Figure 1.
(A) A schematic representation of the vaccinia virus A27 domain structure. A27 comprises three domains, including the heparin binding domain (HBD), the coiled-coil domain (CCD) for hexameric assembly and interacting with A26, and the leucine zipper domain (LZD) for binding with A17. Expressed tA27 for crystallization studies contains residues from 21 to 84, with mutations in two adjacent cysteine residues (C71A and C72A). The magenta cylinder denotes the region of tA27 structure that corresponds to the α-helix. Black dashed lines indicate the disordered regions. The basic residues in HBD (cyan) and the critical segment for specific heparin binding (underlined) are shown. The residues in the N-terminal region (NTR) interface involved in trimer assembly are presented in blue, and residues in the C-terminal region (CTR) interface involved in hexamer assembly are in green. Purple and green dots denote the mutation sites for in vitro and in vivo studies. (B) This representation of hexameric tA27 is composed of trimer 1 (bottom), shown as ribbon diagram and surface model in blue (chain A), cyan (chain B), and magenta (chain C); and trimer 2 (top), presented as surface model and ribbon diagram in light blue (chain A′), light cyan (chain B′), and light magenta (chain C′). The disordered region, including the HBD, is indicated by black dotted lines. Residues 71 and 72 are colored in red. The filled circles indicate the C-terminus and the white circles denote the N-terminus of tA27. (C) The trimeric structure of Influenza virus HA2. A representative trimer-of-hairpins structure of the viral fusion protein of the influenza virus (Flu) HA2 (PDB code 1HTM) is shown [60]. For Flu HA2, the interior N-terminal coiled-coil structures are colored in similar blue and the exterior C-terminal antiparallel helices are in similar green. The right part of the figure shows the model rotated 90°.
Table 1.
Data collection and refinement statistics.
Figure 2.
Interactions of the N-terminal region (NTR) and the C-terminal region (CTR) interfaces.
(A) The residues in the CTR interface are depicted as sphere models (chain A′, light blue; ′ chain B′, light cyan). They are divided into two layers of cross-sections (C-S) for hydrophobic interaction and one hydrogen-bonding interaction. The hydrophobic resides involved in the NTR interface are shown as a cluster of spheres in blue (chain A), cyan (chain B), and magenta (chain C), and divided into 8 layers of C-S. The filled circles denote the C-terminus and the open circles indicate the N-terminus of tA27. The triple L-to-A mutation sites at the NTR interface are boxed. (B) The 2| FO|-|FC| electron density maps are contoured at 1.0σ level as green mesh, and the refined protein model is shown as sticks in blue (chain A′), cyan (chain B′), magenta (chain C), light blue (chain A′), and light cyan (chain B′).
Figure 3.
Analysis of vaccinia A27 orthologs.
(A) Multiple sequence alignment of A27 orthologs. The alignment contains group A from Parapoxvirus, including Bovine papular stomatitis virus (BPSV) and Orf virus (ORFV) group B from Orthopoxvirus including Vaccinia virus (VV), Variola virus (VARV), Monkeypox virus (MPXV), Cowpox virus (CPXV), and Ectromelia virus (ECTV) group C from Capripoxvirus, including Lumpy skin disease virus (LSDV), Goatpox virus (GTPV), Sheeppox virus (SPPV), and Suipoxvirus with Swinepox virus (SWPV); and group D from Leporipoxvirus, including Rabbit fibroma virus (RFV) and Myxoma virus (MYXV). Note the heparin binding domain (HBD, orange), coiled-coil domain (CCD, purple), and leucine zipper domain (LZD, grey) regions. The conserved residues surrounding the CCD are marked with orange boxes for NTR and green boxes for CTR. The two cysteine residues in the CCD are denoted by red boxes and dots. Identical and similar amino acid residues are shaded in black and gray, respectively. Dashes denote the sequence gaps introduced to optimize the amino acid sequence alignment. The accession numbers are: BPSV (NP958013); ORFV (AAR98199); VV (YP233032); VARV (ABF23908); MPXV (NP536566); ECTV (NP671648); CPXV (NP619946); LSDV (NP150551); GTPV (ABS72324); SPPV (NP659689); SWPV (NP570274); RFV (NP052004); and MYXV (NP051829). (B) The protein evolution of A27 orthologs. The respective A27 protein lengths of Groups A, B, C, and D are represented by black, cyan, orange and green lines, respectively. The inserted residues are marked with triangles, and the corresponding conserved regions of CCD are boxed in red dashes. (C) Computer modeling of vaccinia A27 orthologues with the X-ray structures of vaccinia virus tA27 show that the CCD regions are structurally conserved, as predicted by molecular modeling, in group A (BPSV, black), group C (LSDV, orange), and group D (RFV, green). The predicted modeling structures were produced by the Phyre server [61].
Figure 4.
Size exclusion chromatography of tA27-WT protein showing oligomer formation in a concentration-dependent manner.
(A and B) Recombinant tA27-WT, tA27-TM-N, tA27-TM-C, and tA27-6A proteins at concentrations of 1 mg/ml (A) and 9.5 mg/ml (B) were individually injected into a Superdex 75 10/300 GL size exclusion column at pH 7.5, and the protein elution profiles at 280 nm UV absorption were recorded. The apparent molecular mass of each tA27 recombinant protein was determined using standard protein molecular mass markers (Table S3). (C) tA27-WT and tA27-TM-N proteins at concentrations of 1 mg/ml and 9.5 mg/ml were analyzed using the same size exclusion system equilibrated at pH 3.0. Then, the protein elution profiles were recorded. Because protein markers were not suitable for use as standards at low pH, the protein elution volume was used to compare different recombinant proteins.
Figure 5.
