Drosophila melanogaster Limostatin and Its Human Ortholog Promote West Nile Virus Infection

Simple Summary Insect-borne viruses, such as those of the Flaviviridae family, pose a serious risk to global health. WNV, a mosquito-borne flavivirus, is transmitted primarily by the Culex mosquito. Despite the increasing exposure of populations to mosquito-borne flaviviruses and the expanding range of the vector mosquito, there are limited resources available to prevent or treat flavivirus infections. Using the model organism Drosophila melanogaster, commonly known as the fruit fly, we previously found that insulin signaling reduces WNV infection. We translated these finding to mosquitoes and human cells and showed similar mechanisms of insulin-mediated antiviral activity. However, insect and mammalian hormones can regulate insulin signaling. Specifically, decretin hormones suppress insulin secretion, especially during periods of starvation and low glucose intake. In this study, we show that the insect decretin, Limostatin, and its mammalian ortholog, Neuromedin U, can promote WNV infection. These results suggest that the inhibition of decretin signaling may be a novel therapeutic target to control WNV infection. Abstract The arbovirus West Nile virus (WNV) is a danger to global health. Spread primarily by mosquitoes, WNV causes about 2000 cases per year in the United States. The natural mosquito immune response controls viral replication so that the host survives but can still transmit the virus. Using the genetically malleable Drosophila melanogaster model, we previously dissected innate immune pathways used to control WNV infection. Specifically, we showed that insulin/IGF-1 signaling (IIS) activates a JAK/STAT-mediated immune response that reduces WNV. However, how factors that regulate IIS in insects control infection has not been identified. D. melanogaster Limostatin (Lst) encodes a peptide hormone that suppresses insulin secretion. Its mammalian ortholog, Neuromedin U (NMU), is a peptide that regulates the production and secretion of insulin from pancreatic beta cells. In this study, we used D. melanogaster and human cell culture models to investigate the roles of these insulin regulators in immune signaling. We found that D. melanogaster Lst mutants, which have elevated insulin-like peptide expression, are less susceptible to WNV infection. Increased levels of insulin-like peptides in these flies result in upregulated JAK/STAT activity, leading to protection from infection. Treatment of human cells with the insulin regulator NMU results in increased WNV replication. Further investigation of methods to target Lst in mosquitoes or NMU in mammals can improve vector control methods and may lead to improved therapeutics for human and animal infection.


