The Oligomeric Structure of Vaccinia Viral Envelope Protein A27L is Essential for Binding to Heparin and Heparan Sulfates on Cell Surfaces: A Structural and Functional Approach Using Site-specific Mutagenesis

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The soluble domain of the self-assembly vaccinia virus envelope protein A27L, sA27L-aa, consists of a flexible extended coil at the N terminus and a rigid hydrophobic coiled-coil region at the C terminus. In the former, a basic strip of 12 residues is responsible for binding to cell-surface heparan sulfates. Although the latter is believed to mediate self-assembly, its biological role is unclear. However, an in vitro bioassay showed that peptides comprising the 12 residue basic region alone failed to interact with heparin, suggesting that the C-terminal coiled-coil region might serve an indispensable role in biological function. To explore this structural and functional relationship, we performed site-specific mutagenesis in an attempt to specifically disrupt the hydrophobic core of the coiled coil. Three single mutants, L47A, L51A, and L54A, and one triple mutant, L47,51,54A, were expressed and purified from Escherichia coli. The physical properties of the mutants were carefully examined by gel-filtration chromatography, CD, and NMR spectroscopy, and the biological activities were assessed by an in vitro SPR bioassay and three in vivo bioassays: binding to cells, blocking virus infection and blocking cell fusion. We showed that the L47A mutant, which is similar to the parental sA27L-aa in forming a hexamer, is biologically active. L51A and L54A mutants form tetramers and are less active. Notably, in the triple mutant, the self-assembly hydrophobic core structure is uncoiled; as a consequence, the tetrameric structure is biologically inactive. Thus, we conclude that the leucine residues, in particular Leu51 and Leu54, sustain the hydrophobic core structure that is essential for the biological function of vaccinia virus envelope protein A27L, binding to cell-surface heparan sulfate.

Introduction

Vaccinia virus (VV) is a member of the Poxviridae family, the largest known animal virus. It has a double-stranded DNA genome of about 187 kb.1 When VV replicates in host cells, intracellular mature virions (IMVs) form in the cytoplasm and represent the majority of infectious progenies after cell lysis. During IMV infection, the virions attach to cell-surface glycosaminoglycans (GAGs) and enter cells through plasma membrane fusion via an unknown coreceptor.2, 3 IMV contains at least three GAG-binding envelope proteins, two of which, H3L and A27L, bind to cell-surface heparan sulfates, while the other, D8L, binds to chondroitin sulfates.4 In addition to its role in virion attachment, A27L is required for the intracellular wrapping of the IMV into an intracellular enveloped virion (IEV) during virion morphogenesis.5

Wild-type A27L contains 110 amino acid residues that can be divided into four functional domains (Figure 1(a)). Elucidation of the function of the domains of A27L will help us understand the entry mechanism of vaccinia virus. Unfortunately, structure determination by X-ray crystallography has been hampered by intrinsic self-assembly. A27L consists of: a signal peptide for protein processing (residues 1–20);6 a lysine/arginine-rich region, STKAAKKPEAKR (residues 21–32) that is essential for binding to cell surface GAGs (denoted as GAG-binding domain or GBS);7 a coiled-coil domain (residues 43–84) that is involved in self-assembly; and a C-terminal sequence (residues 85–110) that has been shown to interact with another vaccinia viral protein, A17L.8, 9 Although A27L forms an oligomeric coiled-coil structure as commonly found in type I viral fusion proteins,10, 11 such as influenza virus HA2,12 HIV gp41,13, 14 SIV gp41,15, 16 MoMLV p55,17, 18 and ebola GP2,19 however, the role of the coiled-coil region in A27L protein-mediated virus entry has not been addressed. Although A27L is not an essential protein for the vaccinia virus, previous studies have shown that both virus penetration into cells and cell fusion induced by expression of A27L were blocked by a monoclonal antibody recognizing A27L and by soluble A27L.7, 20 These results suggested that A27L may regulate cell fusion either directly or indirectly.

We proposed a molecular model consisting of two distinct structural domains: a flexible, unstructured, extended coil and a more rigid, α-helical, coiled-coil domain.21 Notably, a hydrophobic core (Asn43-Glu55) within the α-helical coiled-coil region was highlighted as responsible for inter-helical interactions. The unstructured coil domain contains the GBS essential for binding to heparan sulfate on the cell surface. However, as shown in this study by in vitro bioassay, the GBS alone is insufficient for binding. It was hypothesized that a cooperative structural and functional relationship exists between the self-assembled coiled coil and the unstructured single strand domains. Although within the hydrophobic core three residues Leu47, Leu51, and Leu54, positioned at a and d in the heptad repeat unit with a high level hydrophobicity, are highlighted as critical for self-assembly,21 the structural contribution of these Leu residues to the biological activity of the protein remains unclear.

To explore the structural and functional relationship, we have constructed four mutants by site-directed mutagenesis: three single mutants L47A, L51A, and L54A, and one triple mutant, L47,51,54A (Figure 1(a)). The physical properties of these mutants were carefully analyzed by CD and NMR spectroscopy, and gel-filtration chromatography. The biological activity of heparan sulfate binding was examined by an in vitro surface plasmon resonance (SPR) assay.22 Our data showed that mutation of these Leu residues effectively disrupts self-assembly such that the degree of oligomerization and the structural integrity of these mutants are affected. In contrast to the single mutants, for the triple mutant, the self-assembly hydrophobic core structure is uncoiled and the mutant has lost all biological activity. Our data provide direct evidence that the hydrophobic core structure is critical for the heparin-binding affinity. Thus, it was concluded that this set of Leu residues (Leu47, Leu51 and Leu54) sustains the self-assembly hydrophobic core structure and is essential for biological function in A27L.

Section snippets

GBS alone is insufficient for heparin binding

To identify whether the GAG-binding site (GBS) is sufficient for heparin binding, we used solid-phase peptide synthesis to generate a 12-mer oligopeptide corresponding to its sequence. In addition, a truncated A27L-aa in which the GBS was deleted (sDA27L) was expressed and purified from Escherichia coli (Figure 1(a) and (b)). An in vitro SPR binding assay was used to examine the heparin-binding affinity of these two samples, with sA27L-aa, serving as a positive control (Figure 2(a)). sA27L-aa

Discussion

A27L is a VV envelope protein that mediates virion attachment during viral infection. Structurally, it contains an extended random coil at the N terminus and a self-assembly coiled-coil rigid segment at the C terminus.21 The random coil region was shown to contain a GAG-binding domain that is essential for A27L binding to cell-surface heparan sulfate; however, the biological role of the coiled-coil region has not been addressed. In this study, aimed at the hydrophobic core of the coiled-coil

Site-directed mutagenesis

The preparation of sA27L-aa and sDA27L has been described.7, 21 Three single mutants, L47A, L51A, and L54A, and one triple mutant, L47,51,54A, were derived from sA27L-aa using a QuickChange XL site-directed mutagenesis kit (Stratagene Inc.). To construct the single mutants, the following primer sets were used in the mutagenesis procedure described by the manufacturer: for the L47A mutant, forward primer A27L-L47A-5′ (5′-gac/gac/aat/gag/gaa/act/GCC/aaa/caa/cgg/cta/act/aat-3′) plus the reverse

Acknowledgements

This work was supported by the National Science Council (grant no. NSC 91-2320-B-001-059) and the Academia Sinica program project “Small Molecule-Biomolecule Interactions in Antiviral Studies”. The authors thank Dr Andrew Atkinson (ESBS, France) for careful reading of the manuscript.

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