Cap-snatching Mechanism Research Articles

Limited high-throughput screening compatibility of the phenuivirus cap-binding domain

Bunyaviruses constitute a large and diverse group of viruses encompassing many emerging pathogens, such as Rift Valley fever virus (family Phenuiviridae), with public and veterinary health relevance but with very limited medical countermeasures are available. For the development of antiviral strategies, the identification and validation of virus-specific targets would be of high value. The cap-snatching mechanism is an essential process in the life cycle of bunyaviruses to produce capped mRNAs, which are then recognized and translated into viral proteins by the host cell translation machinery. Cap-snatching involves cap-binding as well as endonuclease functions and both activities have been demonstrated to be druggable in related influenza viruses. Here, we explore the suitability of the phenuivirus cap-binding function as a target in medium- and high-throughput drug discovery approaches. We developed a range of in vitro assays aiming to detect the interaction between the cap-binding domain (CBD) and the analogue of its natural cap-ligand m7GTP. However, constricted by its shallow binding pocket and low affinity for m7GTP, we conclude that the CBD has limited small molecule targeting potential using classical in vitro drug discovery approaches.

Safety, tolerability, and pharmacokinetics of TG-1000, a new molecular entity against influenza virus: first-in-human study.

Background: The cap-snatching mechanism of influenza virus mRNA transcription is strongly suppressed by TG-1000, a prodrug rapidly metabolized into TG-0527, is a potent cap-dependent nucleic acid endonuclease inhibitor. Herein, we aimed to assess the safety, tolerability, and pharmacokinetics of TG-1000 in healthy participants and the effect of food on the pharmacokinetics and safety of TG-1000. Method: The study was divided into 2 parts: Part A [Single Ascending-Dose (SAD) study, 10-160mg] and Part B [Food-Effect (FE) study, 40mg] were launched sequentially. The study included 66 participants for both investigations. We administered different TG-1000 capsules or placebo doses per the study protocol and collected blood samples for pharmacokinetic assessments at specific times. In plasma, TG-1000 and its active metabolite TG-0527 were assayed, and PK parameters were determined. Results: In SAD, the increase in AUC was less than the proportional increase in dose over the 20-160mg dose range, while the increase in Cmax was proportional to the increase in dose. In the 10-160mg dose range, T1/2, λz and Tmax of TG-0527 were dose-independent; and T1/2 and Tmax were within 33.8-39.4h and 3.02-6h, respectively. In FE, the AUC0-inf, AUC0-last, and Cmax of TG-0527 decreased by approximately 17.52%, 18.76%, and 41.35%, respectively, and the Tmax delay was around 1.50h. No serious adverse events occurred during the studies. Conclusion: Overall, TG-1000 was well tolerated and exhibited an acceptable safety and PK profile, supporting further clinical investigation of TG-1000 for the treatment of influenza.

Chikungunya virus nonstructural protein 1 is a versatile RNA capping and decapping enzyme

Chikungunya virus (CHIKV) non-structural protein 1 (nsP1) contains both the N7-guanine methyltransferase and guanylyltransferase activities and catalyzes the 5’ end cap formation of viral RNAs. To further understand its catalytic activity and role in virus-host interaction, we demonstrate that purified recombinant CHIKV nsP1 can reverse the guanylyl transfer reaction and remove the m7GMP from a variety of capped RNA substrates including host mRNAs. We then provide the structural basis of this function with a high-resolution cryogenic-electron microscopy structure of nsP1 in complex with the unconventional cap-1 substrate RNA m7GpppAmU. We show that the 5’ppRNA species generated by decapping can trigger RIG-I-mediated interferon (IFN) response. We further demonstrate that the decapping activity is conserved among the alphaviral nsP1s. To our knowledge, this is a new mechanism through which alphaviruses activate the antiviral immune response. This decapping activity could promote cellular mRNA degradation and facilitate viral gene expression which is functionally analogous to the cap-snatching mechanism by influenza virus.

Identification of tanshinone I as cap-dependent endonuclease inhibitor with broad-spectrum antiviral effect.

