Fig 1.
Model of A26 protein function and generation of mutant vaccinia viruses containing N-terminal deletions of A26 protein.
(A). Current model of how A26 protein functions as an acid-sensitive fusion suppressor of the WR strain of vaccinia virus MV. Vaccinia MV contains A26 protein that binds to the A16 and G9 components of the viral fusion complex (VFC) to suppress virus membrane fusion activity, which keeps the virus stable at neutral pH. When MV is internalized into endosomes, acidification triggers conformational changes of A26 protein that dissociates itself from the VFC, resulting in activation of the VFC and fusion of the viral and vesicular membranes. However, deletion of the A26L gene results in WR-ΔA26 MV particles lacking fusion suppressor activity, so these MV particles readily fuse to the plasma membrane (PM) at neutral pH. (B). Schematics of full-length WR-A26 and truncated WR-A26 proteins (aa 76–500 or 321–500), as well as each construct containing flag-tag sequences (red) at their N-termini. Locations of A26L mutant genes in the corresponding viral genomes are also shown. WR-A26 and WR-ΔA26 were described previously [32, 33], and the latter was used as the parental virus to generate the WR-A26(76–500) and WR-A26(321–500) recombinant viruses. The J2R locus encodes a non-essential viral thymidine kinase (tk). We inserted A26(76–500) and A26(321–500) gene constructs into the tk locus and then selected blue plaques in agar plates containing X-gal. (C). One-step growth of mutant vaccinia viruses in HeLa cells. Cells were infected with each virus at a multiplicity of infection (MOI) of 5 PFU per cell and cells were washed and harvested at 0 and 24 hpi for virus titer determination. The Y-axis represents MV growth, determined by dividing virus titers at 24 hpi with the respective input virus titer at 0 hpi. We performed three experimental repeats for each virus and used the Student t-test for statistical analyses. *p <0.05, ** p < 0.01. (D). EM images of MV in infected HeLa cells at 24 hpi. Insets in lower right corner represent EM images of CsCl-purified MV. The scale bar represents 1 μm. (E). Immunoblots of N-terminal A26-deletion proteins in CsCl-purified MV particles. A27 and D8 are two viral envelope proteins that served as positive controls.
Table 1.
MV particle infectivity of WR-A26(76–500), WR-A26(321–500), WR-A26-H2R, WR-A26-H3R and the revertant viruses.
Fig 2.
N-terminal region of A26 protein (aa 1–75) is important for endocytic-mediated MV membrane fusion at low pH.
(A). Schematic presentation of vaccinia MV-dependent cell-cell fusion at neutral and acidic pH. L cells expressing GFP and RFP were mixed at a 1:1 ratio during seeding. No cell fusion occurs at either pH in the absence of infection (Mock). When cells were infected with the endocytic WR-A26 virus, cell-cell fusion only occurred at low pH (which mimics endosomal environments) and no cell-cell fusion occurred at neutral pH (which allows plasma membrane fusion). In contrast, WR-ΔA26 virus does not require acidic environments to activate membrane fusion, so cell-cell fusion occurs at both acidic and neutral pH. Truncation of A26 protein to remove the acid-sensing/acid-sensitive domain (WR-A26(76–500)) renders the protein a constitutive fusion suppressor, so no fusion occurred at either pH. Further truncation of A26 protein to remove the fusion suppressor domain (WR-A26(321–500)) resulted in a virus phenotype like that of WR-ΔA26 in that it fuses at both pH. (B). Images of cell-cell fusion induced by various vaccinia MV infections. Single fluorescent cells described in (A) were infected with each virus at an MOI of 50 PFU per cell, washed with neutral or acidic pH buffer for 3 min, and then subjected to live-imaging to monitor cell-cell fusion at 37°C for 2 h. (C). Quantification of % cell fusion induced by each virus at neutral or low pH conditions as described in (B). Five images for each virus were recorded and the % fusion was calculated using the image area of GFP+RFP+ double-fluorescent cells divided by that of single-fluorescent cells. The experiments were repeated three times for each virus and bars represent standard deviation. (D). An “Acid Fusion Index” was calculated to represent the acid-dependence of each A26 deletion protein, i.e., the occurrence of A26 protein conformational change. The index for each virus was obtained by dividing the % of cell fusion at low pH (black bar in C) by that recorded for neutral pH (white bar in C). Endocytic WR-A26 virus demonstrated an Acid-Fusion Index of ~4.6, whereas the value for all other viruses was ~1, meaning these latter exhibit limited dependence on acidic environments for cell fusion. The experiments were repeated three times for each virus and the Student t-test was used for statistical analyses. *** p <0.001. (E). Schematic representation of A26 protein functional regions. The N-terminal region of A26 protein (aa 1–75, in pink) is important for acid sensitivity, whereas the middle region (aa 76–320, in green) is required for the fusion suppressor function. The C-terminal region (in blue) was previously shown to mediate binding to viral A27 protein during MV assembly [34, 35].
Fig 3.
