Vaccinia viral protein A27 is anchored to the viral membrane via a cooperative interaction with viral membrane protein A17

Abstract

The vaccinia viral protein A27 in mature viruses specifically interacts with heparan sulfate for cell surface attachment. In addition, A27 associates with the viral membrane protein A17 to anchor to the viral membrane; however, the specific interaction between A27 and A17 remains largely unclear. To uncover the active binding sites and the underlying binding mechanism, we expressed and purified the N-terminal (18-50 residues) and C-terminal (162-203 residues) fragments of A17, which are denoted A17-N and A17-C. Through surface plasmon resonance, the binding affinity of A27/A17-N (KA = 3.40 × 10(8) m(-1)) was determined to be approximately 3 orders of magnitude stronger than that of A27/A17-C (KA = 3.40 × 10(5) m(-1)), indicating that A27 prefers to interact with A17-N rather than A17-C. Despite the disordered nature of A17-N, the A27-A17 interaction is mediated by a specific and cooperative binding mechanism that includes two active binding sites, namely (32)SFMPK(36) (denoted as F1 binding) and (20)LDKDLFTEEQ(29) (F2). Further analysis showed that F1 has stronger binding affinity and is more resistant to acidic conditions than is F2. Furthermore, A27 mutant proteins that retained partial activity to interact with the F1 and F2 sites of the A17 protein were packaged into mature virus particles at a reduced level, demonstrating that the F1/F2 interaction plays a critical role in vivo. Using these results in combination with site-directed mutagenesis data, we established a computer model to explain the specific A27-A17 binding mechanism.

Keywords: Pox Viruses; Protein Structure; Protein-Protein Interactions; Viral Protein; Virus Assembly.

FIGURE 1.

Illustration of the functional domains in the vaccinia viral envelope protein A27 and the viral membrane protein A17. A, A27 protein used in this study is divided into five functional domains as follows: a flexible Lys/Arg-rich domain (21–32 residues) known as the heparin-binding site (HBS) (6), a flexible spacer (33–42 residues), an α-helical CCD (43–65 residues) critical for A27 oligomerization, DLD (66–84 residues), and LZD (85–110 residues) responsible for anchoring to the viral membrane via interaction with A17. Two adjacent Cys residues (CC) at positions 71 and 72 are labeled by triangles. The three Leu residues within the LZD and part of the DLD responsible for the A17 interaction are shown below (23). Eleven A27(21–110) mutants were used in this study, and the mutated residues are highlighted in the last panel. B, A17 (18–203 residues) consists of an N-terminal domain (18–60 residues), two transmembrane (TM) domains (61–98 and 113–161 residues), and a C-terminal domain (162–203 residues). In the infected cells, A17 is synthesized as a 23-kDa precursor of 203 amino acids and is subsequently cleaved at both the N and C termini during maturation (30), as indicated by arrows. The three A17 fragments used in this study are shown below: A17-N, A17-C, and A17-S, in which the A17-N sequence ²⁴LFTEEQQQSFM³⁴ was scrambled to ²⁴QLESQFQTMEF³⁴. These A17 fragments tagged with thioredoxin were expressed in E. coli, and the tag was removed by factor Xa cleavage (for details, see “Experimental Procedures”). Eight A17-N mutants, including five scrambled-sequence mutants and three single mutants, were synthesized using a solid phase synthesizer. The mutated or scrambled residue(s) are highlighted. To facilitate the HSQC analysis, two residues, Phe³³ and Phe²⁵, were specifically ¹⁵N isotope-labeled. The Coomassie Brilliant Blue staining of A27 and A17 recombinant proteins is shown in C.

FIGURE 2.