Introduction
The flavivirus WNV poses a global health threat [1] and has been present in the United States since it first made landfall in New York in 1999 [2].WNV is spread by mosquitoes, primarily the mosquito species Culex quinquefasciatus [3].Climate change is altering mosquito habitats, feeding activity, and seasonal patterns [4,5].This allows mosquitoes, and the viruses they carry, to move into new areas, spreading those diseases further [6,7].Symptoms and progression of WNV vary in humans due to genetic variation [8,9].WNV causes symptoms in 20% of cases [10], which can include conditions like headache, weakness, and rash [11].Furthermore, 1 in 150 cases may develop the more severe West Nile neuroinvasive disease, which can feature encephalitis [12], meningitis [3], neuronal cell death [13], and death.
Drosophila melanogaster is a useful model to study immunity and the control of viral infection in mosquito vectors like Cx. quinquefasciatus.There is wide genetic diversity between D. melanogaster and mosquitoes [14], but many components of the immune system are conserved [15].The genetic malleability of D. melanogaster provides a tool to study specific mutations that affect insect immunity.Our lab previously used the Kunjin virus strain of WNV (WNV-Kun) to perform a genetic screen in D. melanogaster [16] due to its similarity to the 1999 New York strain [17] and the virulent Linage 1a strain [18][19][20].We found that variants in the Insulin receptor (InR) gene rendered flies more susceptible to WNV-Kun.
Various signaling pathways have an impact on viral infections in D. melanogaster [21][22][23].D. melanogaster and mosquitoes utilize similar antiviral response pathways, specifically RNAi and JAK/STAT, as primary means of protection against viral infection [15,24].RNAi machinery degrades detected cytosolic viral nucleic acids.JAK/STAT induces antiviral cytokines to act upon viral-stimulated ligands.Insulin/insulin-like growth factor 1 (IIS) and MAPK/ERK pathways are also antiviral during infection in insects [16,25].The insulin/IGF-1 signaling (IIS) pathway stimulates the activation of the JAK/STAT antiviral pathway [16], so the role of proteins that control IIS-mediated immunity in insects should be examined.
Decretins are hormones that suppress insulin production and secretion under starvation conditions [26].Decretins exist in insects and mammals [26].Hormonal systems for metabolic regulation, including insulin signaling, are largely conserved in mammals [27].D. melanogaster limostatin (Lst) encodes a peptide hormone decretin, Lst, that suppresses insulin secretion [28].A conserved 15-residue Lst polypeptide is produced in glucosesensing enteroendocrine gut-associated cells.Lst production is suppressed by carbohydrate feeding.Limostatin deficiency leads to hyperinsulinemia, hypoglycemia, and excess fat storage [26].Glucose-stimulated insulin secretion is regulated by different metabolic states due to feeding behavior.Circulating insulin is elevated during times of feeding, leading to increased nutrient storage.In starvation or low-nutrient conditions, insulin is decreased to signal nutrient mobilization [29].Gut-associated hormones play a role in regulating insulin secretion in response to carbohydrate intake.D. melanogaster insulin-like peptides (ilps) produced in neuroendocrine cells regulate nutrient storage in the fly in response to elevated circulating glucose after food intake [26].
The mammalian ortholog of Lst is Neuromedin U (NMU).CG9918 encodes the D. melanogaster Lst receptor, and its mammalian ortholog is the NMU receptor (NMUR).Knockdown of CG9918 in insulin-producing cells (IPCs) decreases insulin secretion.This Lst receptor was identified as a G-coupled protein receptor (GPCR) in IPCs [26].The GPCR NMUR1 is present in mammalian pancreatic beta cells [30], where stimulation by NMU leads to decreased insulin secretion [31,32].Since our lab previously described the mechanism of insulin-mediated immune signaling during WNV infection, we next sought to investigate the role of the insulin regulators Lst and NMU in immunity to WNV infection.In this study, we show that Lst mutant D. melanogaster shows elevated expression of insulin-like peptides (ilps) and elevated expression of genes within the JAK/STAT pathway during infection.These mutants are less susceptible to WNV-Kun infection.Normal human fibroblasts expressing NMU receptor 1 (NMUR1) and treated with NMU-25 peptide show higher viral titer following infection than control cells.This research indicates that suppressors of insulin secretion have a direct impact on susceptibility to WNV-Kun infection.Understanding these immune pathways and factors of susceptibility will have a direct impact on improving therapeutics for diabetic individuals and patients with hypoglycemic symptoms.

Fly Mortality Study
First, 2-5-day-old female D. melanogaster were anesthetized with CO 2 and injected intrathoracically with 23 nl of WNV-Kun at a dose of 200 PFU/fly.Mock infection was performed using PBS injection.For each independent experiment, 40 flies of each genotype, y 1 w 1 (Bloomington Drosophila Stock Center (BDSC) #1495) and y 1 w*; Mi{y +mDint2 = MIC}Lst MI06290 (BDSC #60793), were infected with WNV-Kun or mock infected and kept on vials with standard cornmeal food (Genesee Scientific, Morrisville, NC, USA).Surviving flies were counted every 24 h for 30 days.Vials were changed every three days.Hazard ratios were calculated in the Survival Curve analysis program in Graphpad Prism ver.9.A hazard ratio is an index of effect size and compares the rates of mortality over time between two survival curves [35].Survival curves represent data from three replicate experiments combined together.