NMR analyses show that His48 and His53 are critical residues for acid-dependent conformational change of an A26 protein N-terminal fragment (aa 1–91) in vitro.
(A). Protein sequence of the N-terminal of vaccinia WR-A26 protein (aa 1–91), showing positions of His48 and His53 within aa1-75 (in pink). (B-D). 2D 1H/15N HSQC NMR spectra of TRX-A26(1–91) fusion protein (in B), TRX (in C) and TRX-A26 (1–91)H48.53R (in D). The 2D HSQC spectra were measured at pH 8 (in blue) and pH 6 (in red), respectively. (E and F). CD spectroscopy analyses of recombinant A26(1–91) (in E) and A26(1–91)H48.53R (in F) as a function of pH values ranging from pH 5.1 to 8.5.
Fig 4.
Generation of mutant vaccinia viruses containing His48R and His53R mutations (H2R) of A26 protein.
(A). Schematic representations of full-length WR-A26, A26 His48R and His53R double-mutant (H2R mutant), and A26 His48R, His53R and His92R triple-mutant (H3R mutant) proteins. Each construct contained flag-tag sequences at the N-terminus. (B). Schematics of recombinant viruses showing the WR-A26-H2R and WR-A26-H3R mutant genome arrangements. WR-A26 and WR-ΔA26 were described previously [32, 36], and the latter was used as the parental virus to generate the WR-A26-H2R and WR-A26-H3R recombinant viruses. The A26-H2R and A26-H3R gene cassettes were inserted into the A26L native locus and recombinant virus was selected as blue plaques in agar plates containing X-gal. (C). Immunoblots of viral A26-H2R and A26-H3R proteins expressed in virus-infected cells at 24 hpi. (D). Immunoblots of A26-H2R and A26-H3R proteins in CsCl-purified MV particles. A27 and D8 proteins are two viral envelope proteins that serve as positive controls. (E). EM images of abundant MV from the WR-A26-H2R and WR-A26-H3R viruses in infected HeLa cells at 24 hpi. The scale bar represents 1 μm. (F). One-step growth of the WR-A26, WR-A26-H2R and WR-A26-H3R viruses in HeLa cells. Cells were infected with each virus at an MOI of 5 PFU per cell and then washed and harvested at 0 and 24 hpi for virus titer determination. The Y-axis represents MV growth, which was determined by dividing virus titers at 24 hpi with the respective input virus titer at 0 hpi. The experiments were repeated three times and the Student t-test was used for statistical analysis. ** p < 0.01. (G). Images of cell-cell fusion upon infection by respective MV. Single-fluorescent cells as described in Fig 2A were infected with each virus at an MOI of 50 PFU per cell, washed with neutral or acidic pH buffer for 3 min, and then subjected to live-imaging to monitor cell-cell fusion at 37°C for 2 h. (H). Quantification of % fusion for images described in (G). Five images for each virus were recorded and the % fusion was calculated using the image area of GFP+RFP+ double-fluorescent cells divided by that of single-fluorescent cells. The experiments were repeated three times and the bars represent standard deviations. Student t-test was used for statistical analyses. *** p <0.001. (I). The Acid Fusion Index was calculated by dividing the % of cell fusion at low pH (black bars in H) by that at neutral pH (white bars in H). Endocytic WR-A26 virus demonstrated an Acid Fusion Index of ~7.02, whereas the indexes of all other viruses were much lower, demonstrating their limited dependence on an acid environment for cell fusion. These experiments were repeated three times for each virus and the Student t-test was used for statistical analyses. *** p <0.001.
Fig 5.
Crystal structure of recombinant A26 protein (aa 1–397).
(A). A ribbon diagram of the A261-397 structure. The α-helices and β-strands are colored in cyan and yellow, respectively. Two domains, NTD (aa 17–228) and CTD (aa229-364), are shown and highlighted in cornflower blue and magenta, respectively. (B). The secondary structural elements are shown above the amino acid sequences, with cyan cylinders and yellow arrows representing α-helices and β-strands, respectively. (C). The His-cation and AniAni pairs in the recombinant A261-397 structure. The amino acids that involve in His-cation and AniAni pairs are shown as ball and stick models. Green arrows show the position of His48 and His53.
Table 2.
Data collection and refinement statistics for the crystal structure of A26(1–397) protein.
Fig 6.
Generation of A26 mutant vaccinia viruses containing K47D, R57D, R312D and H314R mutations in A26 protein (A26-H2-CAT).
