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

Abstract

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.

An Integrated Biological Approach to Guide the Development of Metal-Chelating Inhibitors of Influenza Virus PA Endonuclease

“…On the contrary, we noted a 6-times higher V max with MB-U 6 A 2 U 7 -DFO than with MB-het2-DFO. This contrasts with the observation by another group that PA-Nter has a preference for 59-GC-39 in RNA substrates (Datta et al, 2013). However, efficient cleavage of U-rich RNA substrates was already demonstrated by Dias et al (2009).…”

“…1). Among the various PA-Nter substrates that were reported to work (Dias et al, 2009;DuBois et al, 2012;Kowalinski et al, 2012;Parhi et al, 2013;Bauman et al, 2013;Datta et al, 2013;Chen et al, 2014), we chose the M13mp18 single-stranded DNA plasmid since it is commercially available. The active site substitution K134A in the PA-Nter enzyme completely abolished the endonucleolytic cleavage, in agreement with previous reports (Hara et al, 2006;Yuan et al, 2009;Crépin et al, 2010).…”

“…In these two studies, PA-Nter was evaluated against RNA substrates, whereas our MB assay uses DNA-oligonucleotides (which were preferred because of their higher chemical stability). Other groups have successfully developed fluorescent assays for PA endonuclease using totally different DNA or RNA probes (Kowalinski et al, 2012;Noble et al, 2012;Bauman et al, 2013;Datta et al, 2013;Chen et al, 2014), confirming that PA-Nter can cleave different DNA or RNA sequences in enzymatic assays. From a technical viewpoint, our MB assay is superior in having a higher signal-to-noise ratio than fluorescent assays using nonbent oligonucleotide substrates.…”

Identification and characterization of influenza variants resistant to a viral endonuclease inhibitor

“…Translation of viral mRNAs by the host ribosome requires that they be 5′ capped, and this is achieved in cells infected with influenza virus by a "cap-snatching" mechanism in which the endonuclease cleaves 5′ caps from host mRNAs, which then act as transcription primers (19). The endonuclease domain occupies the N-terminal half of PA (PA N ) (20,21) and contains a two-metal (Mn 2+ ) active site that preferentially cleaves the pre-mRNA substrate at the 3′ end of a guanine located 12 nucleotides from the 5′ cap (22). The active site is well conserved (23), and crystal structures have revealed how it…”

The N-Terminal Domain of PA from Bat-Derived Influenza-Like Virus H17N10 Has Endonuclease Activity

“…PB1 binds to the C-terminal domain of PA (14,15), while the N-terminal domain of PA (PAn) can function as an endonuclease to generate small RNA primers that are essential for initiation of the viral gene transcription (16,17). Recently, it has been shown that both PAn (18) and full-length PA (19) cleave the host-derived RNA in a sequence-specific manner.…”