylation (Fearns and Plemper, 2017). Because each of these activities is essential for viral replication, and because the enzymatic domains have features that are distinct from cellular enzymes, the polymerase is a particularly attractive target for antiviral development (Fearns and Deval, 2016). A number of small molecule inhibitors of the RSV polymerase are being explored as therapeutic agents. However, many compounds have failed to advance through clinical trials, due to limited bioavailability for example, and so there is a need to expand the pipeline (Cockerill et al., 2019). High throughput screens for RSV polymerase inhibitors have been developed using cell-based assays that gauge virus-induced cytopathic effect or changes in reporter gene expression (Malykhina et al., 2011; Tiong-Yip et al., 2014). However, these methods measure the endpoints of lengthy cascades of events, including cellular processes that may also be affected by test compounds, meaning that considerable downstream analysis is required to identify the target and mechanism of inhibition. There would be a significant advantage if it were possible to implement assays that measure RSV polymerase activities specifically, as a complementary or alternative approach.

Previously, our group expressed and purified recombinant RSV polymerase (Noton et al., 2012). Naturally, the template for the RSV polymerase is RNA encapsidated along its length with nucleoprotein (Grosfeld et al., 1995). Unfortunately, the N-RNA template is difficult to purify, and so far, it has not been possible to reconstitute it using RNA and recombinant nucleoprotein. However, it was possible to reconstitute RSV RNA synthesis by incubating the RSV L-P complex with a short RNA oligonucleotide template, containing the RSV promoter and radiolabeled nucleotides (Noton et al., 2014; Noton et al., 2012). In this assay, reaction products are separated by denaturing gel electrophoresis and visualized by autoradiography or phosphorimage analysis. However, while this technique yields detailed information on different aspects of RNA synthesis, it is not appropriate for high throughput screening of chemical libraries, and an alternative, non-isotopic approach is required. Non-isotopic assays for other viral polymerases have measured the pyrophosphate by-products of RNA synthesis (Lahser and Malcolm, 2004). However, because the RSV polymerase has evolved to transcribe an encapsidated, rather than naked template, it is not able to elongate long distances in vitro (Morin et al., 2012). This means that the number of RNA polymerization events performed by the RSV polymerase in vitro is relatively limited, and that measures of pyrophosphate output (which typically detect micromolar amounts of pyrophosphate) are probably not sufficiently sensitive to form the basis of a robust assay. Therefore, we considered alternative approaches for measuring RSV polymerase activity. Although the RSV polymerase normally begins RNA synthesis using a de novo (primer independent) initiation mechanism, it has been shown that it is able to carry out templated elongation of a short primer (Deval et al., 2015; Tchesnokov et al., 2018). This suggests a strategy by which a 5´ modified primer could be used in conjunction with a nucleotide analog substrate to assay RSV polymerase activity (Figure 1). For example, a biotin modified primer could be used in combination with a fluorescent nucleotide analog to generate an RNA product that could be immobilized and detected using a fluorescence readout. Alternatively, a FRET-based assay could be envisaged, in which a primer containing a fluorescent moiety is used in conjunction with a fluorescent nucleotide analog. Such assays would have the potential to be developed into a high-throughput format. However, these approaches would require the RSV polymerase to accommodate modified primers and nucleotide analogs. The degree to which the RSV polymerase can tolerate modified substrates has not been well defined. Therefore, the aim of this study was to investigate what primer modifications and nucleotide analogs could be accepted by the RSV RNA polymerase. The results obtained provide a menu of options for downstream assay development.

Figure 1. Design of an assay for RSV polymerase activity.

(A) Schematic diagram illustrating the primer elongation assay. Reactions contain recombinant RSV polymerase, RNA template (dotted line), a primer, and nucleotides. In some experiments, the primer contained a 5´ modification and in others, one of the nucleotides was an analog bearing a detectable or chemically reactive moiety. (B) Analysis of preparations of variant and wildtype RSV L-P preparations. The image shows an SDS polyacrylamide gel stained with colloidal blue stain. The marker is a BenchMark ladder.

