11 research outputs found
The proteasomal pathway is important during WNV infection.
<p><b>A</b>. Hsu cells were pre-treated with MG132 at various concentrations (0.1, 1, 10 μM) for 1 h prior to WNV infection. Total RNA and supernatant medium were collected at 48 hpi. Real-time RT-qPCR was performed using WNV NS1 primers. RpL32 primers were used as an internal control. Error bars represent standard errors from three separate experiments with assays performed in triplicate (Student’s t-test *p < 0.05, comparing between control and MG132 treated cells). <b>B</b>. Viral titer estimation by plaque assays conducted on the supernatant media from cells treated as in <b>A</b>. Error bars represents standard error from three separate experiments with assays performed in triplicate (Student’s t-test *p < 0.05 compared to control). <b>C</b>. Hsu cells were pre-treated with MG132 at various concentrations (0.1, 1, 10 μM) for 1 h prior to infection with WNV at 4°C. Cells were moved to 30°C and total RNA was collected at 6 hpi. Real-time RT-qPCR was performed using WNV NS1 primers. RpL32 primers were used as an internal control. Error bars represent standard error from three separate experiments with assays performed in triplicate (Student’s t-test *p < 0.05, comparing between control and MG132 treated cells). <b>D</b>. Hsu cells were infected with WNV, followed by treatment with 10 μM MG132 at 2, 4 or 6 hpi. Total RNA was collected at 48 hpi and real-time RT-qPCR was performed using WNV NS1 primers. RpL32 primers were used as an internal control. Error bars represents standard error from three separate experiments with assays performed in triplicate (Student’s t-test *p < 0.05, comparing between control and MG132-treated cells).</p
Cullin4 Is Pro-Viral during West Nile Virus Infection of <i>Culex</i> Mosquitoes
<div><p>Although mosquitoes serve as vectors of many pathogens of public health importance, their response to viral infection is poorly understood. It also remains to be investigated whether viruses deploy some mechanism to be able to overcome this immune response. Here, we have used an RNA-Seq approach to identify differentially regulated genes in <i>Culex quinquefasciatus</i> cells following West Nile virus (WNV) infection, identifying 265 transcripts from various cellular pathways that were either upregulated or downregulated. Ubiquitin-proteasomal pathway genes, comprising 12% of total differentially regulated genes, were selected for further validation by real time RT-qPCR and functional analysis. It was found that treatment of infected cells with proteasomal inhibitor, MG-132, decreased WNV titers, indicating importance of this pathway during infection process. In infection models, the <i>Culex</i> ortholog of mammalian Cul4A/B (cullin RING ubiquitin ligase) was found to be upregulated <i>in vitro</i> as well as <i>in vivo</i>, especially in midguts of mosquitoes. Gene knockdown using dsRNA and overexpression studies indicated that <i>Culex</i> Cul4 acts as a pro-viral protein by degradation of <i>Cx</i>STAT via ubiquitin-proteasomal pathway. We also show that gene knockdown of <i>Culex</i> Cul4 leads to activation of the Jak-STAT pathway in mosquitoes leading to decrease viral replication in the