The spread of avian-borne, tick-borne, and rodent-borne pathogens has the potential to pose a serious threat to human health, and candidate vaccines as well as therapeutics for these pathogens are urgently needed. Tanshinones, especially tanshinone I, were identified as a cap-dependent endonuclease inhibitor with broad-spectrum antiviral effects on negative-stranded, segmented RNA viruses including bandavirus, orthomyxovirus, and arenavirus from natural products, implying an important resource of candidate antivirals from the traditional Chinese medicines. This study supplies novel candidate antivirals for the negative-stranded, segmented RNA virus and highlights the endonuclease involved in the cap-snatching process as a reliable broad-spectrum antiviral target.

Computational prediction of phytochemical inhibitors against the cap-binding domain of Rift Valley fever virus.

Rift Valley fever is a zoonotic disease that can spread through livestock and mosquitoes, and its symptoms include retinitis, photophobia, hemorrhagic fever and neurological effects. The World Health Organization has identified Rift Valley fever as one of the viral infections that has potential to cause a future epidemic. Hence, efforts are urgently needed toward development of therapeutics and vaccine against this infectious disease. Notably, the causative virus namely, the Rift Valley fever virus (RVFV), utilizes the cap-snatching mechanism for viral transcription, rendering its cap-binding domain (CBD) as an effective antiviral target. To date, there are no published studies towards identification of potential small molecule inhibitors for the CBD of RVFV. Here, we employ a virtual screening workflow comprising of molecular docking and molecular dynamics (MD) simulation, to identify 5 potential phytochemical inhibitors of the CBD of RVFV. These 5 phytochemical inhibitors can be sourced from Indian medicinal plants, Ferula assa-foetida, Glycyrrhiza glabra and Leucas cephalotes, used in traditional medicine. In sum, the 5 phytochemical inhibitors of the CBD of RVFV identified by this purely computational study are promising drug lead molecules which can be considered for detailed experimental validation against RVFV infection.

Virtual screening of phytochemicals from Indian medicinal plants against the endonuclease domain of SFTS virus L polymerase.

Severe fever with thrombocytopenia syndrome virus (SFTSV) causes a highly infectious disease with reported mortality in the range 2.8% to 47%. The replication and transcription of the SFTSV genome is performed by L polymerase, which has both an RNA dependent RNA polymerase domain and an N-terminal endonuclease (endoN) domain. Due to its crucial role in the cap-snatching mechanism required for initiation of viral RNA transcription, the endoN domain is an ideal antiviral drug target. In this virtual screening study for the identification of potential inhibitors of the endoN domain of SFTSV L polymerase, we have used molecular docking and molecular dynamics (MD) simulation to explore the natural product space of 14 011 phytochemicals from Indian medicinal plants. After generating a heterogeneous ensemble of endoN domain structures reflecting conformational diversity of the corresponding active site using MD simulations, ensemble docking of the phytochemicals was performed against the endoN domain structures. Apart from the ligand binding energy from docking, our virtual screening workflow imposes additional filters such as drug-likeness, non-covalent interactions with key active site residues, toxicity and chemical similarity with other hits, to identify top 5 potential phytochemical inhibitors of endoN domain of SFTSV L polymerase. Further, the stability of the protein–ligand docked complexes for the top 5 potential inhibitors was analyzed using MD simulations. The potential phytochemical inhibitors, predicted in this study using contemporary computational methods, are expected to serve as lead molecules in future experimental studies towards development of antiviral drugs against SFTSV.

Structural insights into the substrate specificity of the endonuclease activity of the influenza virus cap-snatching mechanism

The endonuclease activity within the influenza virus cap-snatching process is a proven therapeutic target. The anti-influenza drug baloxavir is highly effective, but is associated with resistance mutations that threaten its clinical efficacy. The endonuclease resides within the N-terminal domain of the PA subunit (PAN) of the influenza RNA dependent RNA polymerase, and we report here complexes of PAN with RNA and DNA oligonucleotides to understand its specificity and the structural basis of baloxavir resistance mutations. The RNA and DNA oligonucleotides bind within the substrate binding groove of PAN in a similar fashion, explaining the ability of the enzyme to cleave both substrates. The individual nucleotides occupy adjacent conserved pockets that flank the two-metal active site. However, the 2′ OH of the RNA ribose moieties engage in additional interactions that appear to optimize the binding and cleavage efficiency for the natural substrate. The major baloxavir resistance mutation at position 38 is at the core of the substrate binding site, but structural studies and modeling suggest that it maintains the necessary virus fitness via compensating interactions with RNA. These studies will facilitate the development of new influenza therapeutics that spatially match the substrate and are less likely to elicit resistance mutations.