Materials and methods

RNA and nucleotides

RNA oligonucleotides were purchased from Dharmacon or Integrated DNA Technologies. ^AzidoATP (8-azido-adenosine-5’-triphosphate tetralithium salt), ^2-aminoPTP (2-aminopurine-5’ riboside triphosphate tetralithium salt), ^ThienoGTP (2-aminothieno[3,4-d]pyrimidine-ribonucleoside-5’-triphosphate tetralithium salt) and ^Cy3UTP (Cyanine 3-aminoallyluridine-5’-triphosphate tetralithium salt) were from TriLink Biotechnologies (San Diego, CA). ^BiotinUTP (biotin-ε-aminocaproyl- γ-aminobutyryl-[5-(3-aminoallyl)-uridine-5´-triphosphate] tetralithium salt) was from Sigma-Aldrich. ^EthynylUTP (5-ethynyl-uridine-5´-triphosphate sodium salt) was from Abcam. ALS-8112-triphosphate was provided by Merck. Unmodified nucleotides were from Promega and ThermoFisher Scientific.

Purification of RSV RNA polymerase complexes

Recombinant RSV polymerase complex, consisting of codon-optimized polymerase (L) and His-tagged co-factor phosphoprotein P (P) of RSV A2 sequence were co-expressed in insect cells using a recombinant baculovirus (Noton et al., 2012). Sf21 cells were grown in 100-ml cultures in spinner flasks in SF900II medium. Cells were harvested 3 days p.i. by pelleting for 15 min at 1,200 x g, and washed with ice-cold PBS. Cells were lysed in 8 ml of lysis buffer (0.5% NP-40 in 50 mM sodium phosphate buffer, pH 8.0, containing 150 mM NaCl and 20 mM imidazole) for 15 min on ice. Cell extracts were incubated with 400 μl packed volume of Ni-NTA resin (ThermoFisher Scientific), pre-equilibrated in lysis buffer, for 2 h at 4°C with end-over-end rotation. Beads were pelleted by a quick spin to 700 x g at 4°C, washed three times with with 4 ml each of 60 mM imidazole in a buffer containing 50 mM sodium phosphate, pH 8.0, 150 mM NaCl and 0.5 % NP-40, and twice with 4 ml each of 100 mM imidazole in the same buffer. Polymerase complexes were eluted in 2 ml of 250 mM imidazole in the same buffer and dialyzed overnight against a buffer containing 20 mM Tris-HCl, pH 7.4, 150 mM NaCl, and 10% glycerol, and for an additional 2 h against the same buffer containing 1 mM DTT. Concentrations of L protein were estimated by comparing bands to a BSA standard curve on polyacrylamide gels stained with colloidal blue stain, using ImageJ software. Purified polymerase was stored at −150°C or in liquid nitrogen. Mutant polymerase D811A bears an inactivating substitution within the catalytic site of the RNA polymerase domain.

Primer elongation reactions

Primer elongation reactions contained 2 μM template, 20 μM primer, 10 μM native NTPs or 100 μM NTP analogs unless indicated otherwise in figure legends, and a radiolabeled [α-³²P]-NTP reporter nucleotide (0.2 μCi/μl; 0.07 to 0.14 μM) in elongation buffer (50 mM Tris-HCl, pH 7.4, 8 mM MgCl₂, 5 mM DTT and 10% w/v glycerol). Mixtures were equilibrated at 30°C for 5 min before the elongation reaction was initiated with the addition of recombinant polymerase complex at a final concentration of 1.6 ng/μl with respect to L protein. Unless otherwise specified, total reaction volume was 25 μl. After 2 h EDTA was added to a final concentration of 10 mM, and reactions were stored at −20°C. Primer elongation products were monitored by denaturing gel electrophoresis. Primer elongation reactions (3.5 or 10 μl) were mixed with an equal volume of deionized formamide containing 0.01% each bromophenol blue and xylene cyanol dye, and either first heat-denatured for 5 min at 95°C or directly loaded onto 20% acrylamide gels containing 7 M urea in Tris borate EDTA buffer. Reaction products were visualized by exposure to film or phosphor storage screens. Autoradiogram films were scanned using an Epson flatbed document scanner. The films were scanned alongside an autoradiogram that had been exposed to a 2-fold dilution series of radioactive probe, to ensure that the signals obtained were in the linear range. Alternatively, gels were analyzed by phosphorimager analysis using a BioRad Personal Molecular Imager. In both cases, products were quantified using Quantity One software (Version 4.6.6, BioRad).