Virus Replication Assay
Virus replication in flies was measured using a standard plaque assay on BHK21 cells.First, 2-5-day-old female flies were infected with 23 nL of WNV-Kun at a dose of 2000 PFU/fly.Flies were kept on vials with standard cornmeal food, and vials were changed every three days.At 1, 5, and 10 days post-infection, three sets of five flies were collected from each genotype.Flies were homogenized in Phosphate-Buffered Saline (PBS) before virus titration.The homogenate was serially diluted in DMEM with 2% FBS and plated on a 12-well plate of BHK21 cells at 1.5 × 10 5 cells/well.Plates were incubated for 2 h at 37 • C/5% CO 2 and rocked every 15 min.Wells were overlayed with 4% low-meltingpoint agarose (Invitrogen 16520050, Waltham, MA, USA) for a final concentration of 0.75% agarose and 4% FBS in DMEM.Plates were incubated for four days at 37 • C/5% CO 2 before visualization with 0.1% crystal violet (Fisher 548-62-9, Hampton, NH, USA).

Preparation of pcDNA3.1;NMUR1 Plasmid
pcDNA3.1;NMUR1 plasmid was a generous donation from the lab of Dr. Ching-Wei Luo.Plasmid was cloned through transfection into chemically competent E. coli cells and extracted for experimental use using the GeneJET plasmid miniprep kit (Fisher K0503, Hampton, NH, USA) 2.6.Transfection of Plasmids into Cells pcDNA3.1;NMUR1plasmid was transfected into NHF1 cells in a 12-well plate using a concentration of 1 µg of DNA in each well.Transfection was conducted by combining 2.5 µL of lipofectamine (ThermoFisher, 11668019, Bothell, WA, USA) and 125 µL of Optimem (ThermoFisher 31985062, Bothell, WA, USA) in a microcentrifuge tube and 1 µg DNA and 125 µL of Optimem in a separate tube and incubating at room temperature for 5 minutes before mixing and incubating for 20 min.pcDNA3.1+vector was transfected into cells as an empty vector control.

Infection of NMUR1-Expressing Cells
NFH1 cells were cultured at a concentration of 2.0 × 10 5 cells/well in a 12-well plate and transfected with either pcDNA3.1;NMUR1or empty vector plasmid.Six hours later, three biological replicates were supplemented with 100 nM of NMU-25 peptide (Aapptec P002126, Louisville, KY, USA).Then, 24 h later, cells were infected at a dose of 0.01 MOI PFU/cell with WNV-Kun.At 72 h post-infection, cell culture supernatant was collected for use on a viral plaque assay.

Quantification and Statistical Analyses
Results shown are representative of at least three independent experiments.Data points in dot plots represent a biological replicate of a pool of five flies (Figures 1, 3, and  4) or an individual well of cells (Figure 5).Statistical analyses were completed using GraphPad Prism.Two-tailed unpaired t-tests assuming unequal variance were utilized to compare normally distributed pairwise quantitative data.One-way analysis of variance with Tukey's correction for multiple comparisons was used to compare multivariate data.Statistical tests were performed for each independent experiment to verify the robustness of the results.All error bars represent the standard error of the mean.Survival curves (Figure 2) represent three replicate experiments per condition pooled together and analyzed using the log-rank (Mantel-Cox) test using GraphPad Prism to determine p values between infected genotypes.

