Technical Reference #290

290.

Kinesin-dependent movement on microtubules precedes actin-based motility of vaccinia virus Jens Rietdorf; Aspasia Ploubidou; Inge Reckmann; Anna Holmström; Friedrich Frischknecht; Markus Zettl; Timo Zimmermann and Michael Way, European Molecular Biology Laboratory, Nature Cell Biology, 3(290), (2001)
Link To Paper

Abstract:
Vaccinia virus a close relative of the causative agent of smallpox exploits actin polymerization to enhance its cellto-cell spread. We show that actin-based motility of vaccinia is initiated only at the plasma membrane and remainsassociated with it.

Materials & Methods:
Generation of stable GFP–β-actin cell lines. The chicken β-actin gene was amplified by PCR using the primers 5′-CCCGCGGCCGCGGAGGGATGGATGATGATAATGCTGCG- 3′ (called Not-Act-For) and 5′-GGGGAATTCCTATTAGAAGCATTTGCGGTGGAC- 3′ (called Act-RI-Rev). The resulting PCR product was cloned into the NotI–EcoRI sites of the CMV transfection vector CB6N–GFP to generate CB6–GFP–β-actin in which GFP is fused to the N terminus of actin by a short linker of glycine residues. CB6–GFP–β-actin was transfected into NIH3T3 mouse fibroblasts and 143TK– human osteosarcoma cells using a standard calcium phosphate method and maintained in 0.5 μg active units of G418 (Geneticin) per ml or 0.3 μg active units of G418 per ml respectively. In addition 143TK– cells were maintained in 2.5% BMCondimed H1 (Roche Diagnostics Mannheim Germany). Cells were sorted using fluorescenceactivated cell sorting 12 days after G418 selection was applied and clonal cell lines were subsequently isolated by serial dilution. pEL vector constructs. The DNA corresponding to the F13L and A36R open reading frames was amplified from vaccinia genomic DNA by PCR using the primers 5′-GGGAAGCTTACCATGTGGCCATTTGCATCGGTACCTG- 3′ (called F13L-H3-For) 5′-CCCCGCGGCCGCGCCGCCAATTTTTAACGATTTACTGTGGCTAGA- 3′ (called F13L-Not-Rev) 5′-GGGAGATCTACCATGATGCTGGTACCTCTTATC-3′ (called A36R-Bgl-For) and 5′-CCCCGCGGCCGCGCCGCCCACCAATGATACGACCGATGATTC-3′ (called A36R-Not-Rev). The resulting F13L PCR product was cloned into the HindIII–NotI sites of the vaccinia expression vector pEL (ref. 20) into which the gene for GFP had been inserted. The A36R PCR product was cloned into the BglII–NotI sites of the same vector. In the expression clones pEL–F13L–GFP and pEL–A36R–GFP the C termini of F13L and A36R are linked to GFP by a short linker of glycine residues. C-terminal A36R deletion mutants fused to GFP were generated by introducing two glycine codons and a NotI site after the desired residue by PCR and are named based on the remaining sequence from the N terminus to their final C-terminal residue. To generate internal A36R deletions we took advantage of a unique SnaB1 site that immediately follows the transmembrane domain of A36R (residues 1–32; TM). An in-frame SnaB1 site and two glycine codons were introduced by PCR immediately adjacent to the desired residue using A36R N100–GFP as a template. Internal deletion mutants are defined by the presence of TM followed by the residue positions in the cytoplasmic domain of A36R. Residues 155–599 of the mouse KLC2 containing the TPR repeats25 were amplified by PCR using the primers 5′-GGGGCGGCCGCGGCGGCAAGGGTGATGTCCCCAAAGACTCCCT-3′ (called KLC2- Not155-Fr) and 5′-CCCGAATTCAAAGTGCGGCTGTCAGAAAGACCCG-3′ (called KLC2-599 RIRv). The resulting PCR product was cloned into the NotI–EcoRI sites of pELGFP to generate N-terminally GFP-tagged KLC2–TPR. The fidelity of all expression clones used in this study was confirmed by sequencing. Antibodies. IEVs were labelled with antibodies against A33R A34R and A36R (ref. 24) the rat monoclonal antibody 19C2 against B5R (refs 12 61) or polyclonal antibodies raised against the F13L peptides CRLVETLPENMDFRSDHL and CGFVSFNSIDKQLVSEAKK corresponding to residues 14–30 and 339–356 of the protein. Both peptides were coupled via the additional N-terminal cysteine introduced during synthesis to Keyhole Limpet haemocyanin using the Imject activated immunogen conjugation kit (Pierce Chemical Rockford IL USA). The conjugated peptides were separated from uncoupled peptide using a Presto desalting column (Pierce Chemical) and the pooled conjugates injected into rabbits. Anti-F13L antibodies were subsequently affinity purified on their respective peptides which had been coupled via the N-terminal cysteine to SulfoLink columns (Pierce Chemical). The specificity of anti-F13L antibodies