HSQC analyses and in vitro binding assay. Two-dimensional ¹H/¹⁵N HSQC spectra of A27 (A), A17-C (B), A17-N (C), and A17-S (D) at pH 6.5. As observed in A, the HSQC pattern revealed only one-third of the total residues, and the remaining two-thirds of the residues involved in protein self-assembly were absent. B–D, A17 fragments/A27 binding were analyzed by HSQC, and the cross-peaks are colored in red in the absence of A27 and in blue in the presence of A27. B and D, no chemical shift perturbations were found. As indicated in C, two sets of segmental cross-peaks were perturbed due to the presence of A27: ³²SFMPK³⁶ (denoted as the F1 segment) and ²⁰LDKDLFTEEQ²⁹ (F2). The sequential assignments of the A27 and A17 fragments were determined by three-dimensional heteronuclear correlation NMR experiments (47, 48) based on the inter- and intra-residue chemical shift correlations. The single-letter abbreviations for amino acids are used. The numbering is based on the sequences of WT-A17 and WT-A27. E, representative cross-peaks of the F2 residues (Leu²⁰ and Gln²⁷) and the F1 residues (Phe³³ and Met³⁴) reveal a linear diminution as a function of the A17-N-A27 molar ratio (1:0, 1:0.25, 1:0.5, 1:0.75, and 1:1) at pH 6.5 from top to bottom. Notice that the F1 residues decrease faster than the F2 residues, implying that F1 has a stronger binding affinity than F2. In contrast, the cross-peaks of those residues not involved in A27 binding, such as Asp⁵⁰, remained unchanged in the presence of A27. F, two-dimensional HSQC spectra of the synthetic peptides (from left to right) as follows: A17-S1, -S2, -S3, -S4 (upper row), and -S5, -L24A, -Q29A, and -P35A (lower row). As observed, the cross-peaks of the A17-N peptides in the presence (in blue) and absence of A27 (in red) arising from both Phe²⁵ and Phe³³ remain basically unchanged, implying that all of the A17-N mutants lost binding specificity for A27. For comparison, the two spectra were shifted by 0.235 ppm along the horizontal ¹H dimension. Only two residues, Phe²⁵ and Phe³³, have been specifically ¹⁵N isotope-labeled in these synthetic peptides, corresponding to the upfield and downfield cross-peak signals along the vertical ¹⁵N dimension, respectively. For details, see “Experimental Procedures.” G, selective ¹H/¹⁵N HSQC residual intensity ratio determined from the A17-N fragment with uniform ¹⁵N isotope labeling mixed with the A27 mutants (natural abundance). A17-N was mixed with the single mutants A27-E87A and A27-I94A and the double mutant A27-E87A,I94A, respectively, as well as the parental A27 for reference (at a 1:1 molar ratio). The residual intensity ratios were calculated from the intensity ratio of two sets of cross-peaks in the presence and absence of the A27 mutant and calibrated with respect to unperturbed Asp⁵⁰. The effect of mutation upon F1 and F2 binding represented by Phe³³ for F1 (black bar) and by Phe²⁵ for F2 (gray bar) revealed that A27-E87A disrupts F2 binding and partially disables F1 binding, whereas A27-I94A caused a substantial disruption of both F1 and F2 binding. In addition, the double mutation A27-E87A,I94A completely abolished the F1 and F2 binding activities.

FIGURE 3.

In vitro A27/A17 binding assay by SPR. The viral protein A27 was immobilized on a CM5 sensor flow chip. A17-N (A), A17-C (B), and A17-S (C) tagged with thioredoxin were injected onto the immobilized A27 at five different protein concentrations (10, 5, 2.5, 1.25, and 0.625 μm), and the association and dissociation kinetic interactions were monitored as a function of the protein concentration in real time. In the negative control, thioredoxin only gave no response when passing over a CM5 chip onto which A27 was immobilized (supplemental Fig. S2). The kinetic binding constants obtained from the sensorgrams of the A27/A17 fragments are shown (Table 4). For details, see text.

FIGURE 4.

