Insights into the Organization of the Poxvirus Multicomponent Entry-Fusion Complex from Proximity Analyses in Living Infected Cells

Abstract

Poxviruses are exceptional in having a complex entry-fusion complex (EFC) that is comprised of 11 conserved proteins embedded in the membrane of mature virions. However, the detailed architecture is unknown and only a few bimolecular protein interactions have been demonstrated by coimmunoprecipitation from detergent-treated lysates and by cross-linking. Here, we adapted the tripartite split green fluorescent protein (GFP) complementation system in order to analyze EFC protein contacts within living cells. This system employs a detector fragment called GFP1-9 comprised of nine GFP β-strands. To achieve fluorescence, two additional 20-amino-acid fragments called GFP10 and GFP11 attached to interacting proteins are needed, providing the basis for identification of the latter. We constructed a novel recombinant vaccinia virus (VACV-GFP1-9) expressing GFP1-9 under a viral early/late promoter and plasmids with VACV late promoters regulating each of the EFC proteins with GFP10 or GFP11 attached to their ectodomains. GFP fluorescence was detected by confocal microscopy at sites of virion assembly in cells infected with VACV-GFP1-9 and cotransfected with plasmids expressing one EFC-GFP10 and one EFC-GFP11 interacting protein. Flow cytometry provided a quantitative way to determine the interaction of each EFC-GFP10 protein with every other EFC-GFP11 protein in the context of a normal infection in which all viral proteins are synthesized and assembled. Previous EFC protein interactions were confirmed, and new ones were discovered and corroborated by additional methods. Most remarkable was the finding that the small, hydrophobic O3 protein interacted with each of the other EFC proteins. IMPORTANCE Poxviruses are enveloped viruses with a DNA-containing core that enters cells following fusion of viral and host membranes. This essential step is a target for vaccines and therapeutics. The entry-fusion complex (EFC) of poxviruses is unusually complex and comprised of 11 conserved viral proteins. Determination of the structure of the EFC is a prerequisite for understanding the fusion mechanism. Here, we used a tripartite split green fluorescent protein assay to determine the proximity of individual EFC proteins in living cells. A network connecting components of the EFC was derived.

Keywords: green fluorescent protein; membrane proteins; multiprotein complex; proximity analysis; vaccinia virus; virus entry.

FIG 1

Model of the tripartite GFP complementation system. The system consists of one protein tagged with GFP10, another protein tagged with GFP11, and the GFP1-9 sensor. When proteins A and B interact, GFP10 and GFP11 are brought together, allowing complementation with GFP1-9 and green fluorescence.

FIG 2

Adaptation of the tripartite GFP assay for analysis of EFC interactions. (A) Construction of VACV-GFP1-9 and VACV-GFP1-10. Infusion PCR was used to insert DNA encoding GFP1-9 or GFP1-10 into the transfer plasmid pRB21 so as to be regulated by the VACV early-late promoter. The resulting GFP1-9 and GFP1-10 plasmids were transfected into BS-C-1 cells that had been infected with vRB12 to allow homologous recombination and large plaque formation. Virus from large plaques was clonally purified by repeat plaque formation to obtain VACV-GFP1-9 and VACV-GFP1-10. (B) Construction of plasmids expressing tagged EFC proteins. GFP10 or GFP11 sequences were fused to DNA encoding individual EFC proteins with an HA or V5 tag at the ectodomain that are regulated by the late p11 VACV promoter. (C) Expression of EFC proteins with V5 tags in VACV-infected cells was demonstrated by Western blotting. Similar results were obtained with HA-tagged EFC proteins (not shown).

FIG 3

Expression of recombinant GFP1-9 and GFP1-10 by recombinant VACV. (A) RK13 cells were infected with four individual clones of VACV-GFP1-9, VACV-GFP1-10, and a VACV expressing full-length (FL) GFP. Lysates were analyzed by SDS-PAGE, followed by Western blotting with an anti-GFP antibody and a secondary fluorescent antibody. Molecular weight markers are shown on the left. (B) RK13 cells on coverslips were infected with VACV-GFP1-9 or VACV-GFP1-10 and transfected with a plasmid expressing A28-V5-GFP11. After overnight incubation at 37°C, the cells were fixed, permeabilized, and stained with polyclonal antibody (pAb) to A28, followed by fluorescent secondary antibody and DAPI. Green fluorescence was detected in cells infected with VACV-GFP1-10 but not with VACV-GFP1-9. The merge shows DAPI (blue), anti-A28 (red), GFP (green), and overlap of anti-A28 and GFP (yellow).

