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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Virology. Author manuscript; available in PMC 2013 November 10.
Published in final edited form as:
PMCID: PMC3647620

Endonuclease Substrate Selectivity Characterized with Full-Length PA of Influenza A Virus Polymerase


The influenza A polymerase is a heterotrimer which transcribes viral mRNAs and replicates the viral genome. To initiate synthesis of mRNA, the polymerase binds a host pre-mRNA and cleaves a short primer downstream of the 5′ end cap structure. The N-terminal domain of PA has been demonstrated to have endonuclease activity in vitro. Here we sought to better understand the biochemical nature of the PA endonuclease by developing an improved assay using full-length PA protein. This full-length protein is active against both RNA and DNA in a cap-independent manner and can use several different divalent cations as cofactors, which affects the secondary structure of the full-length PA. Our in vitro assay was also able to demonstrate the minimal substrate size and sequence selectivity of the PA protein, which is crucial information for inhibitor design. Finally, we confirmed the observed endonuclease activity of the full-length PA with a FRET-based assay.

Keywords: Influenza A Virus, polymerase, endonuclease


Influenza A virus encodes an RNA-dependent RNA-polymerase responsible for transcription of viral mRNAs and replication of the genomic RNA segments within the host cell nucleus. The viral polymerase is a heterotrimer composed of two basic proteins, PB1 and PB2, as well as the acidic protein PA. Genome replication occurs through primer-independent initiation of (+) strand RNA synthesis from the (−) strand viral genomic segments, which are then used as a template for production of new (−) strand genomic RNA. This process occurs mainly through the active site of the PB1 subunit, which contains the conserved motifs of an RNA polymerase (Biswas and Nayak, 1994; Poch et al., 1989). Viral mRNAs, however, must contain both a 5′ cap structure and a poly(A) tail in order to be recognized by host translational machinery. The polymerase creates a poly(A) tail through a stuttering mechanism (Poon et al., 1999), but the cap structure of viral mRNAs must be derived from cellular pre-mRNAs in a process known as cap-snatching (Beaton and Krug, 1981; Bouloy et al., 1978). The cellular mRNA is cleaved 10-15 nucleotides from the cap structure and is then used as a primer for transcription of the viral mRNA (Plotch et al., 1981) .

The endonuclease activity involved in cap snatching is a novel viral process, making it an attractive target for small molecule inhibition. The cleavage of the host mRNA is catalyzed by endonuclease activity contained within the polymerase complex. This endonuclease active site has been previously reported to be in either the PB1 or the PB2 subunit but was not conclusively demonstrated (Li et al., 2001; Shi et al., 1995). This uncertainty was resolved when crystal structures of the N-terminal domain of the PA subunit revealed an endonuclease active site with similarity to type II restriction endonucleases such as Sda I (Dias et al., 2009; Yuan et al., 2009). The endonuclease activity of the PA subunit was biochemically confirmed using the purified N-terminal truncation (Crepin et al., 2010; Dias et al., 2009). The endonuclease domain of PA has been shown to be active against both single stranded RNA and DNA and is dependent on the binding of divalent cations (Dias et al., 2009). Maximal activity is seen using manganese, but magnesium has been argued to be more biologically relevant (Crepin et al., 2010; Dias et al., 2009; Doan et al., 1999). The active site is highly conserved among influenza viruses with only a single amino acid difference among influenza B and influenza C viruses (Yuan et al., 2009). A similar viral endonuclease has been identified in the L protein of viruses in the Arenaviridae and Bunyaviridae families, though the active site and substrate specificity are distinct from that of influenza PA (Morin et al., 2010; Reguera et al., 2010).

