Effects of silencing the expression of Rae1 or Nup98 on host and viral transcription in VSV-infected cells.

<p>HeLa cells were either not transfected or transfected with Rae1 siRNA (<b>A</b>), Nup98 siRNA (<b>B</b>) or non-targeting (NT) siRNA. At 72 hours post-transfection, cells were either mock or infected with recombinant wild-type (rwt) virus for 6 hours in the presence or absence of actinomycin D (ActD, 5 µg/ml). Cells were labeled with [³H] uridine for 30 minutes. Cells were lysed and RNA was precipitated using trichloroacetic acid, and acid precipitable radioactivity was measured. The graph represents host (ActD sensitive) and viral (ActD insensitive) RNA synthesis expressed as a percentage of total RNA synthesis in mock infected cells as illustrated in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002929#ppat-1002929-t001" target="_blank">Table 1</a>. The data shown are means ± standard deviation from five independent experiments.</p

Effect of silencing Rae1 or Nup98 expression on RNA synthesis in VSV-infected cells.

<p>HeLa cells transfected with the indicated siRNA were either mock-infected or infected with VSV in the presence or absence of actinomycin D. At 6 h postinfection, cells were labeled with ³H uridine for 30 min; RNA was acid-precipitated, and radioactivity was determined by scintillation counting. Data shown are mean ± s.d. for triplicate cultures from a representative experiment.</p>a<p>Abbreviations: ActD - actinomycin D; NT – non-targeting siRNA.</p

Gel filtration and sedimentation velocity analysis of complexes containing Rae1.

<p>(<b>A</b>) Cell lysates were chromatographed on a Superdex 200 column. Fractions were analyzed by immunoblots probed for Rae1 and Nup98. Arrows represent the fractions where standards of the indicated molecular weight eluted under the same conditions. The graph represents quantification of % Rae1 in each fraction normalized to total Rae1 eluting in all fractions. Vo indicates the void volume. (<b>B</b>) Column fractions from the same experiment as in (<b>A</b>) were incubated with wt GST-M protein (M) or GST (G) on glutathione beads for 1 hour. Bound fractions were analyzed by immunoblots probed for Rae1 and Nup98. (<b>C</b>) Cells were transfected with plasmid DNA encoding HA-Rae1. Lysates were chromatographed on a Superdex 200 column. Fractions were analyzed by immunoblots probed for HA. (<b>D</b>) Column fractions from (<b>C</b>) were incubated with wt GST-M protein (M) or GST (G) on glutathione beads for 1 hour. Bound fractions were analyzed by immunoblots probed for HA. (<b>E</b>) Cell lysates were subjected to sucrose gradient centrifugation. Fractions were collected from the top and probed for Rae1 (top panel) and Nup98 (bottom panel). Arrows represent fractions containing standards with the indicated s_20,w value subjected to the same conditions. (<b>F</b>) Sucrose gradient fractions from (<b>E</b>) were incubated with GST-M protein (M) or GST (G) on glutathione beads for 14 hours at 4°C. Bound fractions were analyzed by immunoblots probed for Rae1 and Nup98.</p

Effects of silencing the expression of Nup98 or Rae1 on complexes containing Rae1.

<p>HeLa cells were transfected with Nup98 siRNA (<b>A</b>), Rae1 siRNA (<b>C</b>), or non-targeting siRNA (<b>D</b>). 48 hours post-transfection, lysates were chromatographed on a Superdex 200 column. Fractions were analyzed by immunoblots probed for the indicated proteins. (<b>B</b>) and (<b>E</b>): Column fractions from (<b>A</b>) and (<b>D</b>), respectively, were incubated with GST-M protein (M) or GST (G) on glutathione beads for 1 hour, and bound fractions were analyzed by immunoblots probed for Rae1 and Nup98.</p

Effect of silencing Rae1 expression on the inhibition of host gene expression by M protein expressed in the absence of other VSV components.

<p>HeLa cells were either not transfected or transfected with Rae1 siRNA or non-targeting (NT) siRNA. After 48 hours, cells were re-seeded, then cotransfected with plasmid DNA encoding luciferase under the control of the SV40 promoter together with the indicated amounts of M mRNA. The amount of total RNA was kept constant by addition of yeast tRNA. At 24 hours post-transfection, cells were lysed and luciferase reporter activity was measured. The graph represents activity expressed as a percentage of average luciferase activity in the absence of M mRNA. Data shown are means ± standard deviation from three independent experiments.</p

Complexes of Vesicular Stomatitis Virus Matrix Protein with Host Rae1 and Nup98 Involved in Inhibition of Host Transcription