Quantitative analysis of primer elongation products

In the case of primer elongation reactions comparing different primers, the 23 nt products (or 22 nt products in the case of the 4 mer ACGC primer and its cognate template), which would each contain the same number of radioactive incorporation sites were quantified. These bands are indicated with black arrowheads in the relevant figures. In the case of primer elongation reactions comparing the ability of nucleotide analogs to support elongation, bands representing elongation products containing the second addition of the analog or native nucleotide (indicated with an asterisk in the relevant figures) and all longer products were quantified (we did not quantify shorter products because in some cases the exposure required to detect longer products led to saturation of the signal for shorter products). The signal for each band was adjusted to account for the different amounts of radiolabel incorporation and the sum of the products in each reaction determined.

Results

The RSV polymerase can perform primed RNA synthesis on an artificial template

In the primer-based RSV RNA synthesis assay that was previously described, RNA synthesis was reconstituted using a template derived from the first eleven nucleotides of the RSV promoter and a short primer, complementary to the first four nucleotides of the template (Deval et al., 2015; Tchesnokov et al., 2018). In this situation, high concentrations of primer were used to favor primer-mediated rather than promoter-directed initiation. To increase the versatility of the assay and ensure that RNA synthesis would be primer dependent, we examined if the RSV polymerase could use a 5´ ^OHACGC primer with a template of artificial sequence (Figure 2A, lanes 1–3). A 24 nt template sequence was designed such that it had minimal similarity with the RSV promoter sequence and minimal secondary structure. RNA synthesis reactions were performed with template, primer and NTPs, using conditions that were described previously, and included [α³²P]-GTP, the first incorporated NTP, as the radiolabeled tracer. Analysis of the radiolabeled products by gel electrophoresis showed a different pattern of products in the absence versus presence of primer (Figure 2A, compare lanes 1 and 3). In the absence and presence of primer, products of 25 nt and longer (i.e. longer than the input template) were detected. These products were not readily detectable with a template containing a different 3´ terminal residue (e.g. Figure 2A, lanes 4 and 6) and subsequent experiments using a template containing a 3´ puromycin, rather than a 3´ hydroxyl group, showed that these longer products were due to nucleotide additions to the 3´ end of the template RNA (data not shown). This is possibly due to the propensity of the RSV polymerase to engage in back-priming activity, as described previously (Noton et al., 2012). In contrast, in the presence of primer, products from the first incorporation site up to 24 nt were detected in addition to the longer products (Figure 2A, lane 3). It should be noted that in this experiment, and all others presented in this paper, low molecular weight RNAs, of less than 10 nt, containing a 5´ OH group migrated more slowly than the ladder RNAs, which contained a 5´ monophosphate group, due to the difference in charge. RNA synthesis products were not detectable in reactions containing a polymerase variant with an alanine substitution in the polymerization domain of the L protein (referred to as mut; Figure 2A, lane 2). These results showed that the RSV polymerase could exclusively perform primer elongation, rather than promoter-directed initiation, if it were provided an artificial template and primer.

Figure 2. The RSV polymerase can utilize a non-promoter template in conjunction with 4 or 5 nucleotide primers.

(A) Analysis of RSV RNA elongation of 4, 5, and 8 nt primers in conjunction with a template of non-specific sequence. Reactions contained recombinant wt or variant polymerase deficient in RNA synthesis (mut), and 2 μM template, 10 μM each ATP, CTP, GTP and UTP, with [α³²P]-GTP as tracer. The templates were RNA oligonucleotides of non-specific sequence. Primer (underlined) was included at 20 μM or omitted, as indicated. (B) Analysis of 5, 8 and 11 nt primers. Reaction conditions were as described in (A) except that the template contained a 3´-phosphate group, to prevent it from being modified by 3´ nucleotide addition, and [α³²P]-ATP was included as a tracer. (A and B) Reaction products were separated by gel electrophoresis and visualized by phosphorimager analysis. Note that short primer elongation products, which bear a 5´-hydroxyl group, migrated more slowly in denaturing polyacrylamide gels than the ladder RNAs of corresponding size, which contained a 5´ monophosphate. Bands indicated with arrowheads represent the products that were quantified as presented in Table 1. M, molecular weight ladder.

