Subcellular localization of VSV M proteins.

<p>BHK-T7 cells were transfected for 2 h with pTM1 encoding VSV M1 (A-D), M2 (E-F) or M3 (G-H), and fixed 6 h later. Expression and localization of viral proteins were analyzed by immunofluorescence using specific monoclonal antibodies against M1 that also recognize M2 and M3, and the corresponding mouse secondary antibody conjugated to Alexa 488. Localization of M1 in intracellular membranes and dot-like structures at the nuclear envelope or at the cell surface is shown in panels A, B and C, respectively. Localization of M2 and M3 in intracellular compartments (E and G) or surrounding the nucleus (F and H) is shown. Images were acquired with an Axiovert microscope connected to a digital camera.</p

Effect of VSV M2 and M3 expression on nucleus-cytoplasm transport of mRNAs.

<p>BHK-T7 cells were transfected for 2 h with pTM1 empty (mock) or pTM1 encoding M1, M2 or M3 proteins. Cells were fixed 6 hpt and <i>in situ</i> hybridization with fluorescein-labeled oligo (dT) probe was carried out to detect cellular mRNAs. VSV M proteins were visualized by immunofluorescence using specific monoclonal antibodies against M1 (αM) and the corresponding mouse secondary antibody conjugated to Alexa 555. To-Pro3 was used as a nuclear marker. Images were acquired with a confocal microscope. Merged images are shown on the right.</p

Induction of vesicle budding from the plasma membrane by VSV M proteins.

<p>BHK-T7 cells were transfected for 2 h with pTM1 encoding M1 (A-F), pTM1 empty (G), M2 (H) or M3 (I). Cells were fixed 6 h after transfection and immunodetection of VSV M proteins was performed using specific monoclonal antibodies and the corresponding mouse-secondary antibodies coupled to gold particles. Cells were visualized with a transmission electron microscope. Arrows indicate sites of vesicle budding at the plasma membrane (A-E) where M1 protein is concentrated, as well as vesicles already released from the cells (F). Statistical analyses of the gold granules distribution was carried out by unpaired (two-tailed) Student <i>t</i>-test. p < 0.01 using the Stata Program Version 11.0.</p

VSV M proteins do not alter cell membrane permeability.

<p>(A) BHK-T7 cells were transfected with pTM1 empty plasmid or pTM1 constructs encoding M1, M2 or M3 proteins. As a positive control, cells were transfected with pTM1-2B (encoding poliovirus 2B viroporin). The medium was removed 2 h later and fresh DMEM containing 5% FCS was added. Cells were pre-treated 15 h after transfection with 0.5 mM of the translation inhibitor hygromycin B (HB) for 15 min. Then, cells were metabolically labelled with [³⁵S]Met/Cys for 45 minutes in the presence or absence of HB. Samples were processed by SDS-PAGE (17.5%), fluorography and autoradiography. (B) BHK-21 cells were mock transfected or electroporated with SV-derived mRNA replicons: Rep C, Rep C+M1 or Rep C+6K, obtained by <i>in vitro</i> transcription from their corresponding DNA templates. Cells were pre-treated with HB and metabolically labeled as indicated in (A). Numbers below each lane indicate the percentages of protein synthesis calculated by dividing the densitometric values for HB-treated cells by the values for untreated cells. A cellular protein band in mock transfected cells, or the band corresponding to the SV C protein in replicon transfected cells, was quantified by densitometric scanning, respectively. Bands corresponding to SV C and VSV M1 protein are indicated with arrows. Detection of α-tubulin served as loading control.</p

Cytotoxic effect mediated by expression of VSV M proteins.

<p>BHK-T7 cells were transfected with pTM1 empty (mock), pTM1-M1, pTM1-M2 or pTM1-M3 for 2 h. Cells were then washed and incubated in DMEM containing 5% FCS until they were fixed at 6, 18 and 24 h post transfection (hpt). Cell morphology was examined with a phase-contrast microscope.</p