Analysis of tomato spotted wilt virus RNA-dependent RNA polymerase adaptative evolution and constrained domains using homology protein structure modelling.

Tomato spotted wilt virus (TSWV; genus Orthotospovirus, family Tospoviridae) has a huge impact on a large range of plants worldwide. In this study, we determined the sequence of the large (L) RNA segment that encodes the RNA-dependent RNA polymerase (RdRp) from a TSWV isolate (LYE51) collected in the south of France. Analysis of the phylogenetic relationships of TSWV-LYE51 with other TSWV isolates shows that it is closely related to other European isolates. A 3D model of TSWV-LYE51 RdRp was built by homology with the RdRp structure of the La Crosse virus (genus Orthobunyavirus, family Peribunyaviridae). Finally, an analysis of positive and negative selection was carried out on 30 TSWV full-length RNA L sequences and compared with the phylogeny and the protein structure data. We showed that the seven codons that are under positive selection are distributed all along the RdRp gene. By contrast, the codons associated with negative selection are especially concentrated in three highly constrained domains: the endonuclease in charge of the cap-snatching mechanism, the thumb domain and the mid domain. Those three domains could constitute good candidates to look for host targets on which genetic resistance by loss of susceptibility could be developed.

The Cap-Snatching Mechanism of Bunyaviruses.

In common with all segmented negative-sense RNA viruses, bunyavirus transcripts contain heterologous sequences at their 5' termini originating from capped host cell RNAs. These heterologous sequences are acquired by a so-called cap-snatching mechanism. Whereas for nuclear replicating influenza virus the source of capped primers as well as the cap-binding and endonuclease activities of the viral polymerase needed for cap snatching have been functionally and structurally well characterized, our knowledge on the expected counterparts of cytoplasmic replicating bunyaviruses is still limited and controversial. This review focuses on the cap-snatching mechanism of bunyaviruses in the light of recent structural and functional data.

Alterations in cellular RNA decapping dynamics affect tomato spotted wilt virus cap snatching and infection in Arabidopsis.

RNA processing and decay pathways have important impacts on RNA viruses, particularly animal-infecting bunyaviruses, which utilize a cap-snatching mechanism to translate their mRNAs. However, their effects on plant-infecting bunyaviruses have not been investigated. The roles of mRNA degradation and non-sense-mediated decay components, including DECAPPING 2 (DCP2), EXORIBONUCLEASE 4 (XRN4), ASYMMETRIC LEAVES2 (AS2) and UP-FRAMESHIFT 1 (UPF1) were investigated in infection of Arabidopsis thaliana by several RNA viruses, including the bunyavirus, tomato spotted wilt virus (TSWV). TSWV infection on mutants with decreased or increased RNA decapping ability resulted in increased and decreased susceptibility, respectively. By contrast, these mutations had the opposite, or no, effect on RNA viruses that use different mRNA capping strategies. Consistent with this, the RNA capping efficiency of TSWV mRNA was higher in a dcp2 mutant. Furthermore, the TSWV N protein partially colocalized with RNA processing body (PB) components and altering decapping activity by heat shock or coinfection with another virus resulted in corresponding changes in TSWV accumulation. The present results indicate that TSWV infection in plants depends on its ability to snatch caps from mRNAs destined for decapping in PBs and that genetic or environmental alteration of RNA processing dynamics can affect infection outcomes.

Biochemical characterization of the Lassa virus L protein

The L protein of arena- and bunyaviruses is structurally and functionally related to the orthomyxovirus polymerase complex. It plays a central role in the viral life cycle, as it replicates the virus genome and generates viral mRNA via a cap-snatching mechanism. Here, we aimed to biochemically characterize the L protein of Lassa virus, a human-pathogenic arenavirus endemic in West Africa. Full-length 250-kDa L protein was expressed using a baculovirus expression system. A low-resolution structure calculated from small-angle X-ray scattering data revealed a conformation similar to that in the crystal structure of the orthomyxovirus polymerase complex. Although the L protein did not exhibit cap-snatching endonuclease activity, it synthesized RNA in vitro. RNA polymerization required manganese rather than magnesium ions, was independent of nucleotide primers, and was inhibited by viral Z protein. Maximum activity was mediated by double-stranded promoter sequences with a minimum length of 17 nucleotides, containing a nontemplated 5′-G overhang, as in the natural genome context, as well as the naturally occurring base mismatches between the complementary promoter strands. Experiments with various short primers revealed the presence of two replication initiation sites at the template strand and evidence for primer translocation as proposed by the prime-and-realign hypothesis. Overall, our findings provide the foundation for a detailed understanding of the mechanistic differences and communalities in the polymerase proteins of segmented negative-strand RNA viruses and for the search for antiviral compounds targeting the RNA polymerase of Lassa virus.