Previous studies of the influenza A endonuclease have used either viral ribonucleoparticles isolated from mammalian cells or the truncated N-terminal domain, which can be purified from E. coli. Both of these systems have limitations that make them unsuitable for in-depth enzymology and, ultimately, screening for small molecule inhibitors. Purification of protein from infected mammalian cells is not readily scaled up, making the large-scale production necessary for biochemical studies and high throughput screening not feasible. Assays using these viral ribonucleoprotein particles have also needed to use a capped substrate and the addition of the viral promoter RNA in order to observe endonuclease activity (Hagen et al., 1994). Protein purification from E. coli is amenable to large-scale production of protein, but the use of a truncated enzyme has several drawbacks. It was noted by Yuan et al. that, in their studies, PA N-terminal domain endonuclease activity could not be determined due to nuclease contamination (Yuan et al., 2009). Dias et al. were able to observe endonuclease activity using the truncated PA protein, but only in the presence of manganese and not with any other divalent cation tested (Dias et al., 2009). More recently, Crepin et al. demonstrated that the truncated PA subunit had endonuclease activity in the presence of both magnesium and manganese, but this activity was low and their assay used long incubations (up to 6 hours) and did not quantify activity (Crepin et al., 2010). Given that cell-derived ribonucleoprotein particles have endonuclease activity with several metal ions in addition to magnesium and manganese (Doan et al., 1999), the truncated PA protein seems to behave differently than the full trimeric polymerase. We sought to develop an improved assay for characterizing the endonuclease activity of the influenza A protein using the full-length PA protein. We hypothesize that this intact protein will be more active as well as more biologically relevant than the truncated PA N-terminal domain previously used.

Here we demonstrate that enzymatically active intact PA can be purified from insect cells. Among the strains tested, the avian Nanchang strain was shown to have the highest in vitro endonuclease activity. This in vitro assay was used to show the minimal substrate size and sequence specificity. We also demonstrate that the divalent cation cofactor affects the cleavage pattern and secondary structure of the protein as well as the overall activity. The endonuclease activity of the PA subunit is comparable to that of the full trimeric polymerase but is expressed at much higher levels using our system. Given the difficultly in purifying the polymerase complex, our in vitro assay using the intact PA subunit is an attractive model for studying the endonuclease activity of the viral polymerase and is also a valuable tool for development of new antivirals.


Comparison of PA endonuclease activities from different strains

Previous work has used purified PA from a single influenza A strain and no comparison of endonuclease activity across different strains has been performed. We purified PA from a variety of strains in order to determine the protein with the highest endonuclease activity. Using TAP-tagged constructs (shown in Fig. S1A), we were able to express PA from four different strains: the lab adapted H1N1 WSN (WSN), the pandemic 1918 H1N1 (1918), a high pathogenicity avian H5N1 strain isolated from a human (H5N1), and a low pathogenicity avian strain isolated from a chicken (Nanchang). The PA subunit from the 2009 pandemic strain expressed at very low levels (data not shown) despite being codon optimized, which precluded its use in our study. The intact PA subunits from the four different influenza A strains were purified from Tni insect cells and diluted to equal concentrations (Fig. S1B). To measure endonuclease activity, the proteins were incubated with a 5′ end labeled 33mer RNA. Manganese was added to the reactions, as maximal activity has been reported with this metal cofactor (Crepin et al., 2010). We tested activity at temperatures ranging from 30 to 42°C (data not shown) and found that all of the strains had maximal endonuclease activity at 37°C. All further experiments were carried out at 37°C. After one-hour incubation, products were resolved using 20% urea PAGE. As shown in Fig. 1, distinct cleavage products were observed using all four PA proteins, although the levels of endonuclease activity varied between the strains. The pattern of cleavage is likely to be partially determined by the structured nature of this RNA substrate (predicted by RNAstructure (Reuter and Mathews, 2010)), as the PA endonuclease is only able to cleave single stranded regions (Dias et al., 2009). An additional caveat is that only products retaining the 5′ end label can be visualized on the gel. The smallest product (Fig. 1, *) results from the cleavage of the 5′ terminal G from the RNA (confirmed with size markers, data not shown). Gels in subsequent figures are cropped for presentation purposes, but this cleavage product was observed in all reactions. The presence of cleavage products near both ends of the RNA suggests that there is little directionality with PA binding given this long substrate. In order to cleave very close to the 5 end, the protein must be binding the substrate in the opposite orientation as compared to when cleaving a capped substrate. Overall, this assay revealed that the avian Nanchang strain had the highest PA endonuclease activity of strains tested. Therefore, we chose to conduct all further experiments using PA from the Nanchang strain (Nan PA).

Figure 1
Full-length purified PA protein from insect cells has endonuclease activity. Comparison of PA activity among strains. Protein was incubated with a 5′ 33P-labeled 33mer RNA for 60 minutes at 37°C in the presence of 1 mM MnCl2. A control ...

