1. Volume 6, Issue 5, Pages 1243-1252 (November 2000)
Vesicular Stomatitis Virus Matrix Protein Inhibits Host Cell Gene Expression by Targeting the Nucleoporin Nup98 
Cayetano von Kobbe, Jan M.A. van Deursen, João P. Rodrigues, Delphine Sitterlin, Angela Bachi, Xiaosheng Wu, Matthias Wilm, Maria Carmo-Fonseca, Elisa Izaurralde 
Molecular Cell 
Volume 6, Issue 5, Pages (November 2000)
DOI: /S (00)

2. Figure 1
The N-Terminal Domain of VSV M Is Sufficient for Nuclear Export Inhibition
(A) Purified recombinant proteins indicated above the lanes were injected into Xenopus oocyte nuclei together with a mixture of radiolabeled RNAs. This mixture consisted of DHFR mRNA, histone H4 mRNA, U1ΔSm, U5ΔSm, U6Δss, and human initiator methionyl tRNA. RNA samples from total oocytes (T), cytoplasmic (C), and nuclear (N) fractions were collected 3 hr after injection, or immediately after injection (t0, lanes 1–3) and analyzed on 8% acrylamide/7 M urea denaturing gels.
(B) Purified recombinant proteins indicated above the lanes were injected into Xenopus oocyte cytoplasm. Following a 1 hr incubation, the mixture of labeled RNAs described in (A) was injected into oocyte nuclei. Export was allowed to take place for 3 hr. Symbols are as in (A).
(C) Xenopus laevis oocyte nuclei were injected with recombinant M and a mixture of in vitro transcribed 32P-labeled RNAs consisting of U6Δss RNA, U1ΔSm RNA, and Ad-CTE pre-mRNA. RNA samples were collected 3 hr after injection or immediately after injection (t0) as indicated. Products of the splicing reaction were resolved on 10% acrylamide/7 M urea-denaturing gels. The mature products of the splicing reaction are indicated diagrammatically on the left of the panel. The closed triangle represents the CTE. In all panels, one oocyte equivalent of RNA, from a pool of 10 oocytes, was loaded per lane.
Molecular Cell 2000 6, DOI: ( /S (00) )

3. Figure 2
Substitution of M Residues 52–54 by Alanines Abolishes Its RNA Export Inhibitory Activity
(A) Positions of the alanine scan mutants are shown on the primary amino acid sequence of VSV M. Residues important for the inhibitory effect of M in RNA export are located around position 52. The position of the M51R mutation is indicated by a circle while the APPPY motif implicated in budding is underlined.
(B) Purified recombinant M mutants indicated above the lanes were coinjected into Xenopus oocyte nuclei with the mixture of radiolabeled RNAs described in Figure 1A. RNA samples were collected 3 hr after injection, or immediately after injection (t0, lanes 1–3), and analyzed as indicated in Figure 1A.
(C) Xenopus oocyte nuclei were injected with zz-M or zz-M(D). Protein samples from total oocytes (T), cytoplasmic (C), and nuclear (N) fractions were collected 4.5 hr after injection, or immediately after injection (t0), and analyzed by Western blot.
(D) Human 293 cells were transfected with a mixture of plasmids encoding β-gal, CAT, and either GFP alone or C-terminally fused to M or M(D). Cells were collected 36 hr after transfection and β-gal or CAT activity was determined. Data from three separate experiments were expressed as the percentage of the activities measured when GFP alone was coexpressed. The data are means ± standard deviations. Similar results were obtained in experiments performed with different preparations of plasmid DNA. Protein expression levels were determined by Western blot using anti-GFP antibodies.
Molecular Cell 2000 6, DOI: ( /S (00) )

4. Figure 3 A Fraction of VSV M Localizes to the Nuclear Rim
(A–H) HeLa cells were transfected with pEGFPN3 plasmid derivatives expressing GFP fusions of M or the M(D) mutant. In (B), (D), (F), and (H), HeLa cells were extracted with digitonin prior to fixation. In (C), (D), (G), and (H), HeLa cells expressing M-GFP or M(D)-GFP were double labeled with the monoclonal antibody 414 directed against nucleoporins. Bar, 10 micrometers.
Molecular Cell 2000 6, DOI: ( /S (00) )

