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. Author manuscript; available in PMC: 2012 Feb 16.
Published in final edited form as: Virology. 2008 May 15;377(1):133–142. doi: 10.1016/j.virol.2008.03.025

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

Olga A Kolokoltsova 1, Aaron M Domina 1,2, Andrey A Kolokoltsov 3, Robert A Davey 3,4, Scott C Weaver 3,5,6, Stanley J Watowich 1,4,6,§
PMCID: PMC3280685  NIHMSID: NIHMS57242  PMID: 18485440

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 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.

Introduction

Alphaviruses are arthropod-borne, enveloped, positive sense, single stranded RNA viruses in the Togaviridae family. The Alphavirus genus includes potential biological weapons [e.g. Western (WEEV), Eastern (EEEV), and Venezuelan equine encephalitis viruses (VEEV)] and public health threats [e.g. Chikungunya, Sindbis, Ross River (RRV), and Barmah Forest viruses]. Although aspects of alphavirus assembly, RNA replication, virus binding, and entry have been studied (Strauss and Strauss, 1994), no licensed human vaccine or effective therapeutics are available to combat alphavirus infection (Nagata et al., 2005; Paessler et al., 2006; Phillpotts et al., 2005; Reed et al., 2005; Rulli et al., 2005; Schoepp, Smith, and Parker, 2002). Elucidating virus-host interactions essential for alphavirus pathogenesis should provide insights to help develop novel therapeutics and treatments.

Alphavirus-resistant clonal cell lines were generated from virus-susceptible cells using a combination of insertional mutagenesis and virus selection. A similar approach was previously utilized to identify cellular determinants of susceptibility to SINV infection (Jan, Byrnes, and Griffin, 1999). In that study, Chinese hamster ovary (CHO) cells mutagenized by retroviral insertion were selected for survival following infection with SINV. However, only partially virus-resistant clones were generated, of which the most resistant cell line was shown to lack surface heparan sulfates leading to inefficient SINV cell binding and delayed virus replication. Unfortunately, the cellular gene disrupted by the retroviral integration event was not identified (Jan, Byrnes, and Griffin, 1999).

Extensive human bioinformatics databases may facilitate the identification of potential host cell factors and pathways that promote virus resistance. To take advantage of these databases, the well-characterized human HEK 293 cell line was chosen for this study. Although isolated from embryonic kidney cell culture, this cell line supports replication of diverse viruses and is closely related to differentiating neurons (Graham et al., 1977), a property that may enable identification of cellular factors associated with alphavirus-induced neurological disease.

Retrovirus-based insertional mutagenesis can result in either diminished or increased gene expression, gene truncation, or altered gene processing (Uren et al., 2005). Modified gene expression in host cells may disrupt virus-host interactions required for virus cytopathogenicity, or stimulate host cell responses that protect against cytopathogenicity. In either case, mutagenized cells that are resistant to challenge with a cytotoxic pathogen could be used to identify host factors that provide protection from pathogen-induced cytotoxicity. Sindbis-83 virus (SINV-83), a chimeric virus encoding the structural proteins of VEEV attenuated strain TC-83 and the non-structural proteins of SINV (Paessler et al., 2003), was chosen for pathogen challenge to maximize laboratory safety. This virus is safely handled under BSL-2 laboratory conditions, yet is closely related to pathogenic encephalitic alphaviruses and highly cytopathic in cell culture (Paessler et al., 2003). SINV has been the archetypal alphavirus for studying neurovirulence due to it ability to causes encephalomyelitis in young mice (Griffin, 2005). The utilization of chimeric SINV-83 may improve understanding of neurovirulence if the selected SINV-83-resistant cells arise from changes in host cell factors that interact with the SINV non-structural proteins.

Results

Generation of alphavirus-resistant cells

Retroviral insertional mutagenesis and SINV-83 challenge were used to generate clonal populations of SINV-resistant cells. A homogeneous population of HEK 293 cells, susceptible to alphavirus-induced cytolysis, was modified by infection (MOI of 5 PFU/cell) with replication incompetent retrovirus encoding enhanced green fluorescent protein (EGFP). Approximately 108 modified cells, each containing discrete genome disruptions through retroviral insertion, and 108 unmodified control cells (WT HEK 293) were separately challenged (MOI of 0.1 PFU/cell) with SINV-83. Approximately 100 colonies of retrovirus modified cells survived SINV-83 challenge, and were isolated and cloned. Significantly, no resistant colonies were recovered from WT HEK 293 control cells infected with SINV-83. Retrovirus transduction of cells resulted in SINV-83 resistance at a frequency of 10−6. A subset of the resistant colonies was cloned and characterized in detail as described below.

The possibility that surviving clonal cells harboured infectious virus was investigated. Most supernatants tested from clonal cells harboured very low levels of virus, with titers typically <103-104 PFU/ml. However, when supernatants from these clonal cells were added to WT HEK 293 cells, the WT cells showed CPE and produced one-step virus growth curves similar to those produced when infected with SINV-83 (data not shown). These results indicated that the virus amplified within the selected clonal cells was not attenuated. To further characterize the resistant clonal cells, each was cured of infection by transient treatment with interferon alpha (IFN-α), which stimulates an antiviral response against RNA viruses, including alphaviruses (Weber, Kochs, and Haller, 2004). This treatment effectively eliminated virus from 25 of 27 tested clonal cells lines; the culture supernatant from treated resistant cells no longer caused cytopathic effects (CPE) when added to WT HEK cells (data not shown).

To determine if the IFN-α treated cells were still resistant to virus challenge, they were re-challenged with SINV-83 at an MOI of 0.1 PFU/cell and CPE kinetics monitored. The IFN-α treated cells remained resistant to virus challenge. Sixteen clones had significantly delayed CPE, which occurred between 3 and 6 days post infection compared to day 3 in control, WT cells. Nine clones survived virus challenge with little or no apparent CPE. Eight of the 9 completely resistant clones had morphological changes or grew slowly. However, one clone (termed clone 9) displayed near WT cell growth kinetics and morphology, and was chosen for more extensive analysis as discussed below.

