Dynamin-like MxA GTPase: structural insights into oligomerization and implications for antiviral activity

Abstract

The interferon-inducible MxA GTPase is a key mediator of cell-autonomous innate immunity against a broad range of viruses such as influenza and bunyaviruses. MxA shares a similar domain structure with the dynamin superfamily of mechanochemical enzymes, including an N-terminal GTPase domain, a central middle domain, and a C-terminal GTPase effector domain. Recently, crystal structures of a GTPase domain dimer of dynamin 1 and of the oligomerized stalk of MxA (built by the middle and GTPase effector domains) were determined. These data provide exciting insights into the architecture and antiviral function of the MxA oligomer. Moreover, the structural knowledge paves the way for the development of novel antiviral drugs against influenza and other highly pathogenic viruses.

FIGURE 1.

Phylogenetic tree of Mx proteins. The Mx proteins are grouped into five subgroups according to sequence similarities. Sequences were obtained from GenBank^TM and aligned with ClustalW Version 1.81. The tree was constructed using the DrawGram function of Biology WorkBench 3.2. The following sequences were used for the alignment: human (hu) MxA and MxB; canine (ca) Mx1 and Mx2; porcine (po) Mx1; bovine (bo) Mx1; ovine (ov) Mx; murine (mu) Mx1 and Mx2; rat Mx1, Mx2, and Mx3; chicken (ch) Mx; duck (du) Mx; Atlantic salmon (as) Mx1, Mx2, and Mx3; and Atlantic halibut (hh) Mx. This figure was adapted from Ref. .

FIGURE 2.

Structure and domain composition of human MxA. A, schematic diagram of human MxA and human dynamin 1 (Dyn). MxA consists of three domains: the G domain (blue), the MD (red), and the GED (green). The MD and GED form the MxA stalk. Dynamin features two additional domains, namely a PH domain (yellow) and a proline/arginine-rich domain (PRD; magenta). B, structure of the MxA stalk composed of the MD and GED (amino acids 366–633) combined with the N-terminal globular G domain (blue). The unstructured loops L2 and L4 are indicated by dashed lines. The positions of the antibody 2C12-binding site (positions 432–471) in loop L2 (47) and the proteinase K cleavage site at position 564 (PK, asterisk) in loop L4 (30) are indicated. This figure was adapted from Ref. .

FIGURE 3.

Oligomerization and liposome binding of human MxA. A–C, cryo-transmission EM images of purified MxA dialyzed against low salt buffer in the presence of 1 mm GMP-PCP. Self-assemblies show rings and open arcs (A); higher magnification of the rings reveals a structure of two parallel sets of electron-dense globular domains (B, double arrowheads); and incubation with phosphatidylserine-containing liposomes in the presence of GTP shows MxA ring formation and tubulation (C) (36). Scale bars = 50 nm. D, shown is a model of oligomeric MxA composed of the MxA stalks connected to the G domains of dynamin. Side and front views are shown. E, a complete MxA ring composed of 16 MxA dimers was designed according to cryo-EM reconstructions of dynamin as described previously (46) but with an angle of only 23° between the associating stalks to accomplish formation of a 41-nm oligomeric ring.

FIGURE 4.

Antiviral activity of MxA requires oligomerization. A, influenza A virus minireplicon system. Coexpression of a luciferase reporter minigenome (pPolI-luc) together with the three subunits of the viral polymerase (PA, PB1, and PB2) and the viral NP generates nucleocapsids that transcribe and replicate the minigenome. Luciferase production correlates with the transcriptional and replicative activity of the viral polymerase. vRNPs, viral ribonucleoproteins. B, wild-type (wt) MxA inhibits the polymerase activity of the avian H5N1 influenza A/Vietnam/1203/04 virus in a minireplicon system. Expression plasmids for the indicated MxA constructs were used, and the relative luciferase activity was determined. Assembly-defective MxA variants with mutations either in one of the three interfaces (1, 2, or 3) or in L4 (ΔL4) have lost antiviral activity. Protein expression was analyzed by Western blotting using specific antibodies. This figure was adapted from Ref. .