Hyperinsulinemic D. melanogaster Models Are Less Susceptible to WNV Infection
Lst suppresses the release of ilps from insulin-producing cells, and mutation of lst causes increased expression of ilps [26].Our lab has previously described how ilp signaling mediates the JAK-STAT innate immune response to WNV.We sought to determine if mutation of Lst would cause a hyperinsulinemic phenotype.D. melanogaster encodes for 8 ilps [40].D. melanogaster ilp7 is the most conserved to a mosquito ilp [41][42][43], while D. melanogaster ilps 1-5 are most conserved to human and mouse insulin peptides [44].Lst normally suppresses ilp production and secretion [26].The Lst MI06290 mutant fly line contains a mutation through the insertion of a transposable element to the Lst gene [45].We measured the expression of insulin-like peptides in uninfected Lst MI06290 and y 1 w 1 flies and show that ilp-2, ilp-3, ilp-5, and ilp-7 are significantly upregulated in the Lst MI06290 mutant fly line (Figure 1A-D).The Lst MI06290 mutant fly line expresses ilps at higher levels than control flies.Because insulin signaling in flies results in increased Akt phosphorylation [16], and an increase in ilp gene levels does not necessarily correlate with protein levels [46], we next examined if Lst MI06290 mutant flies showed increased insulin signaling via Akt activation.Indeed, immunoblotting confirms that Lst MI06290 mutant flies exhibit increased Akt phosphorylation compared to control flies (Figure 1E).Together, these results support the use of the Lst MI06290 fly line to model hyperinsulinemia, as the Lst MI06290 flies display phenotypes similar to that described in Alfa et al [26].We can then use this model to test how hyperinsulinemia affects WNV infection.
We next examined the rate of mortality to WNV-Kun infection in the hyperinsulinemic Lst MI06290 mutant fly compared to y 1 w 1 control flies.Over a 30-day period following WNV-Kun infection via intrathoracic injection, Lst MI06290 flies succumbed to infection at a significantly slower rate than control flies (Figure 2A).Comparison of survival was determined using a hazard ratio.A hazard ratio compares the rates of mortality over time between two survival curves, and it is an index of effect size [35].Both the Lst MI06290 line and y 1 w 1 lines showed higher rates of mortality during infection with WNV-Kun than during a mock infection.The y 1 w 1 line exhibits a 2.613 hazard ratio when comparing infection with WNV-Kun to mock infection (Figure 2B), while the Lst MI06290 line exhibits a 2.362 hazard ratio when infected with WNV-Kun compared to mock infection (Figure 2C).This indicates higher survivability of the Lst MI06290 line to WNV-Kun compared to mock infection.When comparing WNV-Kun infection of the Lst MI06290 mutant line to the y 1 w 1 control, there is a 0.4021 hazard ratio, indicating the Lst MI06290 line died at a slower rate than the control (Figure 2A).The results from each independent experiment that were combined for presentation in Figure 2 are presented in Table 1.In summary, the loss of the insulin-regulating Lst peptide led to lower mortality following WNV-Kun infection in hyperinsulinemic flies.We next examined the rate of mortality to WNV-Kun infection in the hyperinsulinemic Lst MI06290 mutant fly compared to y 1 w 1 control flies.Over a 30-day period following WNV-Kun infection via intrathoracic injection, Lst MI06290 flies succumbed to infection at a significantly slower rate than control flies (Figure 2A).Comparison of survival was determined using a hazard ratio.A hazard ratio compares the rates of mortality over time   1) that combined for a final survival curve and statistical analyses.Because control flies exhibited increased susceptibility to WNV infection compared to Lst MI06290 mutant flies, we next examined if WNV replication was correlated to this difference in mortality.Following infection with WNV-Kun, y 1 w 1 and Lst MI06290 mutant flies were collected for use on a standard plaque assay using BHK21 cells.Lst MI06290 flies showed significantly less viral replication at 5 and 10 days post-infection than the control flies (Figure 3).

JAK/STAT Expression Is Upregulated in Lst Mutant D. melanogaster
In D. melanogaster, the RNAi and JAK/STAT pathways are involved in the response to various viruses, including WNV [47,48].The RNAi and JAK/STAT pathways are also used in response to WNV [49] in the mosquito species A. aegypti [50] and C. quinquefasciatus [51,52].Insulin-mediated immunity is also involved in the response to WNV infection in D. melanogaster [16].This pathway is activated through the binding of ilps to the insulin receptor (InR).The InR is expressed in the midgut of both D. melanogaster and mosquitoes