(A). Schematic representation of full-length WR-A26 and mutant A26-H2-CAT proteins. Each construct contains flag-tag sequences at the N-terminus. (B). Genome arrangement of WR-A26-H2-CAT recombinant virus. The A26-H2-CAT gene cassette was inserted into the endogenous A26L locus and selected as blue plaques in agar plates containing X-gal. (C). One-step growth of the WR-A26 and WR-A26-H2-CAT viruses in HeLa cells. Cells were infected with each virus at an MOI of 5 PFU per cell for 60 min and then cells were washed and harvested at 0 and 24 hpi for virus titer determination. The Y-axis represents MV growth, which was determined by dividing virus titers at 24 hpi with the respective input virus titers at 0 hpi. The experiments were repeated three times and the Student t-test was used for statistical analysis. ** p < 0.01. (D). Immunoblots of WR-A26, WR-ΔA26 and WR-A26-H2-CAT mutant proteins in CsCl-purified MV particles. A27 and D8 proteins are two viral envelope proteins that served as positive controls. (E). Images of cell-cell fusion induced upon MV infection. Single-fluorescent cells as described in Fig 2A were infected with each virus at an MOI of 50 PFU per cell, washed with neutral or acidic pH buffer for 3 min, and then subjected to live-imaging to monitor cell-cell fusion at 37°C for 2 h. (F). Quantification of the % fusion of each virus described in E. Five images for each virus were recorded and the % fusion was calculated based on the ratio of image area of GFP+RFP+ double-fluorescent cells divided by that of single-fluorescent cells. *** p <0.001. (G). The Acid Fusion Index was calculated from data (in F) by dividing the % of cell fusion at low pH (the black bars) by that at neutral pH (the white bars). Endocytic WR-A26 had an Acid Fusion Index of ~5.96, whereas the indexes of other viruses were much lower, demonstrating a significantly reduced dependence on acidic environments for cell fusion. The experiments were repeated three times for each virus and the Student t-test was used for statistical analyses. *** p <0.001.
Table 3.
MV particle infectivity of WR-A26, WR-A26-H2-CAT and the revertant virus WR-H2-CAT-Rev1.
Fig 7.
Revertant viruses exhibit a large plaque phenotype and regained MV infectivity by generating intragenic second-site mutations in the A26L ORF that caused a frame-shift and premature termination of A26 protein translation.
(A). Plaque phenotypes of control WR-A26 virus and the small plaque phenotype of various A26 mutant viruses. Low percentages (~2%) of large plaque revertants (Rev) (indicated by red arrows) were isolated from each mutant virus and plaque-purified. (B). Immunoblots of A26 protein from infected HeLa cells at 24 hpi. HeLa cells were infected with each virus as indicated and the cell lysates were harvested at 24 hpi for immunoblot analyses. A27 and D8 proteins are two viral envelope proteins that served as positive controls. (C). Schematic representation of mutant A26 proteins and their revertant A26 proteins, respectively. Revertant A26 proteins contain a second-site deletion that causes a frame-shift and premature termination of A26 mutant protein. Red arrowheads show the designed H2R, H3R and H2-CAT amino acids mutated in each A26 protein. Each revertant A26 protein is color-coded: N-terminal flag tag (red box); translated A26 aa upstream of the second-site deletion (white box); and aberrant downstream aa due to frame-shift (blue dotted box). Second-site mutations are shown in red text. (D). Images of cell-cell fusion induced by MV infections. Single-fluorescent cells as described in Fig 2A were infected with each virus at an MOI of 50 PFU per cell, washed with neutral or acidic pH buffer for 3 min, and then subjected to live-imaging to monitor cell-cell fusion at 37°C for 2 h. (E). The Acid Fusion Index was calculated by dividing the % of cell fusion at low pH by that at neutral pH, similar to what is described in Fig 6F. Endocytic WR-A26 virus has an Acid Fusion Index of 6.53, whereas the indexes of the other viruses were much smaller, indicating limited dependence on an acid environment for cell fusion. The experiments were repeated three times for each virus and the Student t-test was used for statistical analyses. *** p <0.001. (F). One-step growth of WR-A26 and revertant viruses in HeLa cells. Cells were infected with each virus at an MOI of 5 PFU per cell and cells were washed and harvested at 0 and 24 hpi for virus titer determination. The Y-axis represents MV growth, which was determined by dividing virus titers at 24 hpi with the respective input virus titer at 0 hpi. The experiments were repeated three times and there was no significant growth difference among these revertant viruses and control WR-A26 virus (Student t-test).
Table 4.
Whole genome sequencing of A26 revertant viruses derived from WR-A26-H2R, WR-A26-H3R and WR-A26-H2-CAT mutants.
Fig 8.
Vaccinia virus membrane protein A26 is a fusion suppressor of MV.
Data presented in this study reveal an important role for A26 protein in endocytic entry of the WR strain of vaccinia MV. Low pH in endosomes triggers conformational changes of wild type A26 protein to allow membrane fusion within vesicles. However, when mutant A26 protein, such as A26H2R or A26H2-CAT, loses the ability to execute conformational changes at low pH, it becomes a constitutive suppressor that blocks virus membrane fusion, resulting in significant loss of mature virus infectivity. Subsequent generation of the second-site mutations then leads to truncation of A26 protein so the resulting revertant viruses acquire a phenotype similar to that of WRΔA26 virus and initiate virus fusion with the plasma membrane at neutral pH to recover MV infectivity.