Clone 9 did not contain SINV genomes

It was possible that clone 9 contained an attenuated non-cytopathic SINV genome that might have interfered with replication of WT SINV through a “super-infection exclusion” or “defective-interfering” mechanism (Tsiang, Weiss, and Schlesinger, 1988). RT-PCR was performed using RNA isolated from resistant clones and WT cells to detect the portion of the SINV genome encoding the nsP4 RNA polymerase, which is necessary for viral RNA replication and does not readily tolerate mutations. No PCR product was detected from clone 9 or WT cells (Figure 1A). In contrast, clone 26, which was persistently infected with an attenuated mutated SINV virus (data not shown) and used as a positive control, generated a PCR product of the expected size (638 base pairs). The PCR product from clone 26 remained clearly visible after a 125-fold dilution of the initial RNA sample (data not shown), implying that even low levels of SINV virus were not present in clone 9. Western blotting of clone 9, using antibodies against SINV proteins, was negative for the presence of SINV antigens (data not shown). These results demonstrated that clone 9 was unlikely to harbour defective interfering or attenuated virus.

Figure 1. Characterization of alphavirus-resistant clone 9.

Figure 1

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(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.

Clone 9 resisted diverse alphaviruses

Clone 9 was challenged with different alphaviruses to determine if the protective phenotype was specific for SINV-83 or involved a more general antiviral mechanism. Representative alphaviruses from four different lineages (Strauss and Strauss, 1994) were used to challenge resistant clone 9. No CPE was observed in clone 9 following infection with Highlands J virus (HJV; recombinant WEE complex) or with SINV-TE (SIN lineage; Figure 1B, Table 1). In contrast, clone 9 did not survive challenge by VEEV TC-83 (VEE/EEE lineage), or with RRV [Semliki Forest complex]. In these cases, CPE kinetics were similar to those observed in control challenge experiments using WT HEK 293 cells (Table 1). In addition, WT HEK 293 and clone 9 cells showed similar CPE responses when challenged with West Nile flavivirus (WNV; Table 1).

Table 1.

Characterization of the virus-resistant phenotype observed in clone 9 Cell survival was analyzed on 3 days post infection for SINV-83, SINV-TE, HJV, and WNV challenge, 4 days post infection for VEEV TC-83 challenge, and 5 days post infection for RRV challenge.

Virus (MOI [PFU/cell]) Cell survival (%)
WT Clone 9
SINV-83 (0.1) <10 >80
SINV-TE (0.1) <10 >80
HJV (0.5) <10 >80
RRV (0.1) <10 <10
RRV + IFN-α (0.1) <10 >80
TC-83 (0.1) <10 <10
TC-83 + IFN-α(0.1) <10 40-80
WNV (0.1) <10 <10
WNV + IFN-α(0.1) <10 <10
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IFN treatment results in a transient antiviral state in numerous cell types, making them temporarily resistant to infection by many RNA viruses (Weber, Kochs, and Haller, 2004). We examined if sub-protective doses of IFN-α (20 U/ml IFN-α) could synergistically protect clone 9 against the above tested viruses. WT HEK 293 cells challenged with VEEV TC-83, RRV, or WNV, and concurrently treated with sub-protective doses of IFN-α, showed complete CPE (Table 1). In contrast, clone 9 showed no CPE when challenged with RRV and concurrently treated with sub-protective doses of IFN-α (Figure 1C, Table 1). Clone 9 challenged with VEEV TC-83 and concurrently treated with sub-protective doses of IFN-α showed a clear reduction in CPE relative to WT cells (Table 1). Clone 9 challenged with WNV and concurrently treated with sub-protective doses of IFN-α showed extensive CPE, similar to WT cells. As an additional control, WT cells that were repeatedly treated with 200 U/ml of IFN-α at 13 day intervals and then challenged with VEEV TC-83 or RRV showed complete CPE; this result indicated that IFN-induced protection was not due to the initial “curing” procedure (data not shown).

Clone 9 exhibited impaired SINV-83 production

We next determined whether clone 9 survived viral challenge through a mechanism that impaired virus production. Cells were infected with recombinant SINV-83 encoding GFP (Dr. Ilya Frolov, UTMB) at an MOI of 0.1 PFU/cell. GFP expression in infected cells provided a marker to efficiently monitor virus titer. Following infection of WT cells, virus titers rose to ~105 foci/ml at 36 h, and reached a peak of 3 × 105 foci/ml at 48 h. After 48 h, virus titers decreased, coinciding with extensive CPE in the infected WT cells (Figure 1D). In contrast, infected clone 9 cells produced titers of ~3 × 102 foci/ml throughout the experiment, with little or no CPE observed. Thus, the resistant cells could be infected with SINV-83, but had significant deficiencies in virus production.

Analysis of retroviral integration sites

To identify host cell factors responsible for the antiviral phenotype, we analyzed sites of retroviral integration within the genome of clone 9. The resistant cells likely received several independent integration events due to the high MOI used during retroviral insertional mutagenesis. By means of inverse PCR (IPCR), four provirus insertion site sequences (labelled IPCR sites 1-4) were separately recovered and sequenced from clone 9. As expected, each provirus sequence contained the EGFP transgene. Homology BLAST searches (Altschul et al., 1990; Altschul et al., 1997) were performed to identify genes that surrounded the four integration sites (Table 2). No functional genes were identified within the immediate vicinity of IPCR site 1. However, IPCR site 1 was 23 kb downstream of the rragc gene. IPCR sites 2, 3 and 4 were located within the first intron of the nf1, lsm12, and ube2m genes, respectively. All retrovirus integrations were found in the opposite transcriptional orientation (OTO) relative to the disrupted host genes.

Table 2.