The endonuclease activity is PA subunit dependent and inhibited by DPBA

To confirm that the observed nuclease activity was in fact due to the endonuclease activity of PA, we created the E119A mutation in the active site of Nan PA. This mutation was previously shown to ablate endonuclease activity (Crepin et al., 2010). As shown in Fig. S2A, no degradation of the RNA substrate was seen with the E119A Nan PA, indicating that there are no detectable contaminating nucleases. We also tested the previously published influenza endonuclease inhibitor DPBA (Tomassini et al., 1994). As shown in Fig. S2B, there is a dose dependent inhibition of RNA degradation observed with the addition of DPBA. We estimated the IC50 of DPBA to be 2 μM, which is similar to that reported previously in tissue culture systems (Dias et al., 2009; Tomassini et al., 1994). To confirm that the Nan PA subunit alone has comparable activity to the PA subunit present in the trimeric polymerase complex (Nan 3P), equal protein amounts of Nan PA and Nan 3P were incubated with the RNA substrate for 1 hour. Due to the very low level of expression of the Nan 3P, Nan PA was diluted 10-fold in order to be able to use comparable amounts of protein. As shown in Fig. S2C, although Nan 3P is more active than the Nan PA subunit alone, quantification of remaining full-length RNA suggests the difference in activity is only two-fold. Importantly, the pattern of cleavage products is identical for Nan 3P and Nan PA, indicating that the active site of the PA subunit is likely recognizing the substrate in the same way as PA contained within the polymerase complex and therefore is a good model for screening for inhibition. Interestingly, the activity of the trimeric polymerase was not dependent on the presence of the viral promoter RNA as has been previously reported (Hagen et al., 1994). This may be partially due to differences in the sensitivity of the two assays or our assay measuring cap-independent endonuclease activity.

The activity and cleavage pattern with long RNA and DNA substrates

After confirming the specificity of our assay, we next wanted to compare the activity of Nan PA in the presence of manganese and magnesium using the 33mer RNA substrate. A time course over one hour was performed to observe the accumulation of different cleavage products. As shown in Fig. 2, our full-length Nan PA is also active in the presence of magnesium (Fig. 2A), though, as previously reported, significantly less active than in the presence of manganese (Fig. 2B). Surprisingly, the cleavage pattern was also distinct between the two metal cofactors. As previously shown in Fig. 1, PA with manganese produces a distinct pattern of cleavage products, which correspond to single stranded regions within the predicted secondary structure of this RNA. The absence of these specific bands in 2A suggests that PA with magnesium shows less internal cleavage with an apparent preference for cleavage near the ends. These reactions were repeated with DNA of the same sequence using magnesium (Fig. 2C) and manganese (Fig. 2D). With this substrate there was a preference for cleavage near both the 3′ and 5′ ends of the DNA. In the presence of manganese, the cleavage was mainly from the 5′ end of the substrate. This lack of internal cleavage may be because this DNA substrate is also predicted to be structured (Reuter and Mathews, 2010). Overall, these data suggest that the metal cofactor affects not only the level of activity, but, interestingly, also affects the preferred cleavage sites when using partially structured RNA and DNA substrates.

Figure 2
PA endonuclease cleaves both RNA and DNA. Nan PA protein was incubated with the following substrates and salts. The reactions were stopped at 10 min intervals over 60 min: (A) 33mer RNA with 1 mM MgCl2; (B) 33mer RNA with 1 mM MnCl2; (C) 33mer DNA with ...

The activity and secondary structure of PA in the presence of other metal cofactors