FIG 4

Interaction of EFC proteins and complementation of GFP1-9 occur in cytoplasmic virus factories. RK13 cells on coverslips were infected with VACV-GFP1-9 and transfected with plasmids expressing A28-V5-GFP11 and H2-HA-GFP10 (A), H2-V5-GFP11 and A28-HA-GFP10 (B), G3-V5-GFP11 and L5-HA-GFP10 (C), L5-V5-GFP11 and G3-HA-GFP10 (D), G3-HA-GFP10 and L5-HA-GFP10 (E), and G3-V5-GFP11 and G3-HA-GFP10 (F). Cells were stained with DAPI, mouse antibody to V5, rabbit antibody to HA, and secondary fluorescent antibodies and analyzed by confocal microscopy. GFP fluorescence resulting from tripartite interactions that fluoresce due to DAPI and secondary antibodies was determined. Arrows point to cytoplasmic factories in the DAPI images.

FIG 5

Flow cytometry gating strategy. (A) Mock: cells were infected with VACV-GFP1-9 and mock transfected. From left to right are the gated cell population, V5-stained cells from the gated cell population, and GFP fluorescence. (B) Negative interaction. Cells were infected with VACV-GFP1-9 and transfected with A16-HA-GFP10 and A28-V5-GFP11. From left to right: gated cell population, V5-stained cells from gated cell population, GFP expression in V5-stained population. (C) Positive interaction. Cells were infected with VACV-GFP1-9 and transfected with H2-HA-GFP10 and A28-V5-GFP11. The images as described for panel B.

FIG 6

Flow cytometry analysis of EFC interactions. RK13 cells in 48-well plates were infected with VACV-GFP1-9, and triplicate wells of each set were transfected with an EFC-GFP10 plasmid and individual EFC-GFP11 plasmids. The cells were suspended with EDTA and transferred to a 96-well plate where they were fixed, permeabilized, and stained with mouse MAb to V5 and secondary fluorescent antibody. Flow cytometry was carried out by gating on V5-positive cells and determining the GFP mean fluorescence. Each set also contained cells transfected with H2-HA-GFP10 and A28-V5-GFP11, which served as a positive control used for normalization of other values. Mock-transfected cells and cells transfected with A28HA-GFP10 and H2V5-GFP10 and with A28V5-GFP11 and H2V5-GFP served as negative controls. The EFC protein attached to GFP10 is indicated near the top of each panel, and the individual EFC proteins attached to GFP11 are indicated below the x axis. The standard errors of the mean are shown. Z-scores were based on the fluorescence intensities for probe and bait protein pairs in each set of transfections. Filled bars indicate positive Z-scores; “*” and “**” indicate >1 and >2 standard deviations above the mean.

FIG 7

J5, G3, and L5 interactions. RK13 cells were transfected with plasmids expressing HA-J5 and G3-V5 (A), HA-J5 and L5-myc (B), and G3-V5 and L5-myc (C). Lysates were prepared and immunopurified by incubation with beads to which anti-V5, anti-HA, or anti-myc antibody had been bound. Input lysates and proteins following immunoprecipitation (IP) were analyzed by SDS-PAGE and Western blotting with antibodies shown on left of each panel. A table summarizing the data is presented in panel D. Plus signs indicate interaction.

FIG 8

EFC interaction model. Individual EFC proteins are indicated by spheres. Diameters are relative to molecular weight of individual proteins. Paralogs are in identical colors. Dashed lines connect proteins that together complement GFP1-9 in proximity analysis with Z-scores > 1 log above the mean. Blue lines connect protein that copurify on antibody beads. Red lines connect proteins that were chemically cross-linked.