We have established a novel method for measuring the RdRp activity of influenza viruses using RT-PCR. In current methods [17, 20], the amount of mRNA synthesized is quantified using 32P-labeled nucleoside triphosphates (GTP or UTP). Therefore, only the total number of eight-segment mRNAs is calculated. However, in the method we developed, the mRNA of each segment can be analyzed individually, and the copy number of the mRNA produced can be quantified through RT-PCR.
In this study, mRNAs of segments 1, 4, and 5 were examined, but by designing the primer using the primer design reported by Kawakami et al.  as reference, the amount of cRNAs and vRNAs of each of the eight segments present in influenza virus might be also quantified. As described in the result section, the cRNA and mRNA sequences (positive-sense single-strand) and negative-sense single-strand vRNA sequences are complementary. It is possible to quantify the vRNA of each segment by using a tagged RT primer that specifically binds to the vRNA. The amount of cRNA synthesis can also be measured by using RT primers in which a tag (a sequence different from the tag sequence for mRNA or vRNA) is added to the 3’ end sequences transcribed from vRNA without a poly A tail.
The amount of purified RdRp should be evaluated to avoid errors between experiments. We quantified the amount of vRNA in segments 1, 4, and 5 present in RdRp by real-time qPCR using primers designed as well as mRNA (Additional file 1: Fig. S3; Additional file 2: Table S1). The amount of vRNA present in the purified RdRp was almost the same between these segments. This result indicates that the amount of vRNA in any segment present in RdRp can be used as a reference.
In this study, we attempted to quantify the mRNA levels of HA (segment 4) and NP (segment 5), which are highly expressed among the constituent proteins of influenza virus, and of PB2 (segment 1), which is one of the essential components of RdRp. Kawakami et al. had quantified segments 5 and 6 mRNA in MDCK cells infected with influenza virus (A/WSN/33 strain) . They showed that the expression of segment 5 mRNA was significantly higher than that of segment 6 mRNA . Phan et al. analyzed the expression of mRNA, cRNA, and vRNA of all eight segments in infected cells using RNA sequencing . They demonstrated that the mRNA levels of PB2, PB1, and PA were lower than those of the other five segments at any given time after infection . In our results, the mRNA level of PB2 (segment 1) was lower than that of HA (segment 4) and NP (segment 5). (Figs. 5 and 6). This result indicates that the expression of mRNA may differ between segments, even in in vitro experiments. In addition, in the RTP experiment (Fig. 6), the amount of GTP added to the RdRp reaction solution was reduced, as previously reported [18, 19]. Although the low GTP content in the RdRp reaction solution may have strongly affected the transcription of segment 1 mRNA, the GTP content in each segment mRNA sequence without cap structure was not different (GTP content segment 1, 25.5%; segment 4, 23.2%; segment 5, 26.7%). The reason behind no significant difference in the amount of mRNA between the three segments (Figs. 2 and 3) is unclear. To evaluate the mRNA transcription activity of RdRp using the method developed in this study, it is necessary to evaluate the mRNA transcription levels of multiple segments.
In this study, the conditions for the RdRp reaction were same for the three segments. While measuring the other segments, it is possible to work under the same RdRp reaction conditions as in this study. However, as mentioned above, the transcription amount of each segment may be different. Further research is necessary to determine the optimal conditions for the RdRp reaction for each segment. It is also possible to improve the specificity of primer binding by considering the optimization of the RT reaction and real-time qPCR conditions for each segment.
Viral particles were purified from the culture supernatants of infected cells. Although ultracentrifugation is commonly used to prepare influenza virus polymerase solutions, the method is time consuming and unsuitable for screening. In this experiment, we collected influenza viral particles using magnetic beads. Sakudo et al. have shown through immunochromatography that these beads can efficiently capture influenza viruses in cell culture media . Using this method, it is possible to collect viral particles more easily and quickly than that using ultracentrifugation; therefore, viral particles with high RdRp enzyme activity can be collected. However, as these beads may adsorb various viruses through electrostatic interactions, they are not specific for influenza virus, leading to contamination with other components from the cell culture medium. In our case, the virus polymerase activity was not affected by contamination with the culture medium.
The reaction time for polymerase activity was the same as previously reported . The optimum temperature for this method was 37 °C (Fig. 3), but some previous studies have reported the use of 30 °C [9, 15, 23, 24] Even though studies have reported the use of 37 °C [25, 26], it is unclear why 37 °C was optimum for our method.
Regarding the concentration of Mg in the RNA synthesis reaction buffer, the polymerase activity increased (Fig. 4) at Mg concentrations similar to those previously reported [9, 13, 17]. However, the amplification of each segment increased sharply at 3 or 4 mM MgCl2 and showed little increase at concentrations below 3 mM (Fig. 4). Zhang et al. showed a similar rapid increase, although the border concentrations were slightly different .
The presence of ApG, a specific dinucleotide primer, increased mRNA production by approximately tenfold in each segment, but mRNA was synthesized to a certain extent even in its absence (Fig. 5).
RTP inhibits RdRp because it is mistakenly taken up during mRNA synthesis as it is similar in structure to GTP and stops mRNA synthesis [18, 19]. Initially, when the GTP concentration was the same as that of ATP, CTP, and UTP, 100 µM RTP had no inhibitory effect, and 500 µM RTP only inhibited segment 1 by 57.1% and segment 4 by 47.1% (data not shown). Therefore, in this experiment we lowered GTP concentration to show the inhibitory effect of RTP. The inhibitory effect of 100 µM RTP was observed resulting in 20.8% inhibition for segment 1, 39.2%, for segment 4, and 44.5% for segment 5 (Fig. 6). In segments 4 and 5, there was no effect of the GTP concentration on mRNA production, and mRNA production decreased depending on the RTP concentration (Fig. 6b, c). However, in segment 1, mRNA production was significantly reduced by the dilution of GTP, even without RTP. Therefore, the concentration-dependent inhibition of RTP could not be confirmed in segment 1 (Fig. 6a). The concentration of GTP is considered important for the synthesis of segment 1 mRNA.
Since the amount of mRNA synthesis was reduced in the absence of ApG or magnesium, and the inhibition of synthesis by RTP was confirmed, this experimental method proved useful at evaluating the activity of RdRp of influenza viruses.
In the analysis of influenza RdRp activity using the dual-luciferase reporter assay, the 5’ and 3' untranslated regions of vRNA (the region recognized and bound by viral polymerase) were inserted into the luciferase reporter system to detect and quantify RdRp activity [28,29,30,31,32]. The advantage of the dual-luciferase reporter assay is that it is useful for high-throughput screening of many compounds. To apply the method developed in this study to high-throughput screening, further ingenuity is required in the process between the RdRp reaction and real-time PCR. However, as the dual-luciferase reporter assay is a method for evaluating intracellular RdRp activity, it may be affected by various intracellular factors. The method developed in this study is an in vitro RdRp reaction; therefore, it is possible to evaluate the direct effect on RdRp. In addition, the dual-luciferase reporter assay requires a luminometer; however, the method developed in this study involves a more commonly used real-time qPCR measuring device.