Hantavirus RdRp Requires a Host Cell Factor for Cap Snatching.

The hantavirus RNA-dependent RNA polymerase (RdRp) snatches 5' capped mRNA fragments from the host cell transcripts and uses them as primers to initiate transcription and replication of the viral genome in the cytoplasm of infected cells. Hantavirus nucleocapsid protein (N protein) binds to the 5' caps of host cell mRNA and protects them from the attack of cellular decapping machinery. N protein rescues long capped mRNA fragments in cellular P bodies that are later processed by an unknown mechanism to generate 10- to 14-nucleotide-long capped RNA primers with a 3' G residue. Hantavirus RdRp has an N-terminal endonuclease domain and a C-terminal uncharacterized domain that harbors a binding site for the N protein. The purified endonuclease domain of RdRp nonspecifically degraded RNA in vitro It is puzzling how such nonspecific endonuclease activity generates primers of appropriate length and specificity during cap snatching. We fused the N-terminal endonuclease domain with the C-terminal uncharacterized domain of the RdRp. The resulting NC mutant, with the assistance of N protein, generated capped primers of appropriate length and specificity from a test mRNA in cells. Bacterially expressed and purified NC mutant and N protein required further incubation with the lysates of human umbilical vein endothelial cells (HUVECs) for the specific endonucleolytic cleavage of a test mRNA to generate capped primers of appropriate length and defined 3' terminus in vitro Our results suggest that an unknown host cell factor facilitates the interaction between N protein and NC mutant and brings the N protein-bound capped RNA fragments in close proximity to the endonuclease domain of the RdRp for specific cleavage at a precise length from the 5' cap. These studies provide critical insights into the cap-snatching mechanism of cytoplasmic viruses and have revealed potential new targets for their therapeutic intervention.IMPORTANCE Humans acquire hantavirus infection by the inhalation of aerosolized excreta of infected rodent hosts. Hantavirus infections cause hemorrhagic fever with renal syndrome (HFRS) and hantavirus cardiopulmonary syndrome (HCPS), with mortality rates of 15% and 50%, respectively (1). Annually 150,000 to 200,000 cases of hantavirus infections are reported worldwide, for which there is no treatment at present. Cap snatching is an early event in the initiation of virus replication in infected hosts. Interruption in cap snatching will inhibit virus replication and will likely improve the prognosis of the hantavirus disease. Our studies provide mechanistic insight into the cap-snatching mechanism and demonstrate the requirement of a host cell factor for successful cap snatching. Identification of this host cell factor will reveal a novel therapeutic target for combating this viral illness.

Metal chelators for the inhibition of the lymphocytic choriomeningitis virus endonuclease domain

Arenaviridae is a viral family whose members are associated with rodent-transmitted infections to humans responsible of severe diseases. The current lack of a vaccine and limited therapeutic options make the development of efficacious drugs of high priority. The cap-snatching mechanism of transcription of Arenavirus performed by the endonuclease domain of the L-protein is unique and essential, so we developed a drug design program targeting the endonuclease activity of the prototypic Lymphocytic ChorioMeningitis Virus. Since the endonuclease activity is metal ion dependent, we designed a library of compounds bearing chelating motifs (diketo acids, polyphenols, and N-hydroxyisoquinoline-1,3-diones) able to block the catalytic center through the chelation of the critical metal ions, resulting in a functional impairment. We pre-screened 59 compounds by Differential Scanning Fluorimetry. Then, we characterized the binding affinity by Microscale Thermophoresis and evaluated selected compounds in in vitro and in cellula assays. We found several potent binders and inhibitors of the endonuclease activity. This study validates the proof of concept that the endonuclease domain of Arenavirus can be used as a target for anti-arena-viral drug discovery and that both diketo acids and N-hydroxyisoquinoline-1,3-diones can be considered further as potential metal-chelating pharmacophores.

The cap-snatching reaction of yeast L-A double-stranded RNA virus is reversible and the catalytic sites on both Gag and the Gag domain of Gag-Pol are active.

