Structure and Function of A41, a Vaccinia Virus Chemokine Binding Protein

Abstract

The vaccinia virus (VACV) A41L gene encodes a secreted 30 kDa glycoprotein that is nonessential for virus replication but affects the host response to infection. The A41 protein shares sequence similarity with another VACV protein that binds CC chemokines (called vCKBP, or viral CC chemokine inhibitor, vCCI), and strains of VACV lacking the A41L gene induced stronger CD8+ T-cell responses than control viruses expressing A41. Using surface plasmon resonance, we screened 39 human and murine chemokines and identified CCL21, CCL25, CCL26 and CCL28 as A41 ligands, with Kds of between 8 nM and 118 nM. Nonetheless, A41 was ineffective at inhibiting chemotaxis induced by these chemokines, indicating it did not block the interaction of these chemokines with their receptors. However the interaction of A41 and chemokines was inhibited in a dose-dependent manner by heparin, suggesting that A41 and heparin bind to overlapping sites on these chemokines. To better understand the mechanism of action of A41 its crystal structure was solved to 1.9 Å resolution. The protein has a globular β sandwich structure similar to that of the poxvirus vCCI family of proteins, but there are notable structural differences, particularly in surface loops and electrostatic charge distribution. Structural modelling suggests that the binding paradigm as defined for the vCCI–chemokine interaction is likely to be conserved between A41 and its chemokine partners. Additionally, sequence analysis of chemokines binding to A41 identified a signature for A41 binding. The biological and structural data suggest that A41 functions by forming moderately strong (nM) interactions with certain chemokines, sufficient to interfere with chemokine-glycosaminoglycan interactions at the cell surface (μM–nM) and thereby to destroy the chemokine concentration gradient, but not strong enough to disrupt the (pM) chemokine–chemokine receptor interactions.

Chemokine (C-C Motif) Receptor 1 Is Required for Efficient Recruitment of Neutrophils during Respiratory Infection with Modified Vaccinia Virus Ankara

“…Indeed such cross-regulation between CCR1 and CXCR2 has been shown previously in vitro (54), and CXCL2 and CCL3 have been shown to act in a cascade that induces neutrophil recruitment by triggering the release of TNF-␣ and leukotriene B4 (55). In this context, the two VACV CC chemokine binding proteins, A41 (56,57) and the 35-kDa viral CC chemokine inhibitor (58)(59)(60), may play an important role in poxvirus immune evasion, as inhibition of CCR1 ligands, such as CCL5, may be sufficient to affect neutrophil recruitment. However, not all VACV strains, including VACV WR, express the 35-kDa C-C chemokine inhibitor (61), yet many are still able to block chemokine induction and leukocyte recruitment.…”

Deletion of Specific Immune-Modulatory Genes from Modified Vaccinia Virus Ankara-Based HIV Vaccines Engenders Improved Immunogenicity in Rhesus Macaques

“…The experimental vaccines (MVA⌬4-HIV and MVA⌬5-HIV) embody two different genetic modifications to the MVA backbone, which are comprised of deletions of different combinations of MVA genes. MVA⌬4-HIV harbors simultaneous deletions of four poxvirus immune-modulatory genes (MVA008L, MVA153L, MVA159R, and MVA184R), which encode an IL-18 binding protein (75,80), a CC-chemokine binding protein (7,62), a dominant negative Toll/IL-1 signaling adapter, and a soluble IL-1␤ receptor (3,76), respectively. MVA⌬5-HIV harbors a deletion of the viral uracil-DNA glycosylase gene (MVA101R) (30), which reduces late gene expression, in addition to the deletions of the above-described four viral immune-modulatory genes.…”

“…We therefore targeted for deletion those genes encoding a secreted interleukin-18 (IL-18) binding protein (MVA008L) (75,80), a soluble IL-1␤ receptor (MVA184R) (3,76), a CC-chemokine binding protein (MVA153L) (7,62), and a dominant negative Toll/IL-1 signaling adapter (MVA159R) (13,77). The biological activities of these factors can be inferred by the fact that deletions of their orthologs from replication-competent strains of vaccinia virus result in altered pathogenic phenotypes in murine models (2,62,74,77,80).…”

Rodent Herpesvirus Peru Encodes a Secreted Chemokine Decoy Receptor

“…The second group includes vaccinia A41 and ectromelia E163, which are structurally similar to vCCI but appear mechanistically distinct. Chemokine mutational analysis was used to demonstrate that A41 binds directly to the GAG recognition regions of chemokines; however, these proteins are ineffective in blocking chemotaxis (22,23). In addition to blocking chemokine-GAG interactions, E163 itself can tightly associate with GAGs (23).…”

“…In addition to blocking chemokine-GAG interactions, E163 itself can tightly associate with GAGs (23). Thus, it appears that these poxvirus proteins may modulate chemokine networks through the disruption of chemokine gradients rather than the competitive inhibition of chemokine receptor binding (22,23). Although distinct from A41 and E163, myxoma virus encodes M-T7, a secreted glycoprotein that functions both as a species-specific inhibitor of rabbit IFN-␥ and as a chemokine binding protein (21).…”