Retroviral integration sites identified in clone 9

IPCR
fragment		 Targeted genea GenBank
accession #		 Proviral integration
and orientation
1 No hit/Ras-related GTP binding C

(RRAGC)		 NM_022157 23 kb downstream,

OTOb
2 Neurofibromin 1

(NF1)		 NM_000267 1st intron,

OTOb
3 LSM12 homolog (S. cerevisiae)

(LSM12)		 NM_152344 1st intron,
OTOb
4 Ubiquitin-conjugating enzyme E2M
(UBE2M)		 NM_003969 1st intron,
OTOb
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a

Candidate gene identifications are based on proximity to the retroviral integration site.

b

OTO -opposite transcriptional orientation

Nf1, ube2m and lsm12 mRNA levels were reduced in resistant clone 9

Retroviral integration can alter host gene sequences and/or their expression levels (Trono, 2003; Uren et al., 2005). Real-time RT-PCR was performed to determine whether provirus insertions affected RNA transcript levels from the rragc, nf1, lsm12, and ube2m genes in clone 9 relative to unmodified WT HEK 293 cells. Expression levels were normalized relative to gapd gene expression, and these normalized gene expression levels compared between the resistant and WT HEK 293 cells. Retrovirus integrations within the first intron of the nf1, ube2m, or lsm12 genes resulted in a significant reduction of their RNA levels in clone 9 compared to WT HEK 293 cells (Figure 2A). Normalized ube2m, lsm12, and nf1 RNA levels were reduced 47%, 49%, and 30% in the resistant clone relative to WT cells, respectively. There was no significant difference in rragc RNA levels in the resistant clone and WT cells, suggesting the integrated provirus did not affect rragc gene expression or stability.

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

Figure 2

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(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.

NF1 and UBE2M protein levels were reduced in resistant cells

Although quantitative RT-PCR showed that nf1, lsm12, and ube2n RNA expression was reduced in clone 9 relative to WT cells, western blotting was used to determine if the corresponding protein expression levels were affected. Unfortunately, no specific anti-RRAGC and anti-LSM12 antibodies were available, restricting this analysis to NF1 and UBE2M proteins. NF1 and UBE2M protein expression levels were reduced in clone 9 compared to WT HEK 293 cells (Figure 2B). In clone 9, a truncated UBE2M protein was detected using antibody that recognized UBE2M C-terminal residues 169-180. No truncated UBE2M protein was detected with anti-UBE2M antibody specific for residues 11-26 in clone 9 (Figure 2B). These observations suggested that retroviral integration resulted in expression of an N-terminal truncated UBE2M protein.

Truncated nf1 andube2m transcripts were produced in virus-resistant cells

Retroviral integration can alter gene transcription by insertion of a viral promoter within a gene or stimulation of cryptic/alternative cellular promoters via enhancer elements located in retrovirus long terminal repeats (LTR) (Trono, 2003), often producing non-functional or rapidly degraded proteins. To determine if truncated gene products were transcribed in the virus-resistant cells, 5′ RLM-RACE was performed to determine the nucleotide sequence at the 5′ end of nf1, ube2m and lsm12 transcripts.

5′ RLM-RACE with primers specific for nf1 RNA transcripts resulted in products that coded for full-length protein in both the parental HEK 293 cells and in clone 9. Additionally, clone 9 contained a truncated nf1 RNA transcript (Figure 2C). The transcript from clone 9 contained 13 nucleotides of nf1 intron sequence located upstream of exon 3, spliced to exon 3 utilizing a cryptic splice donor site in the intron (Figure 2C). It is likely that provirus integration in clone 9 resulted in LTR enhancer activation of cryptic promoters within nf1 intron/exon sequences upstream of exon 3, thus yielding truncated nf1 transcripts. Sequence analysis showed the truncated transcripts in clone 9 could encode NF1 proteins lacking 68 N-terminal amino acids. Since the NF1 protein contains 2818 amino acids (327 kDa) (Gutmann, Wood, and Collins, 1991), this N-terminal truncation would migrate similarly to full-length NF1 protein using SDS polyacrylamide gel electrophoresis. Mutations that result in deletion of nf1 exons 1 and 2 have been reported among neurofibromatosis type I patients (Ars et al., 2003; Fahsold et al., 2000; Spurlock et al., 2007; Wimmer et al., 2006), suggesting such a truncation would compromise protein function.

5′ RLM-RACE with primers specific for ube2m transcripts identified RNA products sufficient to encode full-length UBE2M protein in WT HEK 293 and resistant cells. However, the resistant cells additionally encoded two RNA products with 5′ deletions (Figure 2C). The 5′ end of the first truncated ube2m product contained three adenine nucleotides preceding exon 2, which might have occurred from incomplete removal of cap structure during 5′ RLM-RACE (Figure 2C). The 5′ end of the second truncated ube2m product contained a nucleotide sequence corresponding to the ube2m second intron preceding exon 3 (Figure 2C). These results suggest that the integrated provirus activated cryptic promoters within the intronic ube2m sequence upstream of exon 2-3, thus yielding truncated transcripts. The first in-frame ATG codon appears at the +188 and +90 positions for the first and second truncated products, respectively, and several out-of-frame ATG triplets occur upstream of the in-frame ATG codons (Figure 2C). The smaller 17 kDa UBE2M antibody-reactive protein observed in virus-resistant clones suggests an alternative initiation of translation at non-ATG codons, since the predicted molecular weight for a protein initiated from the first in-frame ATG triplet is 10.1 kDa. CTG coding for leucine represents the most common alternative translation initiation codon for mammalian mRNA (Touriol et al., 2003). The first in-frame CTG codon occurs at the +15 position of the first truncated product (Figure 2C), and could encode an N-terminally truncated UBE2M protein with a predicted molecular weight of 16.5 kDa, consistent with the observed truncated UBE2M protein.