The yeast L-A double-stranded RNA virus synthesizes capped transcripts by a unique cap-snatching mechanism in which the m7 Gp moiety of host mRNA (donor) is transferred to the diphosphorylated 5' end of the viral transcript (acceptor). This reaction is activated by viral transcription. Here, we show that cap snatching can be reversible. Because only m7 Gp is transferred during the reaction, the resulting decapped donor, as expected, retained diphosphates at the 5' end. We also found that the 5' terminal nucleotide of the acceptor needs to be G but not A. Interestingly, the A-initiated molecule when equipped with a cap structure (m7 GpppA…) could work as cap donor. Because the majority of host mRNAs in yeast have A after the cap structures at the 5' ends, this finding implies that cap-snatching in vivo is virtually a one-way reaction, in favor of furnishing the viral transcript with a cap. The cap-snatching sites are located on the coat protein Gag and also the Gag domain of Gag-Pol. Here, we demonstrate that both sites are functional, indicating that activation of cap snatching by transcription is not transmitted through the peptide bonding between the Gag and Pol domains of Gag-Pol.

Biochemical and structural studies reveal differences and commonalities among cap-snatching endonucleases from segmented negative-strand RNA viruses

Viruses rely on many host cell processes, including the cellular transcription machinery. Segmented negative-strand RNA viruses (sNSV) in particular cannot synthesize the 5′-cap structure for their mRNA but cleave off cellular caps and use the resulting oligonucleotides as primers for their transcription. This cap-snatching mechanism, involving a viral cap-binding site and RNA endonuclease, is both virus-specific and essential for viral proliferation and therefore represents an attractive drug target. Here, we present biochemical and structural results on the putative cap-snatching endonuclease of Crimean–Congo hemorrhagic fever virus (CCHFV), a highly pathogenic bunyavirus belonging to the Nairoviridae family, and of two additional nairoviruses, Erve virus (EREV) and Nairobi sheep disease virus (NSDV). Our findings are presented in the context of other cap-snatching endonucleases, such as the enzymatically active endonuclease from Rift Valley fever virus (RVFV), from Arenaviridae and Bunyavirales, belonging to the His− and His+ endonucleases, respectively, according to the absence or presence of a metal ion–coordinating histidine in the active site. Mutational and metal-binding experiments revealed the presence of only acidic metal-coordinating residues in the active site of the CCHFV domain and a unique active-site conformation that was intermediate between those of His+ and His− endonucleases. On the basis of small-angle X-ray scattering (SAXS) and homology modeling results, we propose a protein topology for the CCHFV domain that, despite its larger size, has a structure overall similar to those of related endonucleases. These results suggest structural and functional conservation of the cap-snatching mechanism among sNSVs.

Characterization of an endonuclease in rice stripe tenuivirus Pc1 in vitro

Rice stripe tenuivirus (RSV) initiates its mRNA transcription by using the cap-snatching mechanism during which an endonuclease activity is required for the cleavage of host mRNA. In this study, we aim to characterize the endonuclease in RSV. Sequence alignment revealed the presence of a cap-snatching endonuclease domain in RSV Pc1. Expression and in vitro enzymatic activity assay demonstrated that this domain indeed had a manganese-dependent endonuclease activity. The enzyme could efficiently degrade ssRNA with preference for unstructured ssRNA, but not DNA. Mutations in the endonuclease domain allowed the identification of four key residues (D547, D567, E585 and K604). The endonuclease of RSV was similar but not identical to other known viral endonucleases, suggesting that RSV endonuclease may have some distinct catalytic characteristics.

Structural insights into reptarenavirus cap-snatching machinery.

