Analysis of infectivity of RVFV virions obtained under conditions of HSP inhibition.

<p>Vero cells were pretreated for 2 hours with equivalent volumes of either DMSO (control) or 17-AGG prior to infection with the MP-12 strain of RVFV (MOI 0.1). Following infection, cells were washed with PBS and media containing either DMSO or 17-AAG was added back to the cells. Twenty-four hours later, culture supernatants were collected and viral RNA levels were analyzed by qRT-PCR to measure the amounts of released virus. The supernatant samples were also functionally analyzed by plaque assays as described in the Methods section, and the plaque data was normalized to the amount of released virus based on the q-RT-PCR results. Data are average values from three independent assays.</p

RVFV virion-associated host proteins represented in canonical entry pathways.

<p>RVFV virion-associated host proteins represented in canonical entry pathways.</p

Ingenuity Analysis of all proteins identified in the study.

<p>(A) The complete data set was used to create the 69 node (largest discovered by our analysis) interaction network depicted in the figure. The nodes colored in blue designate chaperones and those in green designate cytoskeletal components. Solid lines represent proteins with known direct interactions, while indirect interactions are represented with broken lines. (B) The bar graph depicts the ten most enriched canonical pathways as a function of the enrichment score for the pathway (−Log (p-value)) and the number of molecules (proteins) represented within the pathway.</p

Protein-Protein interaction networks in RVFV complexes II and IV.

<p>Proteins in native complexes II and IV are involved in multiple interactions with other proteins in their respective complexes. Interactions were mapped using the Ingenuity knowledge base (orange edges), and the STRING database (red edges). Black edges represent interactions annotated by both sources. (A) Native complex II: <u>ENO1 </u>α-enolase, <u>PKM2</u> pyruvate kinase muscle, <u>ACTN1 </u>actinin-α1, <u>ACTB</u> beta actin, <u>CCT4</u> chaperone containing TCP1, <u>IGHA1</u> Immunoglobulin heavy chain α, <u>ANXA2 </u>annexin A2, <u>TLN1 </u>talin 1, <u>VCP </u>valosin-containing (TER ATPase), <u>HBB</u> hemoglobin beta, <u>HSP90AB1</u> HSP 90 α class B mem 1, <u>FLNA </u>filamin A, <u>FN1</u> fibronectin1, <u>PIGR</u> polymeric immunoglobulin receptor, <u>HSPA5</u> HSP 70, protein 5 (GRP-78), <u>HBA</u> hemoglobin alpha, <u>F2 </u>coagulation factor II, <u>PABPC1 </u>polyA binding protein, <u>A2M </u>α-2-macroglobulin, <u>ALB</u> albumin. (B) Native complex IV: <u>LGALS8</u> galectin-8, <u>CD151 </u>CD151, <u>ITGA3</u> Integrin α 3, <u>ITGB1</u> Integrin β 1, <u>ITGA1</u> Integrin α 1, <u>HBB</u> hemoglobin beta, <u>ACTN4 </u>actinin-α4, <u>HBA</u> hemoglobin alpha, <u>HSP90AA1</u> HSP 90 α class A mem 1, <u>FLNC </u>filamin C, <u>HSPA5</u> HSP 70, protein 5 (GRP-78), <u>ALB</u> albumin, <u>APOA1 </u>apolipoprotein A1, <u>FTH1</u> ferritin polypeptide 1.</p

Effects of siRNA silencing of host chaperones on RVFV levels.

<p>(A) Silencer Select siRNA library (Life Technologies) was used to serially assess the importance of identified chaperones in RVFV infection. HeLa cells were transfected with siRNA molecules targeting chaperone proteins, negative control targets (GAPDH, scrambled siRNA) or “no siRNA” controls. Following 48 hr incubation, cells were infected with the MP-12 strain of RVFV and the extent of viral infection assessed by high content imaging, as described in the Methods section. The down-regulation of chaperone proteins indicated in the figure resulted in statistically significant effect on viral infection when compared to the scrambled siRNA, as indicated by the asterisk symbols (P<0.05). Negative controls corresponding to GAPDH, scrambled siRNA and no siRNA control did not impact RVFV infection. The experiment was repeated in three independent trials with samples assayed in duplicate. The displayed data is a representative of the entire sample set. (B) The effects of siRNA treatments on RVFV titers were also measured using qRT-PCR analysis. For each target HSP with a duplicate siRNA set, the siRNA construct with the highest knockdown efficiency was analyzed. HeLa cells were transfected with the siRNA constructs and after 48 hours the transfected cells were infected with the MP-12 strain. The culture supernatants were analyzed 24 hours after infection. Asterisks designate significant changes in viral levels (P<0.05).</p

Effects of host chaperone inhibitors on RVFV levels and timing of HSP effects.

