Figure 1.
Schematic representation of the recombinant SeV genomes.
1. Genes encoding N, P, M, F, HN and L are flanked by genomic promoter (GP) and complement of anti-genomic promoter (AGPc). 2. Blow up of the F transcription unit as present in wt SeV genome, flanked by the start/stop transcription signals(). F ORF ecto-domain (in grey), trans-membrane (TM in red) and cytoplasmic (ct in green) domains are outlined, with the TYTLE motif. 3. Outline of the F ORF as present in rSeV-F5A where the TYTLE motif is replaced by 5 alanines (5A). 4. Outline of the F gene as present in rSeV-Fgfpt, with insertion in the 5′ UTR of a sequence derived from the green fluorescent protein as target of a specific siRNA (gfpt). 5. Outline of rSeV-HA-F/Fgfpt carrying an additional F gene inserted between the M and F genes. The additional wt-F ORF harbours an HA tag inserted at position aa 405 (HA tag in blue, see also figure S1). The endogenous F gene harbours the gfpt sequence. 6. rSeV-HA-F5A/Fgfpt. As in 5., except that the TYTLE motif of the additional HA-F ORF harbours the TYTLE/5A mutation. 7. rSeV-HA-F5Amvpt/Fgfpt. As in 6., except that a sequence derived from the measles virus P gene (mvpt) has been inserted in the 5′UTR of the additional F gene as target for a specific siRNA. More details about the rSeV's can be found in Materials and Methods and about the F ORF and its modifications in supplemental figure S1. Only highlighted parts (white on black) of the virus denominations were used for the annotation of the viruses in the figures and in the text.
Figure 2.
F-M interactions and movement dependency.
A. Outline of recombinant SeV's harbouring an additional transcription unit expressing the F cytoplasmic tail (ct, green) in its wt (Fctwt) or mutated (Fct5A) configuration fused to a peptide containing a biotinylation site (Biotp, yellow). Corresponding constructs, harbouring the HN cytoplasmic tail (ct, blue) are equally shown, carrying the wt sequence (HNctwt) or a mutation in the SYWST motif (HNctm, [11]) Fgfpt: as in figure 1. Note that the endogenous wt-F is open to conditional suppression by α-gfpt siRNA. B. α-gfpt siRNA expressing MDCK were infected with the indicated viruses in medium supplemented with 1 mg/ml of biotin. Thirty hours post infection cellular extracts were prepared and incubated with streptavidin agarose resins to pull down Fct and HNct fused to Biotp. Upper panel: Western blot analysis recording cellular levels of M (CE). Lower panels: Western blot analysis of the pull down samples probed with α-M, α-Fct or α-HNct (pull down). C. MDCK cells expressing or not α-gfpt siRNA (+/−) were infected with rSeV-Mgfpt, a recombinant virus expressing a M protein open to conditional suppression [15]. The infected cells were either grown in Petri dishes for cellular extract preparation or seeded on coverslips for confocal imaging. Upper panel: Western blot analysis of the cellular extracts prepared 36 hours post infection using an antibody cocktail shown in figure 3 to document M protein suppression. Lower panels: Confocal images of HA-F obtained before (Surface IF) or after (Total IF) cell permeabilization with α-F as primary antibody.
Table 1.
Summary of recombinant virus properties.
Figure 3.
TYTLE/5A mutation of F is detrimental for SeV particle rescue.
A. Coomassie blue stained PAGE analysis of virus purified from egg allantoic fluids. Lanes P2, P3, P4 (HA-F5A/Fgfpt), 2nd to 4th egg to egg serial passages of rSeV-HA-F5A/Fgfpt rescue. Lane P2 (HA-F/Fgfpt), 2nd passage of rSeV-HA-F/Fgfpt rescue. B. Western blot analysis of MDCK cellular extracts obtained 24 hours after mock infection (Mock), infections with rSeV-HA-F/Fgfpt (HA-F/Fgfpt) or rSeV-HA-F5A/Fgfpt (HA-F5A/Fgfpt) in conditions of suppression or not of the endogenous wt-F proteins (+/− α-gfpt siRNA). α-Fct : anti Fct antibody. α-HA: anti HA tag antibody. C. The cDNA fragment containing the HA-F5A gene was purified after MluI digestion of the rSeV-HA-F5A/Fgfpt full length cDNA clone and fully sequenced. D. The viral genomic RNA present in rSeV-HA-F5A/Fgfpt infected cells was reverse transcribed using a primer of positive polarity in the M gene. The RT product was amplified using a primer of negative polarity in HA tag sequence. The RT-PCR product was then purified and fully sequenced. Arrows in C. and D. indicate the G to T substitution position leading to introduction of a STOP at position 37 in the F ORF (see also figure S1).
Figure 4.
Rescue and analysis of rSeV-HA-F5Amvpt/Fgfpt virus.