We then tested the effect of primer length. A 5-nt primer containing an additional G residue at the primer 5´ terminus ^OHGACGC was efficient in supporting RNA elongation (Figure 2A, Lane 6) and yielded more product than the 4-mer primer (Table 1). Further increasing the length of the primer to eight nucleotides did not support elongation (Figure 2A, Lane 9). In this case, bands corresponding to small RNA products could be detected with long exposures. As these bands could be detected in reactions containing variant polymerase, as well as in the absence of template (Figure 2A, lanes 8 and 11), it seems likely that they were produced by terminal transferase modification of the primer by a contaminating enzyme, rather than a bona fide activity of the RSV polymerase. Similar results were obtained with a slightly different template, and primers with different 3´ termini (Figure 2B). While the 5-mer ^OHGACGC primer was efficiently elongated by the RSV polymerase, elongation of a primer that was eight nucleotides in length was weak and no elongation was detected with an 11-nucleotide primer (Figure 2B, compare lanes 6, 8, and 10). Together, these data show that the RSV polymerase can utilize short primers to synthesize RNA from a template with artificial sequence.

Table 1. Relative levels of RNA synthesis products generated with different primers.

Each primer was tested in two experiments and the 23 nt band (or 22 nt band in the case of the experiment using ^OHACGC) was quantified (these bands are indicated with black arrowheads in Figures 2 and 3). The individual experimental results are shown with mean values in parentheses.

Primer	RNA synthesis relative to OHGACGC
OHACGC	0.44, 0.18 (0.31)
BiotinGACGC	0.48, 0.56 (0.52)
DIGGACGC	0.50, 0.57 (0.54)

The RSV polymerase can tolerate primers containing a 5´ modification

To examine whether the polymerase could tolerate large adducts at the 5´ terminus of a primer, primers bearing either biotin (^BiotinGACGC) or digoxigenin (^DIGGACGC), which could potentially be used in solid-phase capture applications or affinity labeling, or cyanine 5 (^Cy5GACGC), as a possible partner for a FRET-based assay, were tested. Each of these modifications adds a bulky side chain to the 5´ terminal nucleotide with the digoxigenin adduct being of the greatest molecular weight, and the biotin adduct being the smallest (Figure 3A). The polymerase was able to generate elongation products from all three modified primers (Figure 3B). The sizes of the products were different from those of the ^OHGACGC primer, consistent with the presence of the adduct. The biotin and digoxigenin linked primers yielded similar levels of full-length products as the hydroxyl primer, demonstrating that, despite their size, they were well tolerated by the RSV polymerase (Figure 3B, lanes 3–5). In contrast, the Cy5 linked primer yielded small products containing approximately 1–4 nt additions (Figure 3B, lane 6) whereas amounts of longer products were at or below the limit of detection (Figure 3B, lane 6 and 6*). This result indicated that although the RSV polymerase was able to utilize primers containing a bulky 5´ terminal moiety, the structure and possibly the hydrophilicity of the modification determined how efficiently the primer could be elongated, with the Cy5 primer being accepted into the active site of the polymerase, but hindering polymerase progression.

Figure 3. RSV polymerase can utilize primers bearing bulky adducts at the 5´ terminus.

(A) Schematic diagram illustrating the primers and their 5´ modifications: biotin, digoxigenin (DIG) and cyanine 5 (Cy5). (B) Analysis of RNA synthesis products generated from the primers. Reactions contained 2 μM template, 20 μM primer, 10 μM each NTP and [α³²P]-GTP, the first incorporated nucleotide, as tracer. Reaction products were separated by gel electrophoresis and visualized by autoradiography (lanes 1–5) or phosphorimager analysis (lanes 6 and 7). Lane 6* is a longer exposure of lane 6. Bands indicated with arrowheads represent the products that were quantified as presented in Table 1. M, molecular weight ladder.

The results presented above differ from those of a previous study showing that the RSV polymerase could only use a primer containing a 5´ phosphate group (Tchesnokov et al., 2018). Therefore, we performed an additional control to ensure that the primer elongation activity that was observed was due to the RSV polymerase. To accomplish this, the primer containing the biotin modification was used in reactions containing ALS-8112-triphosphate, a cytidine triphosphate analog that acts as a chain terminator inhibitor specific to the RSV polymerase (Figure 4A) (Deval et al., 2015; Wang et al., 2015). Inclusion of increasing concentrations of ALS-8112-triphosphate caused a reduction in elongation beyond the CTP incorporation site (Figure 4B, lanes 2–4). This result confirmed that the primer elongation activity detected was a function of the RSV polymerase.

Figure 4. Primer elongation is inhibited by an RSV polymerase inhibitor.