The N-terminally truncated UBE2M protein observed in the resistant cells was likely non-functional. A 12 to 26-residue extension (called docking peptide) at the N-terminus of UBE2M selectively recruits NEDD8 E1 (APPBP1-UBA3 complex) to promote thioester formation between UBE2M and NEDD8 (Huang et al., 2004). Deleting the UBE2M docking peptide substantially reduces UBE2M binding to E1, and impairs the transfer of NEDD8 to substrates of the NEDD8 modification system (Huang et al., 2004).

5′ RLM-RACE with primers specific for lsm12 RNA transcripts resulted in products that coded for full-length protein in both the virus-resistant clone and WT HEK 293 cells; truncated RNA products were not detected (data not shown).

siRNA-mediated NF1 down-regulation inhibited SINV-83 production and prolonged HEK 293 cell survival

To investigate the cellular mechanism(s) responsible for the resistant phenotype, siRNA targeting was used to selectively down-regulate host gene expression in WT cells. siRNA modified cells were challenged with SINV to determine the impact of selective gene down-regulation. Real-time RT-PCR showed that rragc gene expression was similar in clone 9 and WT HEK 293 cells, suggesting RRAGC was not involved in protecting cells from alphavirus challenge. siRNA-mediated down-regulation of lsm12 and ube2m expression in WT HEK 293 cells did not delay or reduce SINV-83-GFP production or SINV-83-GFP induced CPE (data not shown), suggesting LSM12 and UBE2M down-regulation did not contribute to the alphavirus-resistant phenotype.

To determine whether reduced nf1 expression contributed to the alphavirus-resistant phenotype observed in clone 9, siRNA was used to down-regulate nf1 expression in WT HEK 293 cells. Following gene-specific knockdown, decreased NF1 protein expression in WT HEK 293 cells was confirmed by Western blotting (Figure 3A). Non-targeting siRNA and mock transfection had no impact on NF1 protein expression (Figure 3A). NF1 protein expression was down-regulated rapidly, with little protein observed at 2 days post transfection. NF1 protein levels remained reduced for at least 5 days post transfection.

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

Figure 3

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(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.

Two days post transfection, cells were challenged with SINV-83-GFP at an MOI of 0.1 PFU/cell. To quantify the impact of nf1 down-regulation on SINV-83-GFP production, virus titers were determined at different time points post infection. nf1 down-regulation in WT HEK 293 cells substantially reduced virus production, resulting in a 36 h lag (Figure 3B) in virus production relative to infected control cells. Virus production at 48 and 60 h post infection in cells transfected with NF1-targeting siRNAs was also suppressed 10-50-fold relative to virus production of cells transfected with non-targeting siRNA or mock transfected. This experiment suggests NF1, or the protein networks modulated by NF1, plays an important role in alphavirus pathogenesis.

Increased cell survival was observed 5 days post infection for NF1-downregulated cells, consistent with reduced virus production in these cells. MTT-based analysis of cell viability was performed when CPE was clearly observed in control cells. Viability measurements were reported as the percentage of viable cells within the infected sample, with 100% viability being assigned to the same non-infected transfection (Figure 3C). SINV-83-GFP-induced cell death was significantly delayed and reduced in cells transfected with NF1-targeting siRNAs, relative to cells transfected with either control siRNA or mock transfected (FIG 3C). These observations correlated with the reduction in virus production that was observed in cells with NF1 down-regulation.

Hyperactive RAS activity in NF1-defficient alphavirus-resistant cells

NF1 possesses RAS-specific GTPase-activating property (GAP) that accelerates the intrinsic GTPase activity of RAS proteins and promotes the formation of inactive GDP-bound RAS (Wennerberg, Rossman, and Der, 2005). Thus, NF1 functions as a negative regulator of RAS signalling (Wennerberg, Rossman, and Der, 2005). Reduction of NF1 levels and/or NF1-specific GAP activity is commonly associated with hyperactive RAS signalling, either through elevated levels of active RAS proteins or normal levels of RAS protein with hypersensitive, exaggerated and prolonged signalling of active RAS in response to growth factors stimulation (Lau et al., 2000; Weiss, Bollag, and Shannon, 1999). To determine if reduced levels of NF1 protein in the alphavirus-resistant clone 9 resulted in hyperactive RAS signalling, we measured activated RAS in whole-cell extracts of serum-starved WT HEK 293 and resistant cells (Figure 4A). Using a RAS activation assay, increased basal levels of activated RAS were measured in the resistant cells relative to WT HEK 293 cells (Figure 4A). This experiment indicated that hyperactive RAS signalling was associated with reduced NF1 expression in the alphavirus-resistant clone 9.

Figure 4. Hyperactive RAS-signaling in alphavirus-resistance.

Figure 4

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(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.

Expression of constitutively active RAS in HEK 293 cells inhibited SINV-83 production

Since hyperactive RAS-signalling was correlated with reduced NF1 expression in alphavirus-resistant cells, we hypothesized that active RAS signalling may reduce alphavirus replication. To address this question we examined SINV-83 production in HEK 293 cells expressing a constitutively active RAS. The constitutively active mutant HRAS-G12V inhibits GTP hydrolysis, thus enabling RAS-G12V to remain active (Schlichting et al., 1989). As control, the HRAS-S17N dominant negative mutant does not exchange bound GDP and suppresses RAS activity by competing with endogenous cellular RAS for upstream activators (Feig and Cooper, 1988).

To investigate the effects of activated RAS-signalling on SINV-83 production, we infected HEK 293 cells that were previously transfected with constitutively active RAS (HRAS-G12V), dominant negative RAS (HRAS-S17N), parental plasmid, or mock transfected. Expression of HA-tagged HRAS-G12V and HRAS-S17N proteins in HEK 293 cells were confirmed by Western blotting using anti-HA antibody (Figure 4B). To quantify the impact of RAS proteins on SINV-83-GFP production, virus titers were determined at different time points post infection. As shown in Figure 4C, cells transfected with parental plasmid had levels of SINV-83 replication that were similar to that observed in mock-transfected cells. In contrast, cells transfected with HRAS-G12V had significantly reduced SINV-83 replication (Figure 4C) relative to mock-transfected cells. Cells transfected with HRAS-S17N had little effect on SINV-83 replication, similar to that reported previously (Joe et al., 1996). These results indicate that the activated RAS appears to play a significant role in suppressing SINV-83 replication.