JAK/STAT Expression Is Upregulated in Lst Mutant D. melanogaster
In D. melanogaster, the RNAi and JAK/STAT pathways are involved in the response to various viruses, including WNV [47,48].The RNAi and JAK/STAT pathways are also used in response to WNV [49] in the mosquito species A. aegypti [50] and C. quinquefasciatus [51,52].Insulin-mediated immunity is also involved in the response to WNV infection in D. melanogaster [16].This pathway is activated through the binding of ilps to the insulin receptor (InR).The InR is expressed in the midgut of both D. melanogaster and mosquitoes [53][54][55].The IIS pathway impacts both the RNAi and JAK/STAT pathways.Following the phosphorylation of Akt, the transcription factor FoxO is localized outside of the nucleus, decreasing the production of Dicer-2 and Argonaute-2, proteins used in the RNAi complex [56].The activation of Akt also results in the phosphorylation cascade of the MAPK/ERK pathway, which produces upd2 and upd3 [16], the proteins that activate the antiviral JAK/STAT pathway.Activation of the JAK/STAT antiviral pathway leads to the expression of antiviral effectors, including vir-1 and TotM [16,57].
To determine if the JAK/STAT pathway and its downstream cytokines were more highly activated in Lst mutants, expressions of the genes upd-3 and vir-1 were measured in mock-or WNV-infected Lst MI06290 mutant and y 1 w 1 control flies.Expression of upd-1 and vir-1 was significantly higher in infected Lst MI06290 mutant flies compared to mock-infected and control flies (Figure 4).Interestingly, we did not observe induction of upd-3 or vir-1 during WNV infection in control flies.This may be due to the low dose of infection or the timing of sample collection.Additionally, WNV infection alone did not induce upd-3 or vir-1 in Drosophila S2 cells [16].Nevertheless, these results indicate that the hyperinsulinemic Lst MI06290 mutant fly shows upregulation of the JAK/STAT immune signaling pathway during WNV infection.

The Human Ortholog of Lst Promotes WNV Infection in Human Fibroblasts
The human ortholog for limostatin is Neuromedin U, a peptide involved in feeding behavior, insulin regulation, and promoting the expression of inflammatory cytokines in adaptive immune cells [58].The expression of NMUR1 (NMU receptor 1) in HEK-293T cells has been used to demonstrate that NMU signaling suppresses proliferation of SKOV-3 ovarian cancer cells [59].In our experiments, we used the NHF1 cell line, as fibroblasts are infected when a WNV-infected mosquito takes a human bloodmeal [60], and these cells express IGF-1 receptor (IGF-1R) and secrete IGF-1 [61,62].To model the regulation of WNV infection by NMU in NHF1 cells, we first expressed NMUR1 in NHF1 cells (Figure 5A).
NHF1 cells expressing NMUR1 were treated with NMU-25 peptide to activate NMUR1.These cells were then infected with WNV-Kun, and cell culture supernatant was

The Human Ortholog of Lst Promotes WNV Infection in Human Fibroblasts
The human ortholog for limostatin is Neuromedin U, a peptide involved in feeding behavior, insulin regulation, and promoting the expression of inflammatory cytokines in adaptive immune cells [58].The expression of NMUR1 (NMU receptor 1) in HEK-293T cells has been used to demonstrate that NMU signaling suppresses proliferation of SKOV-3 ovarian cancer cells [59].In our experiments, we used the NHF1 cell line, as fibroblasts are infected when a WNV-infected mosquito takes a human bloodmeal [60], and these cells express IGF-1 receptor (IGF-1R) and secrete IGF-1 [61,62].To model the regulation of WNV infection by NMU in NHF1 cells, we first expressed NMUR1 in NHF1 cells (Figure 5A).In summary, insulin plays a key role in the immune response to WNV through the activation of the anti-viral JAK/STAT pathway [16].We have shown that flies that are hyperinsulinemic due to a mutation in the decretin hormone-producing gene Lst are less susceptible to infection and show upregulation of the JAK/STAT pathway during infection.When the insulin-secretion-suppressing human ortholog of Lst, NMU, is supplemented to human fibroblasts expressing NMUR1, the cells show a higher viral titer after infection.These results indicate insulin-suppressing hormones to be important in the innate immune response to WNV infection in insect and mammalian models.