(A) Chemical structures of CTP (cytidine 5’-triphosphate) and ALS-8112-TP (2’-fluoro-4’chloromethyl cytidine-5’-triphosphate). (B) Analysis of primer initiated RNA synthesis products generated in the presence of ALS-8112-triphosphate (ALS-8112-TP). ALS-8112-TP was omitted or added to reaction mixes containing 2 μM template, 15 μM primer and 10 μM each NTP, as indicated. Elongation was initiated by the addition of polymerase. Reactions either contained [α³²P]-UTP as a tracer (lanes 1–4), or [α³²P]-ATP (lanes 5, 6). M, molecular weight ladder.

The RSV polymerase can efficiently substitute ethynyl UTP for UTP

A variety of NTP analogs were tested for their ability to function as non-isotopic reporters of RNA elongation. These included analogs containing an alkyne or azide group, that could be modified by a click chemistry reaction, to add a fluorescent or biotin tag for example, an analog that contained a biotin group, and fluorescent analogs. Three UTP analogs, ^EthynylUTP, ^BiotinUTP and ^Cy3UTP were analyzed (Figure 5A). A template was designed containing multiple UTP incorporation sites, each separated by at least two nucleotides to avoid steric clashes between nucleotide analog side chains. In addition, the template contained a 3´ terminal phosphate group to prevent it from being modified by 3´ nucleotide addition, either by terminal transferase or back-priming activity. This template did not support RNA synthesis in the absence of primer, confirming that it did not have any inherent RSV promoter activity (Figure 5B, lanes 1 and 2). We assumed that the Km for analogs would be higher than for the native UTP, and that although 10 μM UTP was sufficient for primer elongation, the analog NTPs might be required at a higher concentration. Therefore, primer elongation reactions were performed containing either native UTP at 10 μM as positive control, or analog at a concentration of 100 μM, without added native UTP. The reactions contained [α³²P] ATP, the first templated nucleotide, as the radioactive tracer. In the presence of UTP, bands up to 24 nt were detected indicating that the primer was elongated to the end of the template (Figure 5C, lane 3). In contrast, in the absence of UTP, two strong bands could be detected, corresponding to the first two incorporations of ATP (Figure 5C, lane 2). Three much fainter bands could also be detected. These likely result from low level misincorporation of CTP (identified in independent experiments using radiolabeled CTP; data not shown). These data indicate that other NTPs could only substitute for UTP with very low frequency. Analysis of products generated in the presence of analogs showed that ^EthynylUTP was well tolerated, and did not inhibit further nucleotide incorporations (Figure 5C, lane 5; Table 2). Incorporation of ethynyl uridine into the newly synthesized RNA was detected as incremental, cumulative shifts in the electrophoretic mobility of the elongation products. Elongation products with altered mobility were also observed with ^Cy3UTP and ^BiotinUTP (Figure 5C, lanes 7 and 9, respectively). In these cases, it appeared that the polymerase incorporated the analog somewhat inefficiently at the first and second UTP incorporation sites, but elongation beyond the second analog incorporation site was weak. Thus, while these bulky analogs might be incorporated into the nascent RNA, they inhibited elongation.

Figure 5. Incorporation of UTP analogs by the RSV polymerase.

(A) Chemical structures of UTP analogs tested for incorporation into primer elongation products. UTP, uridine 5’-triphosphate; ^Cy5CTP, cyanine 3-aminoallyluridine-5’-triphosphate; ^EthynylUTP, 5-ethynyl-uridine-5’-triphosphate; ^BiotinUTP, biotin-ε-aminocaproyl-γ-aminobutyryl-[5-(3-aminoallyl)-uridine-5’-triphosphate (B) U₆ template designed for use with UTP analogs, with six incorporation sites spaced two to three nucleotides apart, did not direct RNA synthesis in the absence of primer. Reactions were performed with wildtype polymerase, 2 μM U₆ template, 10 μM each NTP and [α³²P]-ATP as a label, either without primer (lane 1) or with 20 μM ^OHGACGC primer (lane 2). (C) Analysis of RNA products generated in the presence of UTP analogs. Reactions contained 10 μM native UTP (lanes 1 and 3), no UTP (lane 2), or UTP analogs at 100 μm each in the absence of native UTP (lanes 4 to 9). mut, RSV polymerase deficient in RNA synthesis. M, molecular weight ladder. The bands indicated with an asterisk show the products extended to the second UTP incorporation site. These and all longer products were quantified, as presented in Table 2.

Table 2. Relative levels of RNA synthesis products generated with different NTP analogs.