Discussion

In this study, alphavirus-resistant HEK 293 cells were generated by a combination of retrovirus insertional mutagenesis and a cell survival selection strategy. This screen utilized SINV-83, a chimeric virus encoding non-structural proteins of SINV and structural proteins of TC-83, a tissue-culture adapted attenuated VEEV (from the VEE/EEE lineage) (Paessler et al., 2003). The observed resistant cells may have resulted from changes to host cell factors that interacted with the SINV non-structural proteins, the VEEV TC-83 structural proteins, or both. One selected cell line, termed clone 9, was resistant to SINV-83, SINV-TE, HJV and RRV. SIN-TE is a neuroadapted SIN virus (Lustig et al., 1988). HJV is closely related to WEEV; the glycoproteins of these viruses are related to those of Sindbis-like viruses, whereas their non-structural proteins are related to those of EEEV (Weaver et al., 1997). RRV is a representative of the Semliki Forest complex (Strauss and Strauss, 1994). In addition, clone 9 was only partially protected from VEEV TC-83. These results suggest that the resistance phenotype observed in clone 9 results from interference with the SINV nonstructural protein functions. Consistent with this interpretation, both WT HEK 293 cells and the resistant cells could be equally infected with a murine leukemia retrovirus pseudotyped with VEEV envelope proteins, as measured by an established luciferase-based virus entry assay (data not shown) (Kolokoltsov and Davey, 2004). This later experiments also suggested that the selected alphavirus-resistant cells had normal virus binding and fusion properties.

Retroviral preference for distinctive regions within a gene, governed by the virus-specific preintegrational complex, has been previously documented (Wu et al., 2003; Wu, Luke, and Burgess, 2006). Our observed gene insertion locations were consistent with the observed preference of murine leukemia virus to integrate in and around promoters (Trono, 2003). Three (i.e., at the nf1, lsm12, and ube2m genes) of the four integration events observed in our insertional mutagenesis study occurred within the first intron of a gene. The ~50% lower RNA transcript levels of ube2m and lsm12 observed in the virus-resistant cells relative to WT cells was not unexpected, since HEK 293 cells were diploid and retroviral integration was monoallelic (i.e., occurred in only one copy of a chromosome pair).

Primers used for real-time RT-PCR analysis detected both truncated and full-length nf1 gene transcripts. The ~30% reduction of nf1 RNA levels observed in the virus-resistant cells relative to WT cells was likely due to the presence of both truncated and full-length nf1 RNA in the virus-resistant cells. The large decrease in NF1 protein levels observed in virus-resistant cells relative to WT cells suggests that the truncated nf1 RNA was either not translated or translated into unstable NF1 protein products. A non-linear relationship between RNA and protein expression levels has been well documented (Mehra, Lee, and Hatzimanikatis, 2003). Decreased NF1 protein levels were shown to be functionally significant in humans, since reduced NF1 protein levels and/or N-terminal deletion mutants were associated with neurofibromatosis disease. Neurofibromatosis patients heterozygous for nf1 mutations develop clinical symptoms of the disease (Upadhyaya et al., 2007) and specific deletions of nf1 exons 1 or 2 have been reported among neurofibromatosis type I patients (Ars et al., 2003; Fahsold et al., 2000; Spurlock et al., 2007; Wimmer et al., 2006). Thus, it is likely that the NF1 protein down-regulation observed in our virus-resistant cells could give rise to a unique and functionally significant phenotype.

SINV-83 production was significantly inhibited when NF1 expression was transiently down-regulated in WT HEK 293 cells. Moreover, these modified cells exhibited resistance to virus-induced cell death. These results suggest that the characterized NF1-deficiency in the virus-resistant cells was largely responsible for the virus-resistant phenotype. Additionally, these studies highlight a previously unknown, yet critical, role of the NF1 host protein (and/or NF1-associated functional pathways) in alphavirus pathogenesis.

NF1 contains several distinct domains, although their functions are largely unknown. The GAP-related domain of NF1 possesses RAS-specific GTPase-activating protein (GAP) activity (Boyanapalli et al., 2006; Corral et al., 2003; D’Angelo et al., 2006; Martin et al., 1990) that accelerates the intrinsic GTPase activity of RAS proteins. Thus, NF1 is a negative regulator of RAS activity (Wennerberg, Rossman, and Der, 2005), and NF1 down-regulation is associated with hyperactive RAS activity and elevated RAS-signalling (Lau et al., 2000; Weiss, Bollag, and Shannon, 1999). The observation that the expression of constitutively active RAS in WT HEK 293 cells inhibited SINV replication was consistent with a role for the NF1 protein and downstream RAS-signalling in alphavirus pathogenesis.

SINV-83 replication was more inhibited in WT cells that were modified by siRNA-mediated NF1 down-regulation compared to WT cells transfected with constitutively active RAS. This observation could result from NF1 possessing an unidentified activity which is required for alphavirus replication, independent from NF1’s RAS-specific GTPase-activating property. Alternatively, NF1 down-regulation may affect the activity of several other RAS isoforms (i.e., KRAS4A, KRAS4B, NRAS) and their downstream pathways (Yan et al., 1998). These pathways, which function independently of HRAS, may be additionally involved in alphavirus pathogenesis. Treatment with IFN-α may have enhanced some hyperactive RAS signalling pathways in the clone 9 cells, thus enabling these treated cells to survive RRV and VEEV TC-83 challenge. Although IFN-α elicits an antiviral response by activating components of the MEK/ERK pathway (David et al., 1995; Lechner and Pfaller, 2001) the detailed interplay between alphavirus inhibition and RAS signalling remains to be determined.