Discussion
Insulin-mediated immune signaling is important in D. melanogaster, the mosquito, and the human cell response to WNV.Insulin has been implicated in insect immune signaling [63].For example, fly mutants of the InR substrate chico have increased resistance to bacterial infection [64].Thor, a gene involved in D. melanogaster host immune defense [65], is upregulated two-fold in infected chico mutants due to activation by higher FoxO activity induced by decreased insulin signaling [66].FoxO is a transcription factor in the RNAi antiviral signaling cascade that is downregulated in the presence of insulin [16] and is also known to induce antimicrobial peptide genes in the fly fat body [66].In the D. melanogaster antiviral response, insulin feeding activates the MAPK/ERK to restrict viral infection [25].Insulin priming activates the JAK/STAT antiviral pathway in D. melanogaster and mosquito cells [26].Feeding insulin to mosquitoes suppresses the RNAi pathway and activates the JAK/STAT antiviral pathway to suppress replication of another flavivirus, Zika virus [67].Here, we demonstrate that D. melanogaster with a mutation in the insulin suppression gene Lst is hyperinsulinemic and shows upregulated expression of genes within the antiviral JAK/STAT pathway during infection.It is likely that upregulated ilps in the Lst MI06290 mutant fly leads to stimulation of the IIS/IGF signaling pathway, resulting in upregulated expression of the JAK/STAT immune signaling pathway.It is possible that the upregulation of this antiviral pathway due to the loss of the insulin-regulator Lst contributes to decreased susceptibility to infection in Lst MI06290 mutant flies.
InR and IGF1-R signaling modulates downstream immune cell processes in human innate immune cells.InR and IGF1-R are expressed in monocytes and macrophages [68,69].Insulin treatment of in vitro human monocytes induces the production of pro- NHF1 cells expressing NMUR1 were treated with NMU-25 peptide to activate NMUR1.These cells were then infected with WNV-Kun, and cell culture supernatant was used for a standard plaque assay with BHK21 cells to measure viral titer.NFH1 cells expressing an active NMUR1 pathway and treated with 100 nM of NMU-25 peptide showed a significantly higher viral titer three days post-infection compared to cells transfected with the empty vector control plasmid or NMUR1-expressing cells not treated with NMU-25 peptide (Figure 5B).Thus, like flies expressing Lst compared to mutant flies, human cells expressing the NMU pathway and treated with the insulin regulator NMU exhibited increased WNV replication.
In summary, insulin plays a key role in the immune response to WNV through the activation of the anti-viral JAK/STAT pathway [16].We have shown that flies that are hyperinsulinemic due to a mutation in the decretin hormone-producing gene Lst are less susceptible to infection and show upregulation of the JAK/STAT pathway during infection.When the insulin-secretion-suppressing human ortholog of Lst, NMU, is supplemented to human fibroblasts expressing NMUR1, the cells show a higher viral titer after infection.These results indicate insulin-suppressing hormones to be important in the innate immune response to WNV infection in insect and mammalian models.

Discussion
Insulin-mediated immune signaling is important in D. melanogaster, the mosquito, and the human cell response to WNV.Insulin has been implicated in insect immune signaling [63].For example, fly mutants of the InR substrate chico have increased resistance to bacterial infection [64].Thor, a gene involved in D. melanogaster host immune defense [65], is upregulated two-fold in infected chico mutants due to activation by higher FoxO activity induced by decreased insulin signaling [66].FoxO is a transcription factor in the RNAi antiviral signaling cascade that is downregulated in the presence of insulin [16] and is also known to induce antimicrobial peptide genes in the fly fat body [66].In the D. melanogaster antiviral response, insulin feeding activates the MAPK/ERK to restrict viral infection [25].Insulin priming activates the JAK/STAT antiviral pathway in D. melanogaster and mosquito cells [26].Feeding insulin to mosquitoes suppresses the RNAi pathway and activates the