AUC-SV and SEC/MALS analysis of tA27 protein showing that tA27 protein formed concentration-dependent dimers.
(A) AUC analyses of tA27-WT protein. Normalized c(s) distribution plots for tA27-WT and mutants. The figure shows the distribution plots for tA27-WT at pH 7.5 (black), tA27-TM-N (green), tA27-TM-C (orange), tA27-6A (purple) and tA27-WT at pH 3.0 (red) obtained from the fitting of SV data using a continuous c(s) distribution model. (B) SEC/MALS profiles of tA27-WT protein at concentrations of 1 mg/ml (red) and 9.5 mg/ml (green). BSA was used as control (black). Thin line segments represent the calculated molar masses; the numbers denote the corresponding molecular weights of each peak (left ordinate axis) in kDa. Solid lines represent normalized UV absorbance (280 nm, right ordinate axis), and dashed lines represent light scattering.
Figure 6.
Immunoblot analysis of A27 proteins in transiently transfected-infected cells.
293T cells were infected with WRΔA27L virus and subsequently transfected with plasmids containing A27-wt, A27-TM-N, A27-TM-C, or A27-6A DNA. Lysates were harvested at 24 h post-infection and separated on 4% to 12% SDS-PAGE gels in reducing (+2ME) (A) or non-reducing (−2ME) conditions (B and C) for immunoblot analysis with anti-A27 (1∶1,000) (A & B) and anti-A26 (1∶5,000) (A & C) antibodies. The arrowheads labeled A27-mono, A27-di, and A27-tri represent A27 protein monomer, dimer, and trimer, respectively. The black and white arrows mark the 90-kDa and 70-kDa A26–A27 protein complexes, respectively. A26-containing bands appeared as doublets because of the incomplete formation of intramolecular disulfide bonding between C43 and C342 in the A26 protein [23].
Figure 7.
WR-A27-TM-N, WR-A27-TM-C, and WR-A27-6A exhibited a small plaque phenotype on BSC40 cells.
(A) Schematic representation of vaccinia virus genomes containing A26–A29 and J2R ORFs. Wild type WR virus is shown at the top, with arrows indicating the direction of transcription. In WRΔA27L, WR-A27R, WR-A27-TM-N, WR-A27-TM-C, and WR-A27-6A, the viral A27L ORF was replaced with a dual expression cassette, Luc-Gpt, containing a luciferase (Luc) gene driven by a viral early promoter and the Eco-gpt (Gpt) gene driven by the viral p7.5 promoter. The J2 locus of the WR-A27R, WR-A27-TM-N, WR-A27-TM-C, and WR-A27-6A genome was inserted with another dual expression cassette containing an A27 ORF driven by a viral late promoter and a lacZ gene driven by the viral p7.5k promoter. (B) BSC40-A27 cells express A27 protein for function complementation in trans. Immunoblot analysis of A27 protein expressed in BSC40-A27 cells. Beta-actin was used as a control. WRΔA27L virus, which formed small plaques on BSC40 cells, produced large plaques on BSC40-A27 cells after cell staining with 1% crystal violet in 20% ethanol. (C) Virus plaque morphology on BSC40 and BSC40-A27 cells. WR-BSC40-A27 and BSC40 cells were infected with 50–100 plaques of WR-A27R, WR-A27-TM-N, WR-A27-TM-C, and WR-A27-6A, fixed at 2 days post-infection and subsequently stained with X-gal and photographed.
Figure 8.
Immunoblot analysis of A27 protein in infected cells and on mature virus particles.
(A) HeLa cells were mock-infected or infected with WR, WRΔA27L, WR-A27R, WR-A27-TM-N, WR-A27-TM-C, and WR-A27-6A at an MOI of 5 PFU/cell. Lysates were harvested at 24 h post-infection and separated on 4% to 12% SDS-PAGE gels in reducing conditions (+2ME) for immunoblot analysis with anti-A26 (1∶1,000), anti-A27 (1∶5,000), and anti-D8 (1∶5,000) antibodies. (B and C) The same infections as in A were processed for immunoblot analysis in non-reducing conditions (−ME) and probed with anti-A27 (B) and anti-A26 and anti-H3 (C) antibodies. A26-monomers (A26-mono) appeared as doublets because of incomplete intramolecular disulfide bonding between C43 and C342 of the A26 protein [23]. (D) Purified MV particles (1µg) of WR, WRΔA27L, WR-A27R, WR-A27-TM-N, WR-A27-TM-C, and WR-A27-6A were separated on 4%–12% SDS-PAGE gels and processed for immunoblot analysis in reducing conditions (+2ME) as described above. (E and F) Immunoblot analysis of purified MV in non-reducing conditions (−2ME) using anti-A27 (E) and anti-A26 and anti-H3 (F) antibodies. The black and white arrows mark 90-kDa and 70-kDa A26–A27 protein complexes, respectively. Arrowheads – labeled mono, -di, and -tri represent protein monomer, dimer, and trimer, respectively.
Figure 9.
WRΔA27L, WR-A27-TM-N, WR-A27-TM-C, and WR-A27-6A induce cell-to-cell membrane fusion on L cells at neutral pH.
(A) L cells expressing GFP or RFP (1∶1 mixture) were either mock-infected or infected with WR, WRΔA27L, WR-A27R, WR-A27-TM-N, WR-A27-TM-C, and WR-A27-6A viruses at an MOI of 50 PFU/cell at 37°C for 30 min and monitored for cell-to-cell fusion at a neutral pH, as described in Materials and Methods. Cell images were photographed at 2 h post-infection. (B) Quantification of cell-to-cell fusion at neutral pH. The percentage of cells containing both GFP and RFP fluorescence was quantified as cell-cell fusion using Axio Vision Rel. 4.8 with a Zeiss Axiovert fluorescence microscope.