Conclusions

This work identified a novel host cell determinant that is involved in alphavirus pathogenesis and demonstrated the power of a rigorous cell survival selection screen as a tool to identify host cell components that impact virus production and virus-induced CPE. Host cell proteins (and their associated pathways) that protect cells from virus infection offer novel therapeutic targets and strategies to combat infectious diseases. Future work will identify in greater detail the NF1 properties and/or NF1-associated pathways that are responsible for inhibiting alphavirus production.

Materials and methods

Generation of resistant clones by retroviral insertional mutagenesis

HEK 293 cells were maintained in DMEM containing 4.5 g/L glucose, 10% (v/v) heat-inactivated FBS (HyClone, Logan, UT) and 1% (v/v) penicillin/streptomycin. Cell genomes were modified by insertional mutagenesis using a replication incompetent retrovirus. To produce recombinant retroviruses encoding enhanced green fluorescent protein (EGFP), HEK 293 cells were cotransfected with 3 separate plasmids: pGag-Pol (Dr. J Cunningham, Harvard Medical School) encoding Gag-Pol proteins of murine leukemia virus, pHCMV-VSV-G plasmid (Clontech, Mountain View, CA) pseudotyping retroviruses with the vesicular stomatitis virus (VSV) G envelope protein, and pFB-EGFP Moloney murine leukemia-based retroviral vector for reporter gene delivery and expression. pFB-EGFP was prepared by cloning the EGFP sequence (Clontech, Mountain View, CA) into the EcoRI and NotI restriction endonuclease sites of the pFB plasmid (Stratagene, La Jolla, CA). One day post transfection, cells were rinsed and fresh media added. Two days post transfection, retrovirus-containing media was collected, filtered (0.45 μM) and stored at −80°C.

Retroviruses encoding EGFP were used to infect ~108 HEK 293 cells at a multiplicity of infection (MOI) of 5 PFU/cell. Two days post infection, >95% of cells expressed EGFP, as determined by fluorescence microscopy. Cells were split and the next day challenged with SINV-83 virus at an MOI of 0.1 PFU/cell. As control, ~108 of WT HEK 293 cells were independently challenged with SINV-83, and maintained under identical conditions as the retrovirus-treated cells. Cell media was changed daily for the next 5 days. Seven days post-challenge surviving colonies were isolated and cloned.

SIN-83 persistent virus infection of the surviving clones was eliminated by treatment with human IFN-α2b (Schering-Plough, Kenilworth, NJ) at a dosage of 200 U/ml for 3 days. After cultivation for an additional 10 days in the absence of IFN-α, resistant cells were tested for residual virus by inoculating WT HEK 293 cells with surviving clone supernatants and monitoring CPE for 5 days.

PCR for viral genome detection

Total RNA was isolated by RNaqueous kit (Ambion, Austin, TX) and its quality and concentration determined by agarose gel electrophoresis and UV spectrophotometry. Ten micrograms of RNA were mixed with 100 pmol of primer (5′-CCACCTCGAGTTTACCCAACTTAAACAGCC-3′) located 600 base pairs downstream of the PCR target in 9 μl of water, heated at 70°C for 10 min, and then transferred to ice. Of this mixture, 2 μl were used to synthesize cDNA using Superscript II (Invitrogen, Carlsbad, CA) and a 1 h incubation at 45°C. The SINV nsP4 gene (polymerase) was amplified from cDNA with Taq polymerase (Sigma-Aldrich, St. Louis, MO), primers 5′-CCACGAGCTCGACCTTGGAGCGCAATGTCC-3′ and 5′-CCACCTCGAGGAACTCCTCCCAATACTCGTC-3′, and 30 cycles of PCR (94°C for 30 sec, 55°C for 30 sec, and 70°C for 1 min). PCR products were separated on 1.5% agarose gels and visualized with ethidium bromide.

Challenging resistant clonal cell lines with heterologous viruses

SINV-83, SINV-TE and SINV-83-GFP were kindly provided by Dr. I. Frolov (UTMB). VEEV strain TC-83, HJV, RRV, and WNV were generously provided from the World Arbovirus Reference Center (Dr. R. Tesh, UTMB).

Virus was added to cells cultivated to greater than 50% confluence (Table 1). After significant CPE was observed in WT HEK 293 cells (typically 3-5 days post-alphavirus infection), cells were fixed with 10% formaldehyde and stained with crystal violet to view residual attached cells. Virus titers were determined for SINV-83-GFP infection. Cells were infected at an MOI of 0.1 PFU/cell, media collected at indicated times post infection, and diluted onto HEK 293 cells. Following 3 h incubation, cells were overlaid with MEM containing 0.6% gum tragacanth (w/v), 2.5% FBS (v/v), and 18 h post infection foci of infection containing GFP expressing cells were counted using a UV microscope.

Inverse PCR

Genomic DNA (5 ug) was purified using GenElute Mammalian Genomic DNA Miniprep Kit (Sigma-Aldrich, St. Loius, MO) and digested with BamHI in a total volume of 200 μl for 7 h. Reactions were purified by DNA Clean and Concentrator-5 (Zymo Research, Orange, CA). Digested DNA was self-circularized using T4 DNA ligase (1200 U; New England BioLabs, Ipswich, MA) and 5% PEG 8000 (w/v) in a total volume of 500 μl at 16°C for 16 h. Circular DNA was purified by DNA Clean and Concentrator-20 (Zymo Research, Orange, CA) and used in the primary PCR in a 50-μl reaction volume containing forward and reverse primers (10 pmol each), PreMix 7 and enzyme mix (2.5 U) in the MasterAmp Extra-Long PCR Kit (Epicentre, Madison, WI). The thermocycler was programmed for 94°C for 2 min, followed by 30 cycles of 94°C for 1 min, 50°C for 1 min, 72°C for 5 min and a final extension step at 72°C for 10 min. The primary PCR product (5 μl) was used as the template in a secondary PCR reaction using the same conditions except the annealing temperature was increased to 52°C. The secondary PCR product was separated on a 1% agarose gel, purified and directly sequenced. Sequences flanking the provirus DNA were BLAST-searched against human genome sequence databases. The following primers were used in the primary PCR reactions: pFB-2149-70-For (5′-CAGAAAAAGGGGGGAATAAAG-3′) and pFB-Rev (5′-GGAGACTAAATAAAATCTTTTAT CGAAC-3′). The secondary PCR primers were: pFB-2665-89-For (5′-CCTCTTGCAGTTGCATCCGA CTTGT- 3′) and pFB-Rev.

