Journal of Molecular Biology
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
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.
References (38)
- et al.
N-terminal amino acid sequences of vaccinia virus structural proteins
Virology
(1994) - et al.
A spring-loaded mechanism for the conformational change of influenza hemagglutinin
Cell
(1993) - et al.
Coiled coils in both intracellular vesicle and viral membrane fusion
Cell
(1998) - et al.
Determination of the secondary structure and global topology of the 44 kDa ectodomain of gp41 of the simian immunodeficiency virus by multidimensional nuclear magnetic resonance spectroscopy
J. Mol. Biol.
(1997) - et al.
Structural analysis of the extracellular domain of vaccinia virus envelope protein, A27L, by NMR and CD spectroscopy
J. Biol. Chem.
(2002) - et al.
Crystals of a fragment of influenza haemagglutinin in the low pH induced conformation
J. Mol. Biol.
(1994) - et al.
Inducible gene expression from vaccinia virus vectors
Virology
(1990) - et al.
Multimerization of the Toxoplasma gondii MIC2 integrin-like A-domain is required for binding to heparin and human cells
Mol. Biochem. Parasitol.
(2004) - et al.
Protein–carbohydrate interactions: learning lessons from nature
Trends Biotechnol.
(2001) - et al.
Native and multimeric vitronectin exhibit similar affinity for heparin. Differences in heparin binding properties induced upon denaturation are due to self-association into a multivalent form
J. Biol. Chem.
(1997)
Design of a novel peptide inhibitor of HIV fusion that disrupts the internal trimeric coiled-coil of gp41
J. Biol. Chem.
A new approach to the calculation of secondary structures of globular proteins by optical rotatory dispersion and circular dichroism
Biochem. Biophys. Res. Commun.
A27L protein mediates vaccinia virus interaction with cell surface heparan sulfate
J. Virol.
Vaccinia virus motility
Annu. Rev. Microbiol.
Vaccinia virus envelope D8L protein binds to cell surface chondroitin sulfate and mediates the adsorption of intracellular mature virions to cells
J. Virol.
Recombinant proteins produced by vaccinia virus vectors can be incorporated within the virion (IMV form) into different compartments
Arch. Virol.
Cell surface proteoglycans are necessary for A27L protein-mediated cell fusion: identification of the N-terminal region of A27L protein as the glycosaminoglycan-binding domain
J. Virol.
Vaccinia virus A17L gene product is essential for an early step in virion morphogenesis
J. Virol.
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