MORC3 Is a Target of the Influenza A Viral Protein NS1

Abstract

Microrchidia 3 (MORC3), a human ATPase linked to several autoimmune disorders, has been characterized both as a negative and positive regulator of influenza A virus. Here, we report that the CW domain of MORC3 (MORC3-CW) is targeted by the C-terminal tail of the influenza H3N2 protein NS1. The crystal structure of the MORC3-CW:NS1 complex shows that NS1 occupies the same binding site in CW that is normally occupied by histone H3, a physiological ligand of MORC3-CW. Comparable binding affinities of MORC3-CW to H3 and NS1 peptides and to the adjacent catalytic ATPase domain suggest that the viral protein can compete with the host histone for the association with CW, releasing MORC3 autoinhibition and activating the catalytic function of MORC3. Our structural, biochemical, and cellular analyses suggest that MORC3 might affect the infectivity of influenza virus and therefore has a role in cell immune response.

Keywords: ATPase; CW; MORC3; NS1; chromatin; histone; immune; reader; structure; virus.

Figure 1.. MORC3-CW Is a Target of NS1

(A) The amino-terminal sequence of H3 (trimethylated at lysine 4) and the C-terminal sequence of NS1 (trimethylated at lysine 229) are shown. (B) Superimposed ¹H,¹⁵N HSQC spectra of ¹⁵N-labeled MORC3-CW collected upon titration with the methylated and unmodified NS1 peptides. Spectra are color coded according to the protein-to-peptide molar ratio. (C) The crystal structure of the MORC3-CW:NS1 complex. The CW domain is shown in a ribbon diagram (green), and NS1 is shown as sticks (yellow). The zinc (gray) atom is shown as a sphere. (D) Electrostatic surface potential of the MORC3-CW domain colored blue and red for positive and negative charges, respectively. The bound NS1 peptide is yellow. See also Figure S1.

Figure 2.. Mechanistic Insight into H3 Mimicry by NS1

(A) Binding affinities of WT and mutated MORC3-CW to indicated peptides as measured by tryptophan fluorescence or NMR (*). The binding affinity of the CW domain to H3K4me3 peptide was determined previously (^a) (Andrews et al., 2016). The experiments were carried out in triplicate (in duplicate for the CW E431A-H3K4me3 interaction). Error bars denote SD. (B) Representative binding curves used to determine the K_d values by tryptophan fluorescence in (A). (C) Superimposed ¹H,¹⁵N HSQC spectra of the ¹⁵N-labeled MORC3-CW mutants collected upon titration with the NS1K229me3 peptide. Spectra are color coded according to the protein-to-peptide molar ratio. (D) Zoom-in views of the NS1 K229- and T225-binding sites of MORC3-CW. See also Figure S2.

Figure 3.. Structural Similarity of the MORC3-CW Complexes

(A) Structural overlay of the MORC3-CW in complex with NS1 peptide (yellow) and H3K4me3 peptide (pink) (PDB: 5SVX). (B) The fold change in H3N2 influenza virus infectivity in a single round of infection in mCherry-MORC3-positive cells compared with mCherry-negative cells from the same sample. Infectivity measured as a percentage of cells positive of expression of the viral NP protein. The results presented are representative of two independent experiments. The experiments were carried out in triplicate (in duplicate for the D67A mutant). Data are represented as means ± SEM. The statistical significance was determined using the unpaired t test, *p < 0.05. See also Figures S3 and S4.