Real-time RT-PCR

DNase-treated RNA was prepared from 70% confluent cells using RNeasy Mini Kit (Qiagen, Valencia, CA) following protocols adapted from manufacturer’s instructions. cDNA was synthesized with ReactionReady™ First Strand cDNA Synthesis Kit (Super Array, Frederick, MD).

To determine if residual DNA were present, control reactions using pooled RNA from all samples were performed with no reverse transcriptase added. A no-template control reaction was also run in parallel with all amplifications to detect contaminating DNA or the formation of secondary products generated by primer interactions. To control for variation in input RNA quantities and reverse transcription efficiencies, reactions were normalized to the glyceraldehyde 3-phosphatedehydrogenase (gapd) housekeeping gene. Real-time PCR master mixes were prepared to the final concentrations of 200 nM primers, and iQ SYBR Green Supermix (Bio-Rad, Hercules, CA). Standard curves were generated for each primer pair using two-fold serial dilution of cDNAs pooled from WT HEK 293.

Real-time PCR was performed on iCycler iQ thermal cycler (BioRad, Hercules, CA) using the following protocol: initial denaturation (1 min 30 s at 95°C); PCR amplification and quantification cycle repeated 50 times (30 s at 95°C; 30 s at 55°C; 30 s at 72°C with a single fluorescence measurement); and a melt curve analysis (55-95 °C) using a heating rate of 0.2°C per 10 s with continuous fluorescence measurement.

The PCR “Base Line Subtracted” mode with manual threshold assignment equal to 300 was used to determine the geometric mean of the threshold cycle (Ct) using iCycler iQ software (version 3.0a, Bio-Rad, Hercules, CA). The relative concentration of gene-specific template in each sample was determined using the appropriate Ct values and the standard curve, normalized relative to the gapd reference gene, and compared to normalized expression levels of the same gene in the appropriate control sample. To establish the statistical significance of each comparison, two-tailed Student’s t-tests assuming unequal variance were performed.

Primers for LSM12, NF1 and GAPD gene transcripts were designed and produced by SuperArray Bioscience Corp., Frederick, MD (Gene Symbol: LSM12, SuperArray Cat#: PPH17720A, Reference position:392-412; Gene Symbol: NF1, SuperArray Cat#: PPH02089A, Reference position:1455-1475; Gene Symbol: GAPD, SuperArray Cat#: PPH00150A, Reference position:362-380). Primers for UBE2M and RRAGC gene transcripts were designed from reference mRNA sequences obtained through GenBank (see Table 2 for GenBank accession numbers) using Primer 3 software (MIT/Whitehead Institute; http://wwwgenome.wi.mit.edu). The following primers were used for UBE2M transcript quantification (UBE2M-5′, 5′-ACGTCTGCCTCAACATCCTC- 3′ and UBE2M-3′, 5′-TCCTTGTTCAGTGGGTCCTC-3′) and for RRAGC transcript quantification (RRAGC-5′, 5′-AATTTTTGGCACTGGTCTGC- 3′ and RRAGC-3′, 5′-CACACCCACCTCAAAAACCT-3′).

Western blotting

Immunoblotting was carried out as previously described (Zou et al., 2004). Antibody incubation and detection was carried out following the protocol for ECL Plus Western Blotting Detection Reagents (Amersham Biosciences, Piscataway, NJ). Primary antibodies were rabbit polyclonal antibody against the C-terminal epitope (residues 2760-2818) of the NF1 protein (antibody BL956, Bethyl Laboratories, Montgomery, TX) and rabbit polyclonal antibodies against N-terminal (residues 11-26) and C-terminal (residues 169-180) epitopes of the UBE2M protein (antibodies AP2169a, Abgent, San-Diego, CA and 600-401-865, Rockland, Gilbertsville, PA). The secondary antibody was a horseradish peroxidase (HRP)-conjugated antibody (donkey anti-rabbit IgG, Amersham Biosciences, Piscataway, NJ). Loading control primary antibody was mouse polyclonal antibodies against beta-actin (antibody ab6276, Abcam, Cambridge, MA). The secondary antibody was a HRP-conjugated antibody (donkey anti-mouse IgG, Amersham Biosciences, Piscataway, NJ).

5′ RNA Ligase Mediated Rapid Amplification of cDNA Ends (5′ RLM-RACE)

Total RNA was isolated from 70% confluent cells, as described for real-time RT-PCR. cDNA containing 5′ RACE Adapters were synthesized according to manufacturer’s instructions (FirstChoice RLM-RACE, Amibon). Outer 5′ RLM-RACE PCR was performed with 2 μl of RT product. The thermocycler was programmed for 94°C for 3 min, followed by 40 cycles of 94°C for 30 sec, 60°C for 30 sec, 72°C for 1 min and a final extension step at 72°C for 7 min. 2 μl of Outer 5′ RLM-RACE PCR product was used in Inner 5′ RLM-RACE PCR using the same conditions except the annealing temperature was increased to 63°C. The secondary PCR product was separated on a 1% agarose gel, purified, and directly sequenced. Sequences were BLAST-searched against human genome sequence databases.