Each analog was tested in two or three experiments, at 100 μM concentration and compared to the unmodified nucleotide which was included in reactions at 10 μM. Products extended to the second analog incorporation site (indicated with asterisks in Figures 5 and 6) and all longer products were quantified and compared to the values obtained for the corresponding reactions containing native NTPs. In each case, the data were normalized to account for the different number of radiolabel incorporation sites in the different sized products. The individual experimental results are shown with mean values in parentheses. The 2-amino purine data show comparison to ATP (rather than GTP).

Analog	RNA synthesis relative to 10 μm native NTP
2-amino purine	1.36, 1.28 (1.32)
8-azido ATP	0.37, 0.42 (0.40)
Thieno GTP	0.69, 0.94 (0.81)
5-ethynyl UTP	1.97, 1.50, 1.81 (1.76)

The RSV polymerase can tolerate purine analogs, but is selective in use of 2-amino purine

Purine analogs (Figure 6A) were analyzed using a similar approach, with either native or analog NTPs included in reactions at 10 or 100 μM, respectively. Templates were designed such that multiple ATP or GTP analogs could be incorporated at spaced intervals, referred to as A₃ and G₃ templates, respectively. Like the other templates used in this study, RNA synthesis from these templates was dependent on addition of a primer (Figure 6B). Analysis of the ATP analog ^AzidoATP and the purine analog ^2-aminoPTP showed that both were tolerated as surrogates for ATP, with ^2-aminoPTP in particular yielding similar levels of full-length product as native ATP at the concentration used (Figure 6C, compare lanes 3, 5, and 7; Table 2). The GTP analog, ^ThienoGTP, was also well tolerated by the polymerase (Figure 6D, compare lanes 3 and 5; Table 2). However, although ^2-aminoPTP could be used in place of ATP, it was not able to substitute for GTP (Figure 6D, compare lanes 8 and 10). In this experiment, we used [α³²P]-UTP as the tracer because an ATP tracer would have been diluted by the high concentration of ^2-aminoPTP, confounding the result. Because the first UTP incorporation site was after the first GTP incorporation site, it was not possible to determine if the RSV polymerase failed to incorporate ^2aminoPTP in place of GTP, or if it incorporated ^2-aminoPTP, but could not elongate it. To distinguish between these possibilities, an experiment was performed in which incorporation of ^2-aminoPTP in place of ATP and GTP was analyzed using the A₃ template (Figure 6E). In the presence of both ATP and GTP, the primer was elongated to the end of the template (Figure 6E, lane 2). If GTP was omitted, a prominent 13 nt band was detected rather than full-length products, indicating that the RNAs were elongated to the residue prior to the GTP incorporation site (Figure 6E, lane 3). Low levels of longer products could be detected, suggesting that the polymerase could utilize ATP in place of GTP, but at low efficiency. A similar pattern of products was detected if reactions contained ^2-aminoPTP in place of GTP (Figure 6E, lane 4). This result showed that the RSV polymerase could not incorporate ^2-aminoPTP in place of GTP. If reactions were performed in which both GTP and ATP were omitted, two bands could be detected, corresponding to addition of the first two (U) residues, confirming that the polymerase could not proceed past the ATP incorporation site (Figure 6E, lane 5). If ^2-aminoPTP was added in the absence of ATP and GTP, this block was alleviated and RNAs were elongated as far as the nucleotide prior to the GTP incorporation site (Figure 6E, lane 6), but no further. This result clearly shows that the active site of the RSV polymerase can accommodate diverse modified NTPs, but discriminates against unfavorable base pairings.

Figure 6. Purine analogs utilized by the RSV polymerase for primer elongation.