Figure 1 .
Figure 1.Lst MI06290 mutant D. melanogaster are hyperinsulinemic and exhibit increased Akt phosphorylation.Ilp expression and Akt phosphorylation were measured in adult (2-5-day-old) female flies (N = 5 flies per biological replicate).Gene expression was normalized to expression of the housekeeping gene rp49.(A) ilp2, (B) ilp3, (C) ilp5, and (D) ilp7 expression in adult Lst MI06290 flies compared to controls (y 1 w 1 ).(E) Fly lysates were subjected to western blot for phospho-Akt, total Akt, and actin.Original gels are provided as Supplementary Figure S1.Results are representative of three independent experiments.* p < 0.05 (unpaired t-test).

Figure 1 .
Figure 1.Lst MI06290 mutant D. melanogaster are hyperinsulinemic and exhibit increased Akt phosphorylation.Ilp expression and Akt phosphorylation were measured in adult (2-5-day-old) female flies (N = 5 flies per biological replicate).Gene expression was normalized to expression of the housekeeping gene rp49.(A) ilp2, (B) ilp3, (C) ilp5, and (D) ilp7 expression in adult Lst MI06290 flies compared to controls (y 1 w 1 ).(E) Fly lysates were subjected to western blot for phospho-Akt, total Akt, and actin.Original gels are provided as Supplementary Figure S1.Results are representative of three independent experiments.* p < 0.05 (unpaired t-test).

Figure 2 .
Figure 2. Lst MI06290 mutant flies are less susceptible to WNV infection.Adult (2-5-day-old, N = 40 flies per experiment) Lst MI06290 mutant and y 1 w 1 control flies were mock-infected or infected with WNV, and survival was monitored for 30 days.(A) Survival of WNV-infected flies of Lst MI06290 and control genotypes.(B) Survival of control fly genotype y 1 w 1 infected with PBS mock or WNV.(C) Survival of Lst MI06290 flies infected with PBS mock or WNV.Each survival curve represents three independent experiments (Table1) that combined for a final survival curve and statistical analyses.

Figure 5 .
Figure 5. Normal human fibroblasts with an active NMUR1 pathway are more susceptible to WNV infection.NHF1 cells (N = 3 wells of cells) were transfected with an NMUR1-expressing plasmid or an empty vector control and treated with either 0 nM or 100 nM of NMU-25 peptide 6 h post-transfection.Then, 24 h later, cells were infected with WNV-Kun (MOI 0.01 PFU/cell).At 72 h post-infection, cells were lysed, and cell culture supernatant was collected.(A) Cell lysate was subjected to western blot for FLAG-tagged NMUR1 and actin.Original gels are provided as supplementary figures.(B) Supernatant used in a standard plaque assay to measure viral titer.Results are representative of three independent experiments.* p < 0.0001 (one-way ANOVA).

Figure 5 .
Figure 5. Normal human fibroblasts with an active NMUR1 pathway are more susceptible to WNV infection.NHF1 cells (N = 3 wells of cells) were transfected with an NMUR1-expressing plasmid or an empty vector control and treated with either 0 nM or 100 nM of NMU-25 peptide 6 h post-transfection.Then, 24 h later, cells were infected with WNV-Kun (MOI 0.01 PFU/cell).At 72 h post-infection, cells were lysed, and cell culture supernatant was collected.(A) Cell lysate was subjected to western blot for FLAG-tagged NMUR1 and actin.Original gels are provided as supplementary figures.(B) Supernatant used in a standard plaque assay to measure viral titer.Results are representative of three independent experiments.* p < 0.0001 (one-way ANOVA).

Table 1 .
Statistics of individual infection trials.