Interaction of poxvirus intracellular mature virion proteins with the TPR domain of kinesin light chain in live infected cells revealed by two-photon-induced fluorescence resonance energy transfer fluorescence lifetime imaging microscopy

Abstract

Using two-photon-induced fluorescence lifetime imaging microscopy, we corroborate an interaction (previously demonstrated by yeast two-hybrid domain analysis) of full-length vaccinia virus (VACV; an orthopoxvirus) A36 protein with the cellular microtubule motor protein kinesin. Quenching of enhanced green fluorescent protein (EGFP), fused to the C terminus of VACV A36, by monomeric red fluorescent protein (mDsRed), fused to the tetratricopeptide repeat (TPR) domain of kinesin, was observed in live chicken embryo fibroblasts infected with either modified vaccinia virus Ankara (MVA) or wild-type fowlpox virus (FWPV; an avipoxvirus), and the excited-state fluorescence lifetime of EGFP was reduced from 2.5 ± 0.1 ns to 2.1 ± 0.1 ns due to resonance energy transfer to mDsRed. FWPV does not encode an equivalent of intracellular enveloped virion surface protein A36, yet it is likely that this virus too must interact with kinesin to facilitate intracellular virion transport. To investigate possible interactions between innate FWPV proteins and kinesin, recombinant FWPVs expressing EGFP fused to the N termini of FWPV structural proteins Fpv140, Fpv168, Fpv191, and Fpv198 (equivalent to VACV H3, A4, p4c, and A34, respectively) were generated. EGFP fusions of intracellular mature virion (IMV) surface protein Fpv140 and type II membrane protein Fpv198 were quenched by mDsRed-TPR in recombinant FWPV-infected cells, indicating that these virion proteins are found within 10 nm of mDsRed-TPR. In contrast, and as expected, EGFP fusions of the IMV core protein Fpv168 did not show any quenching. Interestingly, the p4c-like protein Fpv191, which demonstrates late association with preassembled IMV, also did not show any quenching.

FIG. 1.

Confocal laser scanning microscopy (CLSM) and FRET-FLIM analysis of transfected and/or poxvirus-infected live chicken embryo fibroblasts at 24-h postinfection. Panels A to C" represent infections with recombinant FWPV (rfpv168-EGFP) showing expression of fusion protein Fpv168-EGFP alone as a representative, unquenched, negative control. In panels D to I" cells were transfected with plasmids expressing A36-EGFP and KLC-TPR-mDsRed and infected with MVA (D to F") or parental FWPV (strain FP9; G to I"). MVA- and FP9-infected cells show colocalization (F and I, respectively) and interaction (E′ to F" and H′ to I", respectively) of VACV A36 with kinesin-TPR. In panels J to U" cells were transfected with plasmid expressing KLC-TPR-mDsRed and infected with recombinant FWPV expressing Fpv140-EGFP (rfpv140-EGFP; J to L"), Fpv168-EGFP (rfpv168-EGFP; M to O"), Fpv191-EGFP (rfpv191-EGFP; P to R"), or Fpv198-EGFP (rfpv198-EGFP; S to U"). Color-coded excited lifetimes of EGFP are shown in panels C′, F′, I′, L′, O′, R′, and U′. Panels C", F", I", L", O", R", and U" show areas of strong interaction (red shades) and noninteraction (blue shades). The distribution curves indicate the relative occurrence frequency of the lifetimes within the lifetime image. The distribution curves in panels L′ and U′ describe a distribution for interaction spread from 1.6 to 2.2 ns. Yellow in CLSM images indicates colocalization. To highlight interacting and noninteracting areas in the color-coded image, the green channel was switched off, and the intensity level was adjusted using Adobe Photoshop (CS4 version). Scale bar, 10 μm.

FIG. 2.

Localization of three of the EGFP-tagged FWPV proteins to FWPV virions in close proximity to microtubules in recombinant FWPV FP9-infected chicken embryo fibroblast cells. Cells infected with individual recombinant FWPVs expressing one of the EGFP fusions (green) of FWPV protein Fpv140-EGFP (A and B), Fpv168-EGFP (C and D), or Fpv198-EGFP (E to H) were stained at 24 hpi for microtubules (using mouse anti-tubulin antibody/Alexa Fluor 568 goat anti-mouse IgG; red) and DNA (with ToPRO-3; blue). Merged channels are shown in panels A, C, and E; zoomed sections of merged green and red channels are shown in panels B, D, and F to H. Solid arrows indicate virus particles. Scale bar, 10 μm.

FIG. 3.

Recognition of EGFP fusion of FWPV protein Fpv191-EGFP by monoclonal antibody raised against native protein Fpv191. A cell infected with recombinant FWPV (A to D) expressing both parental Fpv191 and Fpv191-EGFP (green), stained (red) using anti-Fpv191 monoclonal antibody (mDH6) and Alexa Fluor 568 goat anti-mouse IgG, and analyzed by immunofluorescence microscopy, at 24 h postinfection. Colocalization is incomplete; solid arrows indicate incidences of colocalization compatible with virions. DNA staining (blue; labeled with ToPRO-3) reveals the location of the viral factories (indicated by open arrows). Scale bar, 10 μm.

FIG. 4.

Colocalization of FWPV EGFP fusion proteins with FWPV structural proteins recognized by MAbs. Chicken embryo fibroblasts infected with recombinant FWPV FP9 (rFWPV) expressing the native structural proteins and one of the EGFP fusion protein (green; indicated on the figure) were immunolabeled at 24 hpi with anti-Fpv140 (MAb DF6; A, C, and G), anti-Fpv168 (MAb GB9; D, E, and H) or anti-Fpv191 (MAb DH6; B, F, and I) antibody. DNA was labeled with ToPRO-3 (blue; G and H). Merged images are shown, as are zoomed green and red merged channels. Yellow indicates colocalization of EGFP fusion proteins with immunolabeled proteins. Scale bar, 10 μm.