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

Abstract

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.

Structural insights into viral genome replication by the severe fever with thrombocytopenia syndrome virus L protein

“…The next two nts, C4 and A5, sit across two pockets which form the endonuclease metal-coordination site. In influenza virus endonuclease, these are known as the P1 and P0 pockets, respectively ( 59 ). Interestingly, despite mutation of one of the metal-coordinators (D112A), the map suggests there is a single Mg 2+ ion coordinated by E126 and the A5 phosphate.…”

Influenza A virus polymerase acidic protein E23G/K substitutions weaken key baloxavir drug-binding contacts with minimal impact on replication and transmission

“…Based on our modeling, we propose that E23G/K can disrupt BXA-PA N binding through an indirect mechanism that involves destabilizing Arg84–Tyr24 conformations. Stabilization of the inherently flexible Tyr 24 [ 30 , 31 ] contributes to BXA binding, and we observed poses in which Tyr24 interacted with the fluorinated benzene of the upper tricyclic rings of BXA, as well as with the morpholine oxygen of the lower tricyclic rings. Whereas E23G is proposed to disrupt the contact in the upper rings, E23K disrupts both contacts in most of our models and may cause greater reductions in drug affinity.…”

“…When I38T is added, it further decreases drug affinity and increases EC 50 fold-changes, both of which are more severe with E23K. The same E23–stabilized Tyr24 conformations that contribute to BXA binding also bind host RNA substrate, specifically through π-stacking with a nucleotide purine moiety and/or hydrogen bonding with a nucleotide ribose oxygen [ 30 ]. The more disrupted orientation caused by E23K may partly explain the more severe fitness defects caused by this substitution.…”

Alpha-hydroxytropolones are noncompetitive inhibitors of human RNase H1 that bind to the active site and modulate substrate binding

“…The consequences of RNase H1 knockout or loss-of-function mutations are severe ( 2 , 25 ), and therefore, it is important that highly selective antiviral RNase H inhibitors be developed to avoid off-target human RNase H1 inhibition. In addition, αHTs and other metal-chelating compounds can inhibit other DEDD motif–containing viral nucleases, including pUL30/pUL42 of herpes simplex virus 1 and herpes simplex virus 2 ( 26 ), HIV integrase ( 27 ), the influenza and bunyavirus cap-snatching enzymes ( 28 ), and others. Therefore, understanding inhibition of RNase H1 by divalent metal-chelating compounds, including αHTs, will aid development of selective inhibitors of enzymes that share structural or enzymatic similarity with it.…”