Given the distinct cleavage patterns observed with magnesium and manganese, we next tested the activity of Nan PA in the presence of other divalent cations. Dias et al. reported that the N-terminal domain of PA was active with cobalt, but they did not observe endonuclease activity with magnesium. They did find that all divalent cations tested had some effect on the thermal stability as tested by Thermofluor (Dias et al., 2009). This suggests that though these metals can bind, this does not result in a catalytically active protein. As shown in Fig. 3, after one-hour incubation the full-length PA shows at least some activity against RNA (Fig. 3A) and DNA (Fig. 3B) with all metals tested, though there is minimal activity with nickel or zinc. There is also significant degradation in the absence of added salt indicating that the protein is likely purified with a metal, perhaps magnesium, bound in the active site. The addition of the other cations likely competes out the co-purified metal and leads to changes in activity and cleavage pattern. With the exception of manganese, the other cations yielded less activity than that seen with the co-purified cation. Interestingly, calcium yields the same cleavage pattern as that observed for magnesium, while cobalt has the same cleavage pattern as observed for manganese (Fig. 3A). It was previously reported with the truncated PA that the addition of manganese led to a change in protein secondary structure as measured by circular dichroism (CD) (Crepin et al., 2010). To determine if this same effect was present in the full-length PA subunit we repeated this experiment and observed the same trend. As shown in Fig. 4A, there is a small but reproducible change in the CD spectra when comparing protein with manganese to protein with magnesium, which correlates with an increase in helical content (signal from 208 nm to 220 nm). The addition of EDTA (Fig. 4A) caused a large shift in the CD spectra, likely indicating that the protein active site cannot fold correctly in the absence of the divalent cations. To determine if the difference in cleavage pattern correlated with this difference in secondary structure, we compared the CD spectra in the presence of cobalt (Fig. 4B) and calcium (Fig. 4C). Interestingly, cobalt showed a larger shift in CD spectra, but with the same trend as seen for manganese. The increase in helical content fits with the similar cleavage pattern seen for manganese and cobalt. We also observed that the addition of calcium (Fig. 4C) did not cause a shift in this region, which fits with calcium having the same cleavage pattern as magnesium. Nickel was tested as a control (Fig. 4D), however, the low level of activity was not associated with a shift in secondary structure. This lack of activity and the reduced activity with calcium and cobalt are perhaps due to differences in the coordination geometry of these ions as compared to magnesium and manganese. Importantly, this difference observed between magnesium and manganese may also affect the binding of inhibitors that recognize the active site, which would be important for drug design (Parkes et al., 2003)

Figure 3
Effect of metal cofactor on PA endonuclease activity and cleavage pattern. Nan PA protein was incubated for 60 minutes in the presence of 50 mM EDTA, no added salt, 1 mM MgCl2, MnCl2, CaCl2, CoCl2, NiSO4, or Zn(O2CCH3)2 with (A) 33mer RNA substrate or ...
Figure 4
Effect of metal cofactor on PA secondary structure. The secondary structure of the full-length Nan PA was detected using CD spectra in the presence of the metals that yielded endonuclease activity in Fig. 3. (A) 50 mM EDTA, 1 mM MgCl2, or 1 mM MnCl2 (B) ...

Substrate size and sequence specificity of Nan PA endonuclease

Next, the degradation of short unstructured substrates was examined. This would help us to determine the minimal substrate size as well as allow for a more quantitative assay with a single cleavage product. We tested a number of short substrates (data not shown) and found that a 5mer could be cleaved by Nan PA to a 4mer. However, no degradation of this 4mer product to a 3mer was observed, indicating that a 5mer is the smallest substrate that can be efficiently cleaved by Nan PA. To determine the sequence specificity, we tested 5mers and 6mers of both RNA and DNA for three of the bases: C, T/U, and A. Experiments with stretches of guanidine are not possible due to the formation of insoluble complexes, although, based on the cleavage of the long RNA and DNA substrates (Fig. 2) it appears that these guanidines would also be cleaved. Reactions with the short substrates were done in the presence of both magnesium and manganese for one hour. As shown in Fig. 5A for RNA and Fig. 5B for DNA, in the presence of PA there is cleavage of 5mers into 4mers and cleavage of 6mers into 5mers and, in some conditions, 4mers. Data from Fig. 5 are quantified and graphed in Fig. 6 to compare levels of activity. We see the same trend of more endonuclease activity with manganese than with magnesium for all substrates tested though the level of enhancement varies. The 6mer substrates are also cleaved more readily that 5mer substrates, perhaps due to binding affinity. Interestingly, there is not a preference for cleavage of RNA over DNA substrates. For RNA, the rA substrate is cleaved most efficiently with less than 40% of the RNA degraded in an hour while over 60% of the dT substrate is cleaved. Surprisingly, we observed more directionality with these short substrates than for the long substrates. The most abundant product corresponds with cleavage of a single base off the 3′ end.