Cap-snatching was first discovered in influenza virus. Structures of the involved domains of the influenza virus polymerase, namely the endonuclease in the PA subunit and the cap-binding domain in the PB2 subunit, have been solved. Cap-snatching endonucleases have also been demonstrated at the very N-terminus of the L proteins of mammarena-, orthobunya-, and hantaviruses. However, a cap-binding domain has not been identified in an arena- or bunyavirus L protein so far. We solved the structure of the 326 C-terminal residues of the L protein of California Academy of Sciences virus (CASV), a reptarenavirus, by X-ray crystallography. The individual domains of this 37-kDa fragment (L-Cterm) as well as the domain arrangement are structurally similar to the cap-binding and adjacent domains of influenza virus polymerase PB2 subunit, despite the absence of sequence homology, suggesting a common evolutionary origin. This enabled identification of a region in CASV L-Cterm with similarity to a cap-binding site; however, the typical sandwich of two aromatic residues was missing. Consistent with this, cap-binding to CASV L-Cterm could not be detected biochemically. In addition, we solved the crystal structure of the corresponding endonuclease in the N-terminus of CASV L protein. It shows a typical endonuclease fold with an active site configuration that is essentially identical to that of known mammarenavirus endonuclease structures. In conclusion, we provide evidence for a presumably functional cap-snatching endonuclease in the N-terminus and a degenerate cap-binding domain in the C-terminus of a reptarenavirus L protein. Implications of these findings for the cap-snatching mechanism in arenaviruses are discussed.

Functional properties of the novel PA-X endonuclease of human influenza virus

The PA subunit of the RNA dependent RNA polymerase of influenza virus is an endonuclease required for the cap-snatching mechanism leading to the synthesis of viral mRNA. Recently, the PA-X protein has been discovered in human influenza, which is translated from an alternative open reading frame within the PA gene coding sequence, resulting from a ribosomal frameshifting. The PA-X retains the N-terminal endonucleolytic domain of PA, but has a different and shorter C-terminal part. Two isoforms of PA-X are present in circulating human infuenza A strains. It has been proposed that PA-X degrades cellular mRNA, contributing to the attenuation of viral pathogenesis in the host. Here we express the diferent recombinant PA-X proteins from human seasonal influenza virus and show that their endonucleolytic activities have distinct substrate specificities, which depend on the structure of the RNA to be cleaved. Collectively, our results support distinct roles of PA and PA-X in the viral life cycle.

Cap Snatching in Yeast L-BC Double-stranded RNA Totivirus

Yeast L-A double-stranded RNA virus furnishes its transcript with a 5' cap structure by a novel cap-snatching mechanism in which m(7)Gp from a host mRNA cap structure is transferred to the 5'-diphosphate terminus of the viral transcript. His-154 of the coat protein Gag forms an m(7)Gp adduct, and the H154R mutation abolishes both m(7)Gp adduct formation and cap snatching. Here we show that L-BC, another totivirus closely related to L-A, also synthesizes 5'-diphosphorylated transcripts and transfers m(7)Gp from mRNA to the 5' termini of the transcripts. L-BC Gag also covalently binds to the cap structure and the mutation H156R, which corresponds to H154R of L-A Gag, abolishes cap adduct formation. Cap snatching of the L-BC virus is very similar to that of L-A; N7 methylation of the mRNA cap is essential for cap donor activity, and only 5'-diphosphorylated RNA is used as cap acceptor. L-BC cap snatching is also activated by viral transcription. Furthermore, both viruses require Mg(2+) and Mn(2+) for cap snatching. These cations are not only required for transcription activation but also directly involved in the cap transfer process. These findings support our previous proposal that the cap-snatching mechanism of the L-A virus is shared by fungal totiviruses closely related to L-A. Interestingly, L-A and L-BC viruses accept either viral transcript as cap acceptor in vitro. Because L-A and L-BC viruses cohabit in many yeast strains, it raises the possibility that their cohabitation in the same host may be beneficial for their mutual cap acquisition.

Characterization of PA-N terminal domain of Influenza A polymerase reveals sequence specific RNA cleavage

Influenza virus uses a unique cap-snatching mechanism characterized by hijacking and cleavage of host capped pre-mRNAs, resulting in short capped RNAs, which are used as primers for viral mRNA synthesis. The PA subunit of influenza polymerase carries the endonuclease activity that catalyzes the host mRNA cleavage reaction. Here, we show that PA is a sequence selective endonuclease with distinct preference to cleave at the 3′ end of a guanine (G) base in RNA. The G specificity is exhibited by the native influenza polymerase complex associated with viral ribonucleoprotein particles and is conferred by an intrinsic G specificity of the isolated PA endonuclease domain PA-Nter. In addition, RNA cleavage site choice by the full polymerase is also guided by cap binding to the PB2 subunit, from which RNA cleavage preferentially occurs at the 12th nt downstream of the cap. However, if a G residue is present in the region of 10–13 nucleotides from the cap, cleavage preferentially occurs at G. This is the first biochemical evidence of influenza polymerase PA showing intrinsic sequence selective endonuclease activity.