A. BSRT7 cells constitutively expressing or not the α-mvpt siRNA (+/−) were used in rescue protocols with rSeV-HA-F/Fgfpt or rSeV-F5Amvpt/Fgfpt full length cDNA. Western blot analysis of virus particles purified form these supernatants after 3 passages (P3) for rSeV-HA-F/Fgfpt (HA-F/Fgfpt) or after 3, 4 and 5 passages for rSeV-F5Amvpt/Fgfpt (P3,P4,P5, HA-F5Amvpt/Fgfpt). α-HNSDS, α-FSDS, α-NSDS, α-MSDS: antibody preparations raised against the SDS denatured proteins. α-Fct, α-HA: as in figure 2. B. Cellular extracts of BSRT7 cells, constitutively expressing or not the α-mvpt siRNA (+/−) and infected with rSeV-HA-F/Fgfpt or rSeV-F5Amvpt/Fgfpt virus preparations obtained as shown in (A), were analysed by Western blots using the indicated antibody preparations. Actin: used as loading control. C. BSRT7 cells infected as shown in (B) were radio-labelled with 35S methionine and cysteine. Purified virus produced in the supernatants were directly analysed by PAGE and detected by autoradiography (upper panel) or detected by Western blots using α-Fct (lower panel). D. Cellular extracts of MDCK cells constitutively expressing or not α-gfpt siRNA (+/−) and infected with the virus preparations described in (B) plus the rSeV-Fgpt virus were analysed by Western blots using the mixed antibody preparations indicated in (B, upper panel) for the upper panel, α-HA for the middle panel and α-actin for the lower panel.
Figure 5.
Biochemical characterization of the HA-F5A protein.
A. BSRT7 cells expressing or not α-mvpt siRNA (+/−) were infected with the indicated viruses. At 24 hours post infection, cells were biotinylated in situ and a fraction of cellular extracts was analysed directly by Western blotting (left panel). The remaining was submitted to streptavidine bead pull down before analysis (right panel). Western blots were probed with α-Fct and α-N. B. Infections of MDCK cells, expressing or not α-gfpt siRNA. Forty hours post infection total cell immunoprecipitations were performed using or not α-HA antibodies (+/−). Immune-precipitates were analysed by Western blot using α-Fct. Cellular inputs of this experiment can be seen in figure S5. C. Infections of MDCK cells expressing α-gfpt siRNA were performed with the indicated viruses. Sixteen hours post infection, the infected cells were pulse-chase radiolabelled. HA-F proteins were immunoprecipitated using α-HA and then treated or with Endo-H (Endo-H, +/−). Digested samples were analysed by PAGE and detected by autoradiography. HA-Fi (○): initial HA-F recovered at the end of the pulse and not treated with Endo-H. HA-Fs (*): sensitive to Endo-H digestion. HA-Fr (•): resistant to Endo-H and (□): shows lack of resistant HA-F5A.
Figure 6.
Subcellular localization of the wt-HA-F and HA-F5A in the context of infections.
MDCK cells constitutively expressing α-gfpt siRNA were grown on coverslips and infected with rSeV-HA-F/Fgfpt and rSeV-HA-F5Amvpt/Fgfpt or kept mock infected. Twenty-four hours post infection, cells were submitted to (A.) total immunofluorescence (Total IF) or (B.) surface immunofluorescence (Surface IF) protocols. α-HA : primary antibody for both protocols. C. and D. as in (A.), except, α-calnexin (C.) or α-GPP130 (D.) primary antibodies were added with α-HA. DAPI: nuclei staining. Merged channel images are presented for convenience. Separate channel images for C are presented in figure S4.
Figure 7.
Statistical analysis of F, HN and M distribution in infected cells.
Total immunofluorescence confocal images of rSeV-HA-F/Fgfpt (HA-F/Fgfpt) and rSeV-HA-F5Amvpt/Fgfpt (HA-F5A/Fgfpt) infected α-gfpt siRNA expressing MDCK cells as those presented in figures 6 and 8 were analysed through a Metamorph program presented in supplemental figure S3, designed to quantify the fractions of the proteins HA-F, HN and M at the cell border or in the inner compartment. n = x: refers to the number of cells involved. Graphical representation of the data was executed by Graph Pad Prism 6 software which also provided the statistical calculation of the unpaired two-tailed Student t test. Underlined vertical numbers = numerical averages.
Figure 8.
Subcellular localization of the HN and M proteins in the context of wt-HA-F or HA-F5A.
As in figure 6A, but with the use of primary antibodies α-HN in (A) and (B) or α-M in (C) added with α-HA in all the panels.
Figure 9.
Possible model depicting formation of the viral assembly complex dependent on F.
Only the viral membrane proteins F, HN and M are considered for simplicity (see Text for further explanations). A. Wild type F interacts with a fraction of M at the level of the ER membranes. This F-M protein complex is transported to the plasma membrane in a TYTLE (small green rectangle) dependent manner. At the cell surface (PM), F-M defines a nucleation site for more M condensation (a fraction of M positioned at PM without F interaction) and for HN recruitment (likely through M). Nucleocapsids (not shown) may now bind to a stable assembly complex made of the 3 membrane viral proteins leading to virus particle budding. B. When F is mutated (red rectangle), F-M complex remains in ER due to the absence of the TYTLE motif. Absence of assembly nucleation site at PM results in disorganization of M and HN patterns at PM and stable assembly complex capable of binding nucleocapsids is not formed leading to lack of virus particle production.