Figure 5
Sequence specificity and minimal substrate size of PA endonuclease. Nan PA protein was incubated for 60 minutes in the presence of 1 mM MgCl2 or 1 mM MnCl2 and the indicated substrate. Control samples (−) were incubated without protein. (A) Oligo ...
Figure 6
Quantification of PA sequence specificity. Reactions shown in Fig. 5 were done in triplicate and the total degradation was determined by densitometry. Background from no protein control was subtracted. The average and SEM are shown for each condition. ...

Fluorescence-based endonuclease assay

While the gel-based assay is useful for visualization of the cleavage patterns of PA endonuclease activity, it is too labor intensive for large-scale screening of inhibitors. Moreover, our data strongly supports that short DNA substrates can be cleaved by the Nan PA endonuclease. In light of these factors, we designed an assay utilizing Förster Resonance Energy Transfer (FRET) using a short single-stranded DNA. The short oligonucleotide has TAMRA (quencher) and FAM (fluorophore) conjugated to the 5′ and 3′ termini, respectively. When PA cleaves the short single-stranded DNA substrate, the FAM moiety is no longer quenched and therefore cleavage can be detected by an increase in fluorescence (illustrated in Fig. 7A). Incubation of PA with this substrate leads to an increase in fluorescence over time (Fig. 7B). As a control, we tested the E119A Nan PA as well and saw only background signal confirming the absence of contaminating nucleases in our purified protein. Moreover, we further confirmed this cleavage was Nan PA specific by using the known inhibitor DPBA at increasing concentrations. Using the fluorescence-based endonuclease assay, we observe inhibition of cleavage by DPBA and calculated the IC50 to be 5.6 μM (Fig. 7C). This fits well with the data obtained using the gel-based assay (Fig. S2B) and validates the use of full-length Nan PA for screening inhibitors.

Figure 7
Detection of PA endonuclease activity via fluorescence. (A) Schematic of FRET based assay. (B) Nan PA protein was incubated with the DNA probe illustrated and indicated concentration of DPBA in triplicate. Fluorescence was measured every minute for 1 ...


Influenza A virus continues to be a major public health concern due to the yearly burden of seasonal influenza infections and the constant threat of a new pandemic strain. There is already widespread resistance to currently available drugs, making drug discovery an integral part of influenza research. Conserved processes such as viral replication are attractive targets for new inhibitors as resistant strains may be slower to emerge. The endonuclease active site was recently identified in the PA subunit of the polymerase complex and several groups have attempted to screen for endonuclease inhibitors (Baughman et al., 2012; Hastings et al., 1996; Kim et al., 2009). These efforts have been hindered by the use of a truncated form of the protein that, though easily expressed using E. coli, may interact with inhibitors differently than the full-length protein.

Here we sought to develop an improved endonuclease activity assay by using the full-length PA protein, which can be expressed in insect cells. After testing PA subunits from several different strains of influenza A for expression and activity, we chose to further characterize PA from the avian Nanchang strain. Nan PA subunit showed the highest activity in our endonuclease assay at all temperatures tested (Fig. 1). This activity can be inhibited by the previously published inhibitor DPBA (Fig. S2B) and our purified protein had no detectable nuclease contamination (Fig. S2A) We also compared the endonuclease activity of the Nan PA subunit with that of the Nan 3P trimeric polymerase complex and found them to have similar activity (Fig. S2C). Given the higher levels of Nan PA expression and the need for the production and infection with a single baculovirus vector rather than three, the use of the Nan PA subunit alone is clearly more favorable in regards to assay development.

Our biochemical analysis shows that the purified Nan PA subunit is active against both RNA and DNA substrates. It has maximal activity with manganese, as has been previously shown (Crepin et al., 2010; Dias et al., 2009). Moreover, in contrast to previous work with the truncation, the full-length Nan PA subunit is also active with magnesium, calcium, cobalt, though has minimal activity with zinc and nickel (Fig. 2). This indicates that perhaps the C terminal portion of PA stabilizes the active site allowing for activity using these additional cations. We also noted a difference in cleavage pattern using the different metal cofactors (fig. 3). Magnesium and calcium, which have larger ionic radii, have the same cleavage pattern. A different pattern is seen with manganese and cobalt, which have smaller ionic radii. This along with our circular dichroism data (Fig. 4) suggest that the different metal cofactors cause changes in the active site conformation, which then leads to differences in how the protein cleaves the substrate. This effect is most striking using the larger structured RNA and DNA substrates, as compared to short, unstructured substrates (Fig. 5) where the effect of the metal is primarily on level of activity. It remains unclear whether the biologically relevant cofactor is magnesium or manganese. As shown by isothermal calorimetry experiments, there are two metal binding sites, both of which bind manganese with higher affinity (Crepin et al., 2010). While the cellular concentration of manganese (μM range) is significantly lower than that of magnesium (mM range), manganese is a necessary cofactor for many cellular enzymes (Crowley et al., 2000). As either metal could be biologically relevant, it may be important to test both when designing small molecule inhibitors. As suggested by our data, the metal cofactor changes the active site conformation and thus the cleavage pattern. It may also affect the binding of inhibitors that recognize the active site.