<p>(A) Vero cells were pretreated for 2 hours with equivalent volumes of either DMSO (control), 17-AGG (10 µM), BAPTA (10 µM), or KNK437 (10 µM), prior to infection with the MP-12 strain of RVFV (MOI 0.1). Following infection, cells were washed with PBS and media containing either DMSO or each of the compounds was added back to the cells. Twenty-four hours later, supernatants were collected and viral RNA was measured by qRT-PCR as described in the Methods section. Percent viral RNA is expressed as the percent of the DMSO control. The viability of the cells in the presence of 10 µM inhibitor or in DMSO (vehicle) was assessed by MTT assay, as described in the Methods section. Data are average values from three independent assays. (B) Time of addition studies were performed using 17-AAG and BAPTA-AM inhibitors to analyze the timing of HSP effects during RVFV infection. Vero cells were treated with HSP inhibitors, or with vehicle only (DMSO), at the indicated time points relative to MP-12 infection (MOI 0.1). Following infection, cells were washed with PBS and media containing either DMSO or each of the compounds was added back to the cells. qRT-PCR was performed to measure viral RNA levels in the supernatants collected 24 hours post-infection. Data are average values from three independent assays. (C), and (D) Viral inhibition following HSP inhibitor treatment was measured in real time. One hour prior to infection with rMP12-rLuc (MOI 0.1), HepG2 cells were treated with HSP inhibitors 17-AAG, or BAPTA-AM, or a combination of both inhibitors. Controls included no inhibitor treatment and no infection [Untreated (UT)], no inhibitor treatment but with infection using either the regular MP-12 strain that has no luciferase expression (MP12; UT) or with the luciferase expressing rMP12-rluc strain (Mp12-Luc; UT), and treatment with vehicle only, either without any infection (DMSO) or with infection using the rMP12-rluc strain (Mp12-Luc; DMSO). All samples were analyzed either at 4 hours p.i. (C) or at 8 hours p.i. (D), using the Renilla Luciferase assay. Data are average values from three independent assays. Asterisks designate significant changes in luminescence levels (P<0.05).</p

Evaluating the purity of viral preparations by Western blot analysis.

<p>Vero whole cell lysate (positive control) along with detergent solubilized RVFV virions were resolved by SDS PAGE, transferred to PVDF membranes and analyzed by Western blot. (A) Blots probed with antibodies against the RVFV glycoprotein Gn (G2) demonstrate that this protein is only present in solubilized virus. (B) The mitochondrial protein Cytochrome C Oxidase Subunit VIb is detected in whole cell lysate but not in purified virions. (C) The plasma membrane protein Annexin A1 is detected in whole cells but not in the viral preparation.</p

siRNA silencing of target host chaperones.

<p>Knockdown efficiencies of Silencer Select siRNA library (Life Technologies) were quantified, using qRT-PCR to measure the transcript levels of the respective target genes. HeLa cells were transfected with siRNA molecules targeting chaperone proteins, or negative control targets (GAPDH, scrambled siRNA), and RNA samples were purified 48 hours post transfection for analysis by qRT-PCR. Analysis was performed for both pre-validated single siRNA constructs and the constructs in the duplicate sets that represent independent siRNAs targeting the same gene and had not been previously validated. siRNA constructs that showed significant down-regulation compared to siRNA control are designated by asterisk (P<0.05). Data are average values from three independent assays.</p

Blue native PAGE separation of high molecular weight protein complexes in solubilized RVFV virions.

<p>Purified virions were solubilized under native conditions by diluting virus into a buffer containing 1% Trition X-100 and removing insoluble material by centrifugation. Sample buffer containing Coomassie Blue G-250 was then added to supernatants and samples were subjected to Blue native polyacramide gel electrophoresis on 4–12% gradient gels. Native protein standards (thyroglobulin 669 kDa, apoferritin 443 kDa, beta amylase 200 kDa, alcohol dehydrogenase 150 kDa, and bovine serum albumin 66 kDa) were resolved in adjacent lanes. The position of the fastest migrating band for each protein standard is indicated on the figure. Four high molecular weight protein species were observed in solubilized virions; they are labeled I–IV on the figure.</p