(A) Chemical structures of the ATP and GTP analogs used by the RSV polymerase for primer elongation: adenosine-5´-triphosphate (ATP); 8-azido-adenosine-5’-triphosphate (^AzidoATP); 2-aminopurine-5´ riboside triphosphate (^2-aminoPTP); 2-aminothieno[3,4-d]pyrimidine-ribonucleoside-5´-triphosphate (^ThienoGTP); guanosine-5´-triphosphate (GTP). (B) RNA synthesis from templates A₃ or G₃, containing three incorporation sites for ATP or GTP analogs, respectively, is not observed in the absence of primer. Wildtype polymerase was incubated with 2 μM A₃ or G₃ template (lanes 1 and 2, or 3 and 4, respectively), 10 μM each NTP and [α³²P]-UTP as a tracer. Reactions were performed either without primer (lanes 1 and 3) or with 20 μM ^OHGACGC primer (lanes 2 and 4). (C) Analysis of RNA products generated in the presence of ATP analogs. Reactions contained 2 μM of A₃ template and 20 μM ^OHGACGC primer, and either 10 μM native ATP (lanes 1 and 3), no ATP (lane 2) or 100 μM ATP analog (lanes 4 to 7). The radioactive tracer was [α³²P]-UTP. (D) Analysis of RNA products generated in the presence of GTP analogs. Reactions contained the G₃ template (2 μM) and ^OHGACGC primer (20 μM). Incorporation of ^ThienoGTP was tested using [α³²P]-ATP as a label and reactions testing incorporation of ^2-aminoPTP were carried out with [α³²P]-UTP. Control reactions contained template G₃, and 10 μM GTP (lanes 1, 3, 6 and 8) or no GTP (lanes 2 and 7). GTP was replaced by 100 μM ^ThienoGTP in lanes 4 and 5, and by 100 μM ^2aminoPTP in lanes 9 and 10. (E) The polymerase can discriminate between ^2-aminoPTP and GTP. Reactions contained template G₃, [α³²P]-UTP, and ATP, GTP or ^2aminoPTP as indicated. mut, RSV polymerase deficient in RNA synthesis. M, molecular weight ladder. The bands indicated with an asterisk show the products extended to the second incorporation site of the analog (or respective native NTP). These and all longer products were quantified, as presented in Table 2.

Discussion

The goal of this study was to identify primer modifications and analogs that could be tolerated by the RSV polymerase to facilitate future development of customized primer elongation assays suitable for use in high throughput screens. In addition to providing this information, the results obtained also provide some insight into the biology of the RSV polymerase that might help inform our mechanistic understanding of this key protein.

In its native state, the RSV polymerase does not use a primer to initiate RNA synthesis (Cressey et al., 2018; Noton et al., 2012; Tremaglio et al., 2013). Our results show for the first time that the RSV polymerase is capable of using primers in conjunction with templates consisting of artificial sequences and lacking an intact promoter. Thus, it is possible to design templates specifically tailored to direct incorporation of reporter nucleotide analogs at well-spaced sites, to avoid steric hindrance due to the bulkier side chains. The polymerase was able to use primers of four or five nucleotides in length to produce full-length templated elongation products. In these experiments, the 5-nt primer was more efficient than the 4-nt primer. The reason for this is unclear as the 4-nt primer was relatively efficient at generating smaller products, so it could be due to the template containing an additional 3´ nucleotide being preferred for elongation. In contrast, only low levels of elongation products were detected with one 8-mer primer tested, and products generated from a second 8-mer, or an 11-mer primers were below the limit of detection. In the case of the 8-mer primers, this was the case irrespective of whether the 5´ or 3´ end of the primer contained the same sequence as the 4 and 5-mer primers (Figure 2A and B, lane 8). Predicted melting temperatures for the 8-mer and 11-mer primers (47.3 to 53.8°C) were well above the reaction temperature of 30°C, whereas the predicted melting temperatures for the 4 and 5-mer primers were 12.6 and 27.8°C, respectively. It is possible that the polymerase could interact with single stranded template and primer, with annealing between the two RNA strands occurring after each had been threaded into the polymerization active site, but that the polymerase was unable to accept pre-formed RNA duplexes. Alternatively, it is possible that the polymerase could bind to a pre-formed primer-template duplex, but that the melting temperature of the duplex needed to be low enough to allow dissociation of the template and primer/ product strands after the polymerase began elongation of the primer. These findings have implications for understanding RSV polymerase behavior during infection as they suggest that if a polymerase were to disengage prematurely from the template/transcript complex, the transcript could not be subsequently elongated by the same or another re-associating polymerase. It was recently reported that a 5´ phosphate group on the ACGC primer was essential for templated primer elongation on an 11-mer template derived from the RSV genomic promoter (Tchesnokov et al., 2018). This was not the case in the present study. It is possible that the longer templates used in this study (17 to 25 nt) accounts for the difference in dependence on a 5´ phosphate group, with the phosphate being required to allow stabilization of the primer/template/polymerase complex in a situation with a relatively short (11 nt) template. The recently published structure of the RSV L-P complex suggests that the polymerase can adopt an elongation conformation, in which the exit channels for template and nascent RNA are apparent (Gilman et al., 2019). This could explain why the RSV polymerase is able to accept and utilize an artificial primer, even though it naturally initiates RNA synthesis by a de novo (primer independent) initiation mechanism. It might also explain why the polymerase can accommodate bulky modifications at the 5´ terminus of a primer.