We attempted to conduct more in depth enzymological studies using full-length Nan PA to determine the steady state kinetic parameters and substrate binding affinity. We were not able to saturate the rate of the endonuclease reaction when using up to 25 μM substrate (data not shown), which made accurately determining the Km or Vmax for this enzyme not feasible. Similarly, using surface plasmon resonance, we were not able to detect any specific binding of Nan PA subunit to a DNA substrate using 1 μM protein (data not shown). This indicated that the KD or binding affinity of this enzyme was likely above 100 μM. While problematic for characterization of the enzyme, these results are not surprising when considering the polymerase as a whole. In the context of the trimeric complex, the PA subunit does not need to bind substrate with high affinity as the capped substrate is bound by the PB2 subunit, which presumably orients it in the PA active site. After cleavage, the substrate must then be released in order to relocate the primer to the polymerase active site in the PB1 subunit. Tight binding affinity of the PA subunit could then be detrimental to the overall process of transcription as product release could be rate limiting. As the PA subunit is also a non-specific nuclease, it is possible that a highly active PA endonuclease could cleave viral and cellular RNAs in a cap-independent manner, which would inhibit viral replication and, perhaps, lead to apoptosis.

The data obtained from this study will be helpful in guiding the design of assays to screen for PA endonuclease inhibitors. While the preferential degradation of short DNA substrates is not biologically relevant, it is advantageous for assay design, as DNA is less expensive to synthesize and more stable than RNA. This is observation is also not unprecedented as the hepatitis C virus helicase NS3 has also been shown to be more active against DNA than RNA (Frick et al., 2004; Pang et al., 2002). The ability to use short DNA substrates as shown by our fluorescence-based assay (Fig. 7) is a promising platform for screening of chemical libraries. In addition, the minimal substrate size of five bases also may be helpful in modeling how the protein binds RNA to position it in the active site.

Overall, we have demonstrated that our in vitro endonuclease assay with full-length PA subunit is a useful tool for characterizing the substrate specificity and activity of the influenza A polymerase endonuclease. These studies also suggest further questions that remained to be explored. There was an unexpected discrepancy in activity between the PA proteins tested from different strains of influenza A. Surprisingly, the most active protein was obtained from the low pathogenicity avian Nanchang strain rather than the more virulent pandemic 1918 strain or the high pathogenicity avian H5N1 strain. It is possible that through the process of host adaptation the virus selects for a less active endonuclease. This is supported by the recent finding that a frameshift product encoding the endonuclease domain of PA modulates virulence and host response (Jagger et al., 2012). Further work is needed to determine if endonuclease activity is strain specific and if there are any host adaptive residues that affect PA enzymology. This may give further insight into the role of the polymerase in viral adaption.

