Structural Conservation and Functional Diversity of the Poxvirus Immune Evasion (PIE) Domain Superfamily

Abstract

Poxviruses encode a broad array of proteins that serve to undermine host immune defenses. Structural analysis of four of these seemingly unrelated proteins revealed the recurrent use of a conserved beta-sandwich fold that has not been observed in any eukaryotic or prokaryotic protein. Herein we propose to call this unique structural scaffolding the PIE (Poxvirus Immune Evasion) domain. PIE domain containing proteins are abundant in chordopoxvirinae, with our analysis identifying 20 likely PIE subfamilies among 33 representative genomes spanning 7 genera. For example, cowpox strain Brighton Red appears to encode 10 different PIEs: vCCI, A41, C8, M2, T4 (CPVX203), and the SECRET proteins CrmB, CrmD, SCP-1, SCP-2, and SCP-3. Characterized PIE proteins all appear to be nonessential for virus replication, and all contain signal peptides for targeting to the secretory pathway. The PIE subfamilies differ primarily in the number, size, and location of structural embellishments to the beta-sandwich core that confer unique functional specificities. Reported ligands include chemokines, GM-CSF, IL-2, MHC class I, and glycosaminoglycans. We expect that the list of ligands and receptors engaged by the PIE domain will grow as we come to better understand how this versatile structural architecture can be tailored to manipulate host responses to infection.

Keywords: PIE domain; SECRET domain; chemokine and cytokine decoy receptors; poxvirus; viral immune evasion.

Figure 1

The poxvirus immune evasion (PIE) domain structures adopt a strikingly similar fold despite considerable sequence diversity. Strands in the β-sandwich core domain (cyan) are numbered the same for all four structures to aid comparison. The decorations unique to each structure (dark blue) are labeled (h for helix, β for strand). CPXV203 does not contain a strand β11 in sheet I. Similarly, strand β8 of sheet II is absent from vCCI and A41. The disulfide bonds are labeled in red (A–E). All ribbon diagrams are shown in the same orientation and at the same scale. Structures displayed include: rabbitpox vCCI (2FFK) [20], cowpox CPXV203 (4HKJ) [28], vaccinia A41(2VGA) [22], and ectromelia CrmD C-terminal SECRET domain (3ON9) [25]. Figure made in PyMol [47].

Figure 2

PIE domain connectivity diagrams highlighting the conserved core β-sheet architecture (cyan) and unique connecting decorations (dark blue). The disulfide bonds are labeled in red (A–E). Ligand contact regions are annotated with magenta stars for those PIE domains with structurally defined interactions.

Figure 3

Structure based sequence alignment for the PIE domains of Figure 1. Numbering of the core β strands (cyan) is given above the sequences. The decorations are indicated (dark blue), with new strands as arrows, helices as cylinders, and extended coil as a blue line above the sequence. The decorations occur as insertions primarily to the β6–β7 loop, the β7–β9 loop, and at the C-terminus. Disulfide bond cysteines are marked above the alignment, with red circles containing letters of the different disulfide-bond pairs (A, B, C, D, or E) as indicated in Figure 1 and Figure 2. The contacts made by ligand are marked with stars (magenta) under each sequence.

Figure 4

Comparison of PIE domain surface properties. The molecules and orientations are the same as in Figure 1. (a) Electrostatic potential surfaces calculated using APBS [52]. Negative charge in red and positive charge in blue from −3kT/e to +3kT/e. Crystallographically observed contact surfaces for ligand are circled; (b) Sequence conservation within individual families was mapped to the molecular surface and colored magenta for highly conserved and green for variable. Because so few CrmD exist and CrmB and CrmD are closely related, sequences for CrmB and CrmD SECRET domains were aligned and conservation mapped to the CrmD molecular surface.

Figure 5

PIE domain proteins use unique determinants to engage ligands. vCCI primarily employs sheet II to bind CC-chemokines (2FFK) [20], CPV203 (T4) uses the edge of the β-sandwich plus part of sheet I to bind MHC class I/peptide complexes (4HKJ) [28], and CrmD appears to use sheet I for the binding of a low-affinity chemokine (3ON9) [25]. All ribbon diagrams are shown with the PIE domain in the same orientation.

Figure 6

Sequence alignment of representative members of the PIE families. The positions of the vCCI core strands (cyan) are shown above the sequences. Cysteines are boxed in yellow. The predicted disulfide bonds are lettered in red. The predicted signal peptides are shown under the red bar. The PIE family name is given before the ORF name when they differ.

Figure 7

Dendrogram of PIE domain sequences showing relatedness among representative members of the PIE domain families. The tree is midpoint rooted for purposes of illustration. Values in percent at internal nodes indicate posterior probabilities calculated for the Bayesian inference of phylogeny from the alignment in Figure 6 using MrBayes v3.2.0 [81]. The scale bar relates branch lengths to the number of expected substitutions per site. The family names are shown at the terminal nodes.