5. Figure 4 Identification of Nup98 as a Cellular Target of VSV M
(A) Solubilized nuclear envelope (NE) proteins from HeLa cells were incubated with IgG-Sepharose beads coated with zz-tagged M or the M(D) mutant. After extensive washes, bound proteins were eluted stepwise with 500 mM and 1 M MgCl2 as indicate above the lanes. One half of the eluted fractions was analyzed by SDS-PAGE followed by silver staining. Lane 1 shows 1/200 of the input fraction. Lanes 7 and 8 show proteins eluted from the M or M(D) coated beads with 1 M MgCl2 when the HeLa extracts were omitted. The asterisk indicates the position of Nup98.
(B) Samples from a similar experiment described in (A) were analyzed by Western blot using a polyclonal antibody directed against Nup98. One tenth of the input and one half of the eluted fractions were analyzed. The lower panel shows that tubulin can be selected on beads coated with M or M(D) when HeLa S-100 extracts are used.
(C) Recombinant purified zz-M (lanes 1–4) or zz-M(D) mutant (lane 5) were immobilized on IgG-Sepharose beads and incubated with solubilized NE proteins (lanes 1–5). In lanes 2 and 3, WGA was included in the binding reactions. In lanes 3 and 4, N-acetylglucosamine (GlcNac) was included. After incubation and extensive washes, bound proteins were eluted with SDS sample buffer and analyzed by Western blot using anti-Nup98 antibodies.
Molecular Cell 2000 6, DOI: ( /S (00) )

6. Figure 5 VSV M Interacts with the FG Repeat Domain of Nup98
(A) Full-length Nup98 and fragments 222–920 or 66–515 were expressed in 293 cells as GST fusions. Cells were lysed with 1% Triton X-100, and the presence of each protein in the lysates was detected by Western blot using a polyclonal anti-GST antibody. Schematic representation of Nup98: numbers indicate the amino acid position. The vertical bars show the position of the FG repeats. The black rectangle represents the GLEBS-like motif, and the branches the position of putative glycosylation sites.
(B) Lysates from human 293 cells expressing full-length Nup98 or fragments 222–920 or 66–515 were incubated with IgG-Sepharose beads coated with purified zz-M. Binding reactions were performed in the presence (+) or absence (−) of WGA, as indicated above the lanes. After extensive washes, bound proteins were eluted with SDS sample buffer. One tenth of the inputs (lanes 1, 4, and 7) and one quarter of the bound fractions (lanes 2, 3, 5, 6, 8, and 9) were analyzed by Western blot using anti-GST antibodies.
(C) Nup98 fragment 66–515 fused to GST was expressed in either 293 cells or E. coli. The corresponding cell lysates were incubated with IgG-Sepharose beads coated with zz-M or the M(D) mutant as indicated above the lanes. Binding to zz-M coated beads was performed in the presence (+) or absence (−) of WGA. Note that E. coli lysates were supplemented with lysates prepared from nontransfected 293 cells.
Molecular Cell 2000 6, DOI: ( /S (00) )

7. Figure 6 Nup98 Is the Major Binding Site for VSV M at the NPC
(A–F) Wild-type or Nup98−/− mouse cells were transfected with pEGFPN3-M. Nup98−/− cells were also cotransfected with pEGFPN3-M and a plasmid encoding Nup98 (C and D). The GFP signal was detected throughout the nucleoplasm and cytoplasm (A, C, and E). A nuclear rim staining was also detected in the wild- type cells (A) or the knockout cells coexpressing Nup98 (C). In (B), (D), and (F), transfected cells were extracted with Triton X-100 prior to fixation.
(G) Schematic representation of the transactivation assay.
(H) Wild-type or Nup98−/− cells were cotransfected with a mixture of plasmid DNA encoding the luciferase reporter, a Gal4-VP16 fusion protein, and VSV M. The data from three separate experiments were quantitated and expressed as fold inhibition relative to the luciferase activity in the absence of M. The data are means of three experiments ± standard deviations. M expression was analyzed by Western blot.
Molecular Cell 2000 6, DOI: ( /S (00) )

8. Figure 7
VSV M Inhibits Host Cell Gene Expression Primarily by Blocking Nuclear Export
(A–D) HeLa cells expressing wild-type M or mutant M(D), C-terminally fused to GFP, were double labeled for poly(A) RNA.
(E) Oocytes were injected with U1ΔSm gene followed by recombinant M or M(D) proteins and α-32P-GTP as indicated diagrammatically on the top panel. RNA was analyzed as in Figure 1. Bands indicated by asterisks on the edge of the figure correspond to endogenous transcripts.
Molecular Cell 2000 6, DOI: ( /S (00) )