Materials and Methods

Expression and purification of PA protein

The PA gene from the pandemic 1918 strain (H1N1 A/Brevig Mission/1/1918) was codon optimized and synthesized by Epoch Biolabs (GenBank Accession Number DQ208311.1). H5N1 PA (A/Vietnam/1203/04) was codon optimized and synthesized by GeneArt (GenBank Accession Number AY818132). The pandemic 2009 strain (H1N1 A/California/04/2009) was a gift of Dr. R. Webby, St. Jude Children's Research Hospital, Memphis, TN; the PA gene was codon optimized and synthesized by Epoch Biolabs. The WSN strain (H1N1 A/WSN/33) was a gift of Dr. Y. Kawaoka, University of Wisconsin-Madison, WI. The Nanchang strain (A/chicken/Nanchang/3-120/01 H3N2) was a gift of Dr. R. Webster, St. Jude Children's Research Hospital, Memphis, TN (Liu et al., 2003). The Nanchang PA gene was codon optimized and synthesized by GeneArt. The trimeric polymerase complex of the Nanchang strain was also prepared using the PB1 and PB2 genes. Cloning and purification for all proteins were as previously described for the polymerase complex (Aggarwal et al., 2010). Briefly, the genes were cloned into the pVL1392 baculovirus expression plasmid (Invitrogen) and a TAP tag was added at the C-terminus of PA. Recombinant viruses were produced in Sf9 cells and used to infect Tni cells for protein expression. Cells were harvested 72 hours post infection and purification was performed as previously described using the TAP tag (Bradel-Tretheway et al., 2008). Protein concentration was determined through comparing to a BSA standard (Sigma Aldrich) on SDS-PAGE. Proteins were all used at a final concentration of 50 ng/μl in the reactions. For comparison with the trimeric polymerase, Nan PA was diluted 10-fold and the polymerase complex was concentrated (Millipore Micron Filters) to yield comparable levels of PA protein. The level of PA protein in the complex was comparable to the levels of the other two subunits. The E119A mutant was created using site-directed mutagenesis (Agilent) of the Nan PA gene.

RNA and DNA substrates

The 33mer RNA substrate (GGG GUC CUA AGC CAG UGC CAG AAG AGC CAA GGA) was synthesized from a DNA template using a T7 transcription kit (Ambion). All other DNA and RNA substrates were purchased from Integrated DNA Technologies. All substrates were 5′ end labeled using KinaseMax (Ambion) and γ-32P-ATP (Perkin Elmer).

Endonuclease reactions

PA protein was incubated with 10 nM substrate for the indicated times at 37°C. The reaction buffer contains 50 mM HEPES (pH 7.8), 150 mM KCl, and 1 mM of the indicated salt. Reactions were stopped with the addition of 40 mM EDTA in 99% formamide. Reactions were denatured at 70°C for RNA or 95°C for DNA. Products were resolved using 20% urea PAGE. Gels were dried and analyzed using a PhosphorImager (Perkin Elmer). Band intensities were quantified using Quantity One Software (BioRad). Remaining full-length substrate was normalized to the total amount of signal in the lane and background in the samples incubated without protein. The remaining full-length product was used to determine the activity. 2,4-dioxo-4-phenylbutanoic acid (DPBA was a gift from Alios Biopharma. DPBA was resuspended and diluted in DMSO.

Circular dichroism

Nan PA protein was dialyzed overnight in buffer containing 20 mM Na2PO4 and 30 mM NaCl2. Indicated salts were added to 1 mM final concentration. A 0.1 mm path length quartz cuvette (Starna) was used for all readings. Wavelength scans were recorded from 260 nm to 200 nm (1 nm increment, 1 nm bandwidth, 10 seconds averaging time) at 37°C. Data from three scans were averaged, background from buffer was subtracted, and curves were smoothed using the weighted average of nine nearest neighbors.

Fluorescence based assay

PA protein was incubated with a DNA probe (FAM-TCT CTA GCA GTG GCG CC-TAM) at a concentration of 200 nM (Integrated DNA Technologies). The reaction buffer contains 50 mM HEPES (pH 7.8), 150 mM KCl and 1 mM MnCl2. Reactions utilized 25 ng/μl of either Nan PA or the Nan E119A protein in 20 μl volume reactions and were performed in triplicate on a Biorad CFX96 at 37°C for 60 min. EDTA (50 mM) was used as an additional negative control. DPBA was added at the indicated concentrations and an IC50 was determined using a linear regression model.


  • Full-length PA has endonuclease activity against both RNA and DNA.
  • The divalent metal cofactor affects both level of activity and cleavage pattern.
  • The minimal substrate size for the PA endonuclease is five bases.
  • A FRET-based endonuclease assay is well suited for screening novel antivirals.

Supplementary Material



This work was supported, in whole or in part, by a National Institutes of Health (NIH) contract granted to the New York Influenza Center of Excellence (NIH/NIAID HHSN 266200700008C), the Medical Scientist Training Program funded by NIH T32 GM07356 (AC), and the Oral Cellular and Molecular Biology Training Grant (T32 DE007202 (EN)). We would like to thank Dr. Joseph Hollenbaugh for critical reading of this manuscript.


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