<div><p>Vesicular stomatitis virus (VSV) suppresses antiviral responses in infected cells by inhibiting host gene expression at multiple levels, including transcription, nuclear cytoplasmic transport, and translation. The inhibition of host gene expression is due to the activity of the viral matrix (M) protein. Previous studies have shown that M protein interacts with host proteins Rae1 and Nup98 that have been implicated in regulating nuclear-cytoplasmic transport. However, Rae1 function is not essential for host mRNA transport, raising the question of how interaction of a viral protein with a host protein that is not essential for gene expression causes a global inhibition at multiple levels. We tested the hypothesis that there may be multiple M protein-Rae1 complexes involved in inhibiting host gene expression at multiple levels. Using size exclusion chromatography and sedimentation velocity analysis, it was determined that Rae1 exists in high, intermediate, and low molecular weight complexes. The intermediate molecular weight complexes containing Nup98 interacted most efficiently with M protein. The low molecular weight form also interacted with M protein in cells that overexpress Rae1 or cells in which Nup98 expression was silenced. Silencing Rae1 expression had little if any effect on nuclear accumulation of host mRNA in VSV-infected cells, nor did it affect VSV's ability to inhibit host translation. Instead, silencing Rae1 expression reduced the ability of VSV to inhibit host transcription. M protein interacted efficiently with Rae1-Nup98 complexes associated with the chromatin fraction of host nuclei, consistent with an effect on host transcription. These results support the idea that M protein-Rae1 complexes serve as platforms to promote the interaction of M protein with other factors involved in host transcription. They also support the idea that Rae1-Nup98 complexes play a previously under-appreciated role in regulation of transcription.</p> </div

Effects of silencing the expression of Rae1 on host and viral protein synthesis.

<p>(<b>A</b>) HeLa cells were transfected with Rae1 siRNA or non-targeting (NT) siRNA. 72 hours post-transfection, cells were mock infected or infected with rwt virus for the indicated times and labeled with [³⁵S ] methionine for 10 min. Lysates from Rae1 siRNA cells or NT siRNA cells were analyzed by SDS-PAGE and phosphoimaging. Shown is a phosphoimage with the viral proteins indicated on the right. (<b>B</b>) Quantification of host protein synthesis expressed as a percentage of mock infected cells. Data shown are the means ± standard deviation of three separate experiments. (<b>C</b>) Quantification of viral protein synthesis expressed as a percentage of synthesis at six hours postinfection. Data shown are the means ± standard deviation of three separate experiments.</p

Effects of silencing the expression of Rae1 on mRNA expression in VSV-infected cells.

<p>Cells were transfected with Rae1 siRNA or NT siRNA, then mock-infected or infected with rwt virus. At 6 h postinfection, total RNA was isolated and analyzed using Affymetrix Human Genome U219 Array strips. Data shown are gene symbols of probe sets that were reproducibly decreased (<b>A</b> and <b>B</b>) or increased (<b>C</b> and <b>D</b>) by greater than 3-fold in VSV-infected versus mock-infected siNT cells (<b>A</b> and <b>C</b>) or siRae1 cells (<b>B</b> and <b>D</b>). The selection criteria were that the combined variance in the probe set in repeat experiments gave p<0.005 to minimize the false discovery rate. The probe sets, definition of gene symbols, and numerical data are provided in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002929#ppat.1002929.s004" target="_blank">Table S1</a>.</p

Interaction of M protein with Rae1 and Nup98 in nuclear chromatin-associated fractions.

<p>HeLa cells were lysed in hypotonic buffer and separated into nuclei and cytoplasm (Cyto). Nuclei were fractionated as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002929#ppat.1002929-Matunis1" target="_blank">[36]</a>. (<b>A</b>) Supernatant (S) and pellet (P) fractions were analyzed by immunoblots probed for the presence of Rae1, Nup98, and TATA-binding protein (TBP). (<b>B</b>) Supernatant fractions from (<b>A</b>) were incubated with GST-M protein (M) or GST (G) on glutathione beads. Bound fractions were analyzed by immunoblots probed for Rae1 and Nup98.</p

Specificity of interaction of M protein with Rae1 and Nup98.

<p>Recombinant wild type or mutant M protein GST fusion proteins or GST alone on glutathione beads were incubated for 1 hour with lysates from HEK 293 cells. Bound and unbound fractions were analyzed by immunoblotting and probed for Rae1 (<b>A</b>) or Nup98 (<b>B</b>). Arrow in unbound fraction in (<b>A</b>) indicates unrelated protein cross-reactive with Rae1 antibody (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002929#ppat.1002929.s001" target="_blank">Figure S1</a>).</p