We tested a number of nucleotide analogs for use as reporter nucleotides in downstream applications. Nucleotides bearing larger substitutions, ^Cy3UTP and ^BiotinUTP, appeared to be incorporated, as indicated by a significant shift in the mobility of the product following the first uridine incorporation site (i.e. the product migrating at 10 nt), but inhibited further elongation. If these analogs were indeed incorporated, this would suggest that the RSV polymerase could accommodate a nucleotide analog able to form appropriate base-pair interactions with the template residue, even if the analog contained a bulky side-chain. However, these results should be interpreted with caution. The products obtained with ^Cy3UTP or with ^BiotinUTP, had similar electrophoretic mobilities, and it is possible that these products resulted from incorporation of a contaminating 5-aminoallyl UTP reactant that is typically used in the synthesis of ^Cy3UTP and ^BiotinUTP, rather than the intended analog. Unfortunately, we were unable to detect incorporation of ^Cy3UTP by fluorescence imaging to confirm if it was incorporated, and so there is no definitive evidence one way or another. In any case, the low levels of elongation with these analogs indicate that they would not be effective in an assay.

In contrast to the larger analogs, nucleotides bearing small modifications were well-tolerated by the polymerase, including the fluorescent nucleotides ^ThienoGTP and ^2-aminoPTP, as well as chemically reactive ^AzidoATP and ^EthynylUTP. There were differences in the relative levels of elongation products in reactions containing nucleotide analogs. For example, the 14 and 17 nt bands were relatively stronger in reactions containing thieno-GTP compared to GTP (Figure 6D). As the 14 and 17 nt positions were GTP incorporation sites, these data suggest that the polymerase could incorporate the analog relatively efficiently, but then had a greater tendency to pause or dissociate prior to incorporation of the next nucleotide. The polymerase could incorporate the intrinsically fluorescent purine analog ^2-aminoPTP in place of ATP but not in place of GTP even though research with DNA polymerases has shown that 2-aminopurine is able to pair with cytidine (Reha-Krantz et al., 2011). Our findings are similar to results obtained with T4 and RB69 bacteriophage DNA polymerases, which exhibit strong preferences for the incorporation of thymidine over cytidine of ≥100-fold opposite 2-amino purine (Fidalgo da Silva et al., 2002; Reha-Krantz et al., 2011). Crystal structure analyses of base pairs within the active site of RB69 polymerases suggested that wobble base-paired thymidine and 2-aminopurine adopted conformations similar to authentic A-T base pairs, but pairing of 2-aminopurine with cytidine significantly increased the distance between the bases and forced the cytidine residue to adopt a tilted conformation (Reha-Krantz et al., 2011). The base-pairing interactions which allow the RSV polymerase to incorporate 2-amino PTP in place of ATP but not in place of GTP are not known, but it is possible that the active site of the polymerase would not readily allow the template contortion required for a 2-amino PTP-cytidine base-pairing interaction. Together these data suggest that the RSV polymerase could incorporate a nucleotide analog if it was able to form appropriate base-pair interactions with the template residue without requiring template repositioning, and could continue to elongate the RNA, provided that the analog was not too bulky.

The data presented here provide information on the feasibility of different approaches for non-isotopic detection of RSV polymerase activity. The simplest, a FRET-based assay using the Cy5 modified primer paired with incorporation of Cy3-UTP appears to be unfeasible due to the low yields of elongation products. However, assays with a fluorescent read-out could be developed using the more readily incorporated fluorescent analogs ^2-aminoPTP or ^ThienoGTP with biotin or digoxigenin linked primers, using streptavidin or antibodies to capture elongation products on a solid support, as necessary. ^ThienoGTP has been shown to be particularly well-suited for detection after incorporation into DNA oligonucleotides (Sholokh et al., 2015). In addition, Cy3 or biotin could be covalently bound to the RNA product post-RNA synthesis by taking advantage of the efficient incorporation of ^Azido-ATP and ^EthynylUTP, which could be substrates in a click chemistry reaction. Thus, although the natural properties of the RSV polymerase, such as its use of an encapsidated template, a de novo initiation mechanism and selectivity against non-native nucleotides, present challenges in developing RNA synthesis assays, the polymerase has flexibilities that could be exploited to design non-isotopic assays.