Secondary structure of A17 fragments based on CD and CSI analyses. Far-UV CD spectra of A27 (A), A17-N (dark line) and A17-S (gray line) (B). The A17-N spectrum indicated a superimposition of the major random coil and minor α-helix patterns, where the former exhibited ellipticity at 200 nm, and the latter exhibited ellipticity at 208 and 222 nm. Furthermore, the A17-S spectrum revealed a similar spectral pattern with the exception of a slightly lower value at ∼220 nm. In contrast to C, the A27 spectrum indicated a structural pattern distinct from that of a typical α-helix. ¹³C^α (C) and ¹³CO (D) chemical shift propensity index analysis of the A27 binding domain of A17-N and A17-S. The chemical shift index was determined as (δ − δ_random)/(δ_α − δ_random), where δ is the experimental chemical shift and δ_α and δ_random are the chemical shift values reported in the literature for a α-structure and random coil, respectively. The propensity index for the EEQQQ segment of A17-N is >0.3, suggesting a mild α-helical conformation. However, in the CSI analysis of A17-S, no such conformation is observed. The sequence numbering for A17 (single-letter code) is shown on the horizontal axis. C, respective F1- and F2-binding sites are indicated by an arrow bar.

FIGURE 5.

Chemical shift assignments and conformational analysis of the A17-N peptide by two-dimensional TOCSY and NOESY. A, sections of the ¹H^N-¹H^α region of the NOESY and TOCSY spectra of the 33-mer A17-N peptide that binds to A27. The ¹H chemical shift assignments were successfully determined using the inter- and intra-residual correlations in these spectra (supplemental Table S1). B, sections of ¹H^N-¹H^β and ¹H^N-¹H^γ of the A17-N NOESY and TOCSY spectra. Consecutive inter-residual (i, i + 1) NOE signals were resolved as follows: between the ¹H^β of Asp²¹ and the ¹H^N of Lys²²; between the ¹H^β of Lys²² and the ¹H^N of Asp²³; between the ¹H^N of Asp²³ and the ¹H^γ of Leu²⁴; between the ¹H^β of Asp²³ and the ¹H^N of Leu²⁴; between the ¹H^β of Leu²⁴ and the ¹H^N of Phe²⁵; between the ¹H^γ of Leu²⁴ and the ¹H^N of Phe²⁵; between the ¹H^β of Phe²⁵ and the ¹H^N of Thr²⁶; between the ¹H^γ of Thr²⁶ and the ¹H^N of Glu²⁷; between the ¹H^β of Gln³⁰ and the ¹H^N of Gln³¹; and between the ¹H^β of Lys³⁶ and the ¹H^N of Asp³⁷. Additionally, (i, i + 2) NOE signals were found between the ¹H^N of Thr²⁶ and the ¹H^β of Glu²⁸, between the ¹H^β of Ser³² and the ¹H^N of Met³⁴, and between the ¹H^β of Met³⁴ and the ¹H^N of Lys³⁶.

FIGURE 6.

Computer modeling of A27 and A17 viral protein complex formation. A, representative of the F1-binding site, including the linker; the Gln²⁹–Asp³⁷ of the F1-binding site of A17 interacts with Arg⁹⁰–Asp¹⁰¹ of A27. The amine group of Lys⁹⁸ interacts with the aromatic ring of Phe³³ to form a specific cation-π interaction. Intermolecular hydrogen bonds were found between the amine group of Lys⁹⁸ and the hydroxyl group of Met³⁴, between the hydroxyl group of Ser⁹⁵ and the oxygen of Ser³², and between the guanidinium group of Lys³⁶ and the carboxyl group of Asp¹⁰¹. B, representative F2-binding site. Leu²⁰–Gln²⁹ of A17 interact with Phe⁸⁰–Glu⁸⁷ of A27. Notably, A17 presents an α-helical zigzag backbone conformation. At one side, the aromatic ring of Phe²⁵ interacts with the aromatic ring of Phe⁸⁰ to form a π-π stacking interaction. At the opposite side, it interacts with the guanidinium group of Arg⁸¹ to form a cation-π interaction. In addition, the carboxyl group of Leu²⁴ forms main chain interactions with the guanidinium side chain group of Arg⁸¹ to form a hydrogen bond. In this computer simulation, we confined the ¹H-¹H inter-residual distance constraints to within 5 Å for those revealing NOE signals (see Fig. 5B). The hydrogen bonds are highlighted by yellow solid lines and the cation-π interactions are labeled by orange dotted lines. The single-letter abbreviations of the amino acids of A27 and A17 are labeled in white and gray, respectively. For clarity, some of the side chains that are not involved in the A27-A17 interaction are not shown.

