Alphavirus production is inhibited in neurofibromin 1-deficient cells through activated RAS signalling

Abstract

Virus-host interactions essential for alphavirus pathogenesis are poorly understood. To address this shortcoming, we coupled retrovirus insertional mutagenesis and a cell survival selection strategy to generate clonal cell lines broadly resistant to Sindbis virus (SINV) and other alphaviruses. Resistant cells had significantly impaired SINV production relative to wild-type (WT) cells, although virus binding and fusion events were similar in both sets of cells. Analysis of the retroviral integration sites identified the neurofibromin 1 (NF1) gene as disrupted in alphavirus-resistant cell lines. Subsequent analysis indicated that expression of NF1 was significantly reduced in alphavirus-resistant cells. Importantly, independent down-regulation of NF1 expression in WT HEK 293 cells decreased virus production and increased cell viability during SINV infection, relative to infected WT cells. Additionally, we observed hyperactive RAS signalling in the resistant HEK 293 cells, which was anticipated because NF1 is a negative regulator of RAS. Expression of constitutively active RAS (HRAS-G12V) in a WT HEK 293 cell line resulted in a marked delay in virus production, compared with infected cells transfected with parental plasmid or dominant-negative RAS (HRAS-S17N). This work highlights novel host cell determinants required for alphavirus pathogenesis and suggests that RAS signalling may play an important role in neuronal susceptibility to SINV infection.

Figure 1. Characterization of alphavirus-resistant clone 9

(A) Detection of SINV genome in cells. Total RNA was prepared and used for RT-PCR with primers specific for amplification of a 638 nucleotide portion of the SINV nsP4 (polymerase) gene, which is required for genome replication. (B, C) Cross-virus resistance of clone 9. The data shown is representative of 2 or more independent experiments. Surviving cells were fixed with 10% formaldehyde and stained with crystal violet. (B) WT HEK 293 cells and clone 9 were infected with SINV-TE at an MOI of 0.1 PFU/cell or HJV at an MOI of 0.5 PFU/cell. Cells were fixed and stained on day 3 post infection. (C) WT HEK 293 cells and clone 9 were treated with IFN-α (200 U/ml) one day before RRV was added at an MOI of 0.1 PFU/cell. Cells were fixed and stained on day 5 post infection. (D) Production of progeny virus by clonal cell line 9. Clone 9 and WT HEK 293 cells were infected with SINV-83-GFP at an MOI of 0.1 PFU/cell. At the times indicated, supernatants were removed and titrated onto HEK 293 cells. The data shown is the mean±SD from 3 independent experiments.

Figure 2. Characterization of retroviral mutagenesis gene disruption in alphavirus-resistant clone 9

(A) Real-time RT-PCR analysis was performed by using total RNA derived from WT HEK 293 cells and clone 9 with primers specific to ube2m, nf1, lsm12 and rragc transcripts. Gene-specific transcript amounts were normalized to gapd levels. The data shown is the mean±SD from 2 independent experiments. ** indicates P≤0.001 and * indicates P≤0.05 compared to WT control. (B) Western blotting analysis of protein lysates from WT HEK 293 cells and clone 9 using α-UBE2M C-terminus, α-UBE2M N-terminus, and α-NF1 antibodies. Anti—beta-actin (α-ACTB) antibody was used to verify protein loading. Arrow indicates N-terminally truncated UBE2M protein. (C) Characterization of truncated transcripts in alphavirus-resistant clone 9. Analysis of the 5′ end of truncated ube2m and nf1 transcripts by 5′ RLM-RACE. Diagram of ube2m and nf1 gene structures and products identified in clone 9. Hatched boxes represent untranslated regions of the gene. Introns are not drawn to scale. Horizontal arrows show PCR products generated with gene-specific primers; vertical arrows indicate sites of provirus integration.

Figure 3. Effect of siRNA-mediated down-regulation of NF1 expression in WT HEK 293 cells on SINV-83-GFP production and cellular viability

(A) Down-regulation of NF1 protein expression. Western blot analysis of protein lysates from WT HEK 293 cells transiently transfected with NF1-specific siRNA compared with non-targeting control siRNA (siCONTROL), mock-transfected cells (Mock) and non-transfected cells (NT). Cell lysates were tested 2-5 days after transfection using α-NF1 antibody. Anti—beta-actin (α-ACTB) antibody was used to verify protein loading. (B, C) WT HEK 293 cells transiently transfected with NF1-specific siRNA were infected 2 days after transfection with SINV-83-GFP, MOI of 0.1 PFU/cell. The data shown is the mean±SD of triplicate cultures that is representative of 3 separate experiments. (B) Production of SINV-83-GFP by transfected cells. At the times indicated, supernatants were removed and titrated onto HEK 293 cells. ** indicates P≤0.05 and * indicates P≤0.1 compared to siCONTROL. (C) Cell viability was assessed 5 days post infection using an MTT-based assay. * indicates P<0.001 compared to siCONTROL.

Figure 4. Hyperactive RAS-signaling in alphavirus-resistance

(A) Hyperactive RAS-signaling in alphavirus-resistant clone 9. RAS activity in whole-cell extracts from clone 9 and WT HEK 293 cells. Cells were serum-starved for 5 h prior to cell lysis and analysis. The data shown is the mean±SD from 3 independent experiments. * indicates P≤0.001 compared to WT control. (B, C) Effect of constitutively active RAS expression on SINV-83 production in WT HEK 293 cells. HEK 293 cells transiently transfected with constitutively active (G12V) or dominant-negative (S17N) form of pcDNA3.1-HA-HRAS plasmid, parental plasmid (pcDNA3.1), or mock-transfected (Mock). (B) Western blot analysis of RAS proteins expression 24 h post transfection using anti-HA antibody (α-HA). Anti-beta-actin antibody (α-ACTB) was used to verify protein loading. (C) Production of SINV-83 by transfected cells. Transfected cells were infected 24 h post transfection with SINV-83, MOI of 0.1 PFU/ml. At the times indicated, supernatants were removed and titrated onto HEK 293 cells. The data shown is the mean±SD of triplicate cultures from 1 experiment that is representative of 3 separate experiments. ** indicates P≤0.01 and * indicates P≤0.05 compared to parental plasmid.