was confirmed by western and immunofluorescence analysis on HeLa cells infected with WR-strain virus expressing F13L–GFP. Conventional kinesin was visualized with the monoclonal antibodies SUK4 (Covance Richmond CA USA) or MAB1614 (H2) (Chemicon International Temecula CA USA) directed against the heavy chain or MAB1616 (L1) (Chemicon International) directed against the light chain. In addition polyclonal sera against the kinesin light chain25 a pan-KLC serum (35.1 Covance) and polyclonal sera against the heavy chain53 were also used. The pan-kinesin heavy-chain polyclonal antibody HIPYER (ref. 62) was used to detect kinesin-family motors. Actin was visualized with either the anti-β-actin antibody AC-74 (Sigma Deisenhofen Germany) or Alexa-488– or Texas-Red–phallicidin (Molecular Probes Eugene OR USA). Microtubules were visualized by expressing mouse β-tubulin fused at its C terminus to EGFP using the expression vector pBactin-mb5tubulin-EGFP (A. Matus unpublished). Infection pEL-driven expression and immunofluorescence on fixed preparations. Cells were infected with WR and the recombinant vaccinia strain ΔA36R which lacks the A36R gene63. Infected cells were fixed 6–8 h after infection and processed for immunofluorescence as described previously2124. All pEL expression constructs were transfected 4–6 h after infection with WR ΔA36R and A36R-YdF using lipofectin (Gibco–BRL) and processed for western blot or immunofluorescence analysis 2–4 h later. Alternatively confocal sections of fixed non-permeabilized infected cells expressing GFP–β-actin and labelled with wheat-germ agglutinin Alexa Fluor 594 (Molecular Probes) were taken on a Leica TCS SP2 confocal microscope using a 1.4 NA PlanApo objective lens obtaining volume elements (voxels) measuring 66 × 66 × 200 nm (x × y × z). Live cell imaging. Cells were grown in glass-bottomed dishes (MatTek AshlandMaryland USA) and maintained at 37 °C in CO2-independent medium (MEM without phenol red but containing 30 mM Hepes (Gibco–BRL)) throughout the recording. Images were taken at 5 frames s–1 using an Olympus IX70 microscope with 1.2 NA UPlanApo PSF water immersion lens a monochromator a Piezzo stepper (Physik InstrumenteWaldbronn Germany) and a camera controlled by TillVision software (Till PhotonicsMartiensried Germany). The pixel size was 111 nm2. For volume imaging stacks consisting of five planes spanning 10 μm above the coverglass were taken every second. For live cell confocal microscopy images were taken from infected cells at 1.25 frames s–1 using an UltraView real-time confocal system (Perkin Elmer) using a Nikon 1.3 NA PlanFluar objective lens. The pixel size was 136 nm2. Image analysis. For deconvolution and image reconstruction image stacks were processed on a SGI Octane workstation running Huygens (Scientific Imaging Leiden The Netherlands) and Imaris (Bitplane Zürich Switzerland) software. Semi-automated particle tracking was performed using macros and routines for NIHimage (http://rsb.info.nih.gov/nih-image/) and Interactive Data Language (IDL Research Systems Boulder Colorado USA). The NIHimage macros used for this work can be downloaded from http://www.embl-heidelberg.de/~rietdorf/nihmacros.html. To calculate virus-particle velocities subtraction images were automatically created for each time point of a time series by subtracting the preceding frame. Thus particles changing their position between frames were highlighted while non-moving components of the image were suppressed. Virus particle trajectories were obtained by subsequent maximum intensity projection from the subtracted images. Along these trajectories lines of grey value were read out for every image of a series and recombined into a new ‘time–space plot’64. Velocities are read as changes in grey value over time from the time–space plots. Velocities were confirmed by manual tracking. A similar approach was used to image virus particles on microtubules more clearly. The highlighted moving particle images were overlaid on the original images in red to facilitate virus tracking on microtubules. To enhance the contrast of the microtubule signal (essentially static compared with the virus particles) a weighted walking average of three successive frames was used. This ‘averaged’ microtubule image was then recombined with the highlighted moving virus particles.

Microscopic Technique
Confocal Microscopy

Cell Type(s)
3T3, 143TK– human osteosarcoma cells