FIGURE 7.

Generation of recombinant vaccinia viruses expressing mutant A27 proteins in the infected cells. A, schematic representation of recombinant vaccinia virus genomes containing various A27L mutant ORFs. The wild-type WR virus is shown above each viral ORF, and the arrows indicate the direction of transcription. J2R represents the nonessential thymidine kinase locus. In all of the recombinant viruses except the WR virus, the viral A27L ORF is substituted with a dual-expression cassette, Luc-Gpt, which contains a luciferase (Luc) gene driven by a viral early promoter and the Eco-gpt (Gpt) gene driven by the viral p7.5k promoter. WR-A27-Rev, WR-A27-E87A, WR-A27-I94A, and WR-A27-E87A,I94A also contain another expression cassette inserted in the J2R locus in which the WT-A27, A27-E87A, A27-I94A, or A27-E87A,I94A mutants are driven by a synthetic late promoter, and a lacZ gene is driven by the viral p11k promoter. The asterisks mark the mutant residues. B, morphology of plaques produced on BSC40 and BSC40-A27 cells by WR, WR-ΔA27L, WR-A27-Rev, WR-A27-E87A, WR-A27-I94A, and WR-A27-E87A,I94A infections. BSC40 and BSC40-A27 cells were infected with each virus, fixed at 2 days p.i., stained with 1% crystal violet in 20% ethanol, and photographed. The tiny plaques are shown by triangles. C, immunoblot analysis of A27 protein in the infected cells. HeLa cells were infected with each virus at an m.o.i. of 5 pfu/cell, and the lysates were harvested at 24 h p.i. for immunoblot analyses with anti-A26 (1:1,000), anti-A27 (1:5,000), anti-A17 (1:1,000), and anti-H3 (1:1,000) antibodies.

FIGURE 8.

Coimmunoprecipitation analyses of the A27 and A17 interaction in virus-infected cells. HeLa cells were infected with WR, WR-ΔA27L, WR-A27-Rev, WR-A27-E87A, WR-A27-I94A, and WR-A27-E87A,I94A at an m.o.i. of 5 pfu/cell and harvested 24 h p.i. The cell lysates were used for immunoprecipitation (IP) experiments using anti-A27 antibody (A) or anti-A17 antibody (B). The immunoprecipitates were subsequently separated through SDS-PAGE and analyzed by immunoblot analyses using anti-A27 (1:5,000), anti-A17 (1:1,000), anti-H3 (1:1,000), and anti-A14 (1:500) antibodies.

FIGURE 9.

Structure and function analyses of A27 mutant viruses. A, silver staining of SDS-polyacrylamide gels containing 1 μg of purified mature virions of WR, WR-ΔA27L, WR-A27-Rev, WR-A27-E87A, WR-A27-I94A, and WR-A27-E87A,I94A viruses. B, immunoblot analyses of WR, WR-ΔA27L, WR-A27-Rev, WR-A27-E87A, WR-A27-I94A, and WR-A27-E87A,I94A MV particles (1 μg) with anti-A27 (1:5000), anti-A17 (1:1000), and anti-A26 (1:1000) antibodies. The protein bands were scanned, and their relative intensity is shown at the bottom of each panel. C, cell-cell fusion at neutral pH induced by virus infections. L cells expressing GFP or RFP (1:1 mixture) were either mock-infected or infected with WR, WR-ΔA27L, WR-A27-Rev, WR-A27-E87A, WR-A27-I94A, or WR-A27-E87A,I94A viruses at an m.o.i. of 50 pfu/cell at 37 °C for 30 min and monitored for cell-to-cell fusion at neutral pH. The cell images were photographed at 2 h p.i. D, quantification of cell-cell fusion at neutral pH. The percentage of cells containing both GFP and RFP fluorescence was determined using Axio Vision Rel. 4.8 with a Zeiss Axiovert fluorescence microscope.