Primers for UBE2M and NF1 gene transcripts were designed from reference mRNA sequences obtained through GenBank (see Table 2. for GenBank accession numbers) using Primer 3 software (MIT/Whitehead Institute; http://wwwgenome.wi.mit.edu). Outer 5′ RLM-RACE PCR was performed with gene-specific primers UBE2M-1307-1327-R (5′-GCCGACCTTAATCACATGGT-3′), NF1-936-956-R (5′-CTTTTCTGCACATTCAGCCA-3′) and LSM12-477-497-R (5′-TCCACTTGATATGGGGGTGT-3′). Inner 5′ RLM-RACE PCR was performed with gene-specific primers LSM12-477-497R, UBE2M-931-951-R (5′-CTGGCTTCCAGTCCTCTCTG-3′) and NF1-785-805-R (5′-GCTGTTTCCTTCAGGAGTCG-3′).

Transient transfection of siRNA into HEK 293 cells

Silencer™ Firefly Luciferase (GL2 + GL3) siRNA (Ambion, Austin, TX) and siCONTROL Non-Targeting siRNA #2 (Dharmacon, Inc., Lafayette, CO) targeting Firefly Luciferase were used independently to determine optimal conditions for siRNA transfection, including siRNA titration, using HEK 293 cells infected with replication defective retrovirus expressing Firefly Luciferase (R. Davey, UTMB). Firefly Luciferase protein knockdown was assayed by Luciferase Assay System, Promega, Madison, WI (data not shown). In addition, siCONTROL Non-Targeting siRNA #2, having no perfect matches to known human genes, was used as a negative control.

Transfection procedures were carried out according to manufacture’s instructions (“Transfecting Stealth RNAi or siRNA into HEK 293 Cells Using Lipofectamine2000,” Invitrogen, Carlsbad, CA). Briefly, 24 h before transfection, 5×104 HEK 293 cells were seeded in each well of a 24 well plate in MEM-Eagle media containing 2 mM glutamine, 0.1 mM MEM non-essential amino acids, 1 mM sodium pyruvate, 1.5 g/L sodium bicarbonate, and 10% heat-inactivated FBS (v/v). Transfections were carried out in triplicate using 2 ul of Lipofectamine2000 reagent and 50 pmol of siRNA per well.

siRNA targeting NF1 transcript (siNF1-D; sense sequence 5′-AAACGAUGCUGGUCAAACAtt-3′) was designed from reference mRNA sequences obtained through GenBank (see Table 2 for GenBank accession numbers) using Dharmacon siDESIGN™ Center software (http://www.dharmacon.com/sidesign/) and custom synthesized by Dharmacon Inc. (Lafayette, CO). In addition, Silencer Pre-designed siRNA named siNF1-A (siRNA ID #121252; sense sequence 5′-GCU AAUCCUUAACUAUCCAtt-3′) targeting NF1 transcript were purchased from Ambion (Austin, TX).

Cell viability assay

Cell viability was assessed 72 h post infection using a (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) or (MTT)-based assay (In Vitro Toxicology, Sigma-Aldrich, St. Loius, MO) according to the manufacturer’s instructions. To control for differences in cell propagation rates, infected sample viability levels were normalized by non-infected sample viability levels for the same transfection. The normalized viability data for each transfected group were compared to normalized viability data for non-targeting siRNA transfection samples.

RAS activity assay

105 cells were plated in each well of 24-well plate in triplicates. Cells were serum-starved for 5 hrs prior to lysis. Total protein was determined by Bio-Rad Protein Assay (Bio-Rad, Hercules, CA). 25 μg of whole-cell extracts were assayed for RAS activity using the RAS GTPase Chemi ELISA Kit (Active Motif, Carlsbad, CA) according to the manufacturer’s instructions.

Transient transfection of plasmid DNA into HEK 293 cells

pcDNA3.1-HRAS-G12V and pcDNA3.1-HRAS-S17N plasmids express 3xHA tagged (N-terminus) constitutive active RAS mutant and dominant negative RAS mutant, respectively (UMR cDNA Resource Center, Rolla, MO). Parental vector pcDNA3.1+ was purchased from Invitrogen. HEK 293 cells were maintained in MEM-Eagle containing 2 mM glutamine, 0.1 mM MEM non-essential amino acids, 1 mM sodium pyruvate, 1.5 g/L sodium bicarbonate, 10% FBS (v/v). Transfection procedure was carried out according to manufacture’s instructions (“Lipofectamine2000 Transfection Procedure for DNA”, Invitrogen, Carlsbad, CA). Briefly, 24 hrs before the transfection 1×105 HEK 293 cells were seeded in each well of 24 well plates. Transfections were carried out in triplicate using 2 μl of Lipofectamine2000 reagent and 0.8 μg of plasmid per well in 1 ml of medium. Plasmid-Lipofectamine2000 complexes were removed 4-6 hrs after transfection.

Statistical analysis

To establish the statistical significance of each comparison, two-tailed Student’s t-tests, assuming unequal variance, were performed.

Acknowledgements

We thank Drs. I. Frolov, A. Barrett, and R. Shope for helpful advice, Drs. R. Tesh and R. Shope for access to the World Arbovirus Reference Collection, Dr. M. Holbrook for the WNV work, and the UTMB molecular biology core facility for sequencing. This work was supported by grants from the Defense Advanced Research Projects Agency (DAAD19-01-1-0361, S.J.W.), the Texas Advanced Technology Program (004952-0033-2001, S.J.W.), the National Institutes of Health (AI53551-01, S.J.W. and AI48807, S.C.W.), and the Department of Homeland Security (subcontract from the National Center for Foreign Animal and Zoonotic Disease Defense, Texas A&M, PI: Dr. Neville Clark). A.M.D. was the recipient of a NIH/NIAID-supported postdoctoral training fellowship (2 T32 AI007536-06).

Footnotes

Authors’ contributions

OAK and SJW planned the experiments and drafted the manuscript. OAK performed the experiments. AMD and AAK participated in experimental design and experiments. RAD, SCW and SJW planned the project and edited the manuscript. All authors read and approved the final manuscript.

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