Antiviral activity and RNA polymerase degradation following Hsp90 inhibition in a range of negative strand viruses

Abstract

We have analyzed the effectiveness of Hsp90 inhibitors in blocking the replication of negative-strand RNA viruses. In cells infected with the prototype negative strand virus vesicular stomatitis virus (VSV), inhibiting Hsp90 activity reduced viral replication in cells infected at both high and low multiplicities of infection. This inhibition was observed using two Hsp90 inhibitors geldanamycin and radicicol. Silencing of Hsp90 expression using siRNA also reduced viral replication. Hsp90 inhibition changed the half-life of newly synthesized L protein (the large subunit of the VSV polymerase) from >1 h to less than 20 min without affecting the stability of other VSV proteins. Both the inhibition of viral replication and the destabilization of the viral L protein were seen when either geldanamycin or radicicol was added to cells infected with paramyxoviruses SV5, HPIV-2, HPIV-3, or SV41, or to cells infected with the La Crosse bunyavirus. Based on these results, we propose that Hsp90 is a host factor that is important for the replication of many negative strand viruses.

Figure 1. Effect of Hsp90 inhibition of VSV replication

A) viral titers at 12hpi from cells infected with VSV at an MOI=0.01 and treated with increasing concentrations of geldanamycin. B) growth curve of VSV in the presence (△) or absence (■) of geldanamycin (5μM) at high MOI (=10) and C) growth curve at low MOI (=0.1). D) growth curve of VSV in the presence (△) or absence (■) of radicicol (5μM). Virus titers for all panels represent the average of three different experiments. E) Western blot analysis of 25ug of lysate from cells that were mock-transfected, transfected with GAPDH targeting siRNA or transfected with Hsp90 targeting siRNA. Top image shows Hsp90 levels, middle panel shows GAPDH levels, bottom panel shows actin as a loading control. F) growth curve of mock-transfected and siRNA-treated cells infected with VSV at an MOI of 0.01. Error bars reflect standard deviation for three experiments.

Figure 2. Effect of geldanamycin on viral protein synthesis

A) Schematic of experimental procedure showing times of geldanamycin treatment after viral infection (top) and total times of geldanamycin treatment (right). B) Phosphorescence image of cell lysates from cells that were infected with VSV and treated with geldanamycin as described, followed by labeling with ³⁵S Methionine for 1 hour.

Figure 3. Assembly of VSV in geldanamycin treated cells

Cells were infected with VSV for 5 hours, and then proteins were radiolabeled and followed using a one hour pulse and one hour chase protocol. Upon addition of radioactive methionine, different concentrations of geldanamycin were added to individual samples. Following a 1 hour chase in the presence of drug A) intracellular radiolabeling of protein was determined by SDS-PAGE electrophoresis phosphorescence imaging. B) The amount of newly synthesized protein that was incorporated into the virus was determined by virion purification from the media followed by SDS-PAGE and phosphorescence analysis.

Figure 4. Effect of geldanamycin on viral protein stability

A) BHK cells infected with VSV for 5 hours were starved for methionine, and then protein synthesis and stability were determined by pulse chase analysis (see materials and methods). Results were determined by SDS-PAGE and phosphorescence imaging. Viral proteins are identified to the right of the gel. Levels of protein remaining are quantities for viral L (B) and M (C) proteins presence (△) or absence (■) of 5μM geldanamycin. Values represent the average of three separate experiments +/- standard deviation

Figure 5. Geldanamycin destabilization of VSV L protein requires proteosome function

A) BHK cells were infected with VSV, and 5hpi left untreated, treated with geldanamycin, or treated with both geldanamycin and proteosome inhibitor. Pulse-chase results for 10 minute and 30 minute chases are shown. B) Stability of L protein in the proteins presence (△) or absence (■) of 5μM geldanamycin and in the presence of geldanamycin and a proteosome inhibitor (◆, dotted line). Levels are expressed as a percentage of the signal that was present at the 10 minute chase. Values are the average of three separate experiments +/- standard deviation.

Figure 6. Effect of Geldanamycin on Paramyxovirus growth and L stability

A and B) Cells were infected at an MOI of 0.05 with rSV5-GFP or HPIV-2 and incubated with or without 0.5 μM Geldanamycin. At 72 hpi, virus titers were determined by plaque assay (A) or cells were examined for GFP expression (B). Results are representative of three independent experiments. C and D) Cells that were mock-transfected or transfected with either GAPDH targeting siRNA or Hsp90 targeting siRNA were infected with SV5-GFP at an MOI of 0.05. C) shows SV5-GFP growth curve (done in triplicate) untransfected (■) GAPDH siRNA transfected (◆), and Hsp90 siRNA transfected (■), D) shows representative images showing cell spread of the virus in different transfection conditions.

Figure 7. Hsp90 inhibition destabilizes newly synthesized L proteins from multiple paramyxoviruses

A) Cells were mock infected (M lane) or infected at an MOI of 10with rSV5-GFP. At 13 h pi, cells were treated for 1 hr with or without 1μM Geldanamycin. Cells were radiolabeled for 20 min with ³⁵S-amino acids and incubated in nonradioactive media for the indicated times before lysis and analysis by immunoprecipitation with antibodies specific for NP, P or L. Geldanamycin was included at 1 μM during radiolabeling and chase periods. B) Stability of L proteins. Cells were mock infected or infected at an MOI of 10 with SV5, HPIV-2, SV41 or HPIV-3. 13hpi, cells were treated with 1μM Geldanamycin, radiolabeled for 20 minutes and lysed (Pulse) or chased using unlabeled methionine for 1 hour (Chase). Following cell lysis, samples were immunoprecipitated using L-specific antibodies. C) Experiments similar to B were carried out, but 1μM radicicol was used instead of geldanamycin

Figure 8. Geldanamycin inhibition of La Crosse virus replication

A) La Crosse virus titers at 24hpi from cells infected at an MOI=0.01 and increasing concentrations of geldanamycin at time of infection. B) Light microscope images of cells infected with La Crosse virus in the absence (left) and presence of 0.5uM geldanamycin. C) Protein synthesis and stability of La Crosse L and G1 proteins. 16hpi with Lacrosse virus at an MOI of 1, cells were labeled with ³⁵S-methionine for 20 min and then lysed (P) or incubated in non-radioactive media for 1 hour. Cells were then lysed, and incorporation of radioactivity into viral proteins was determined by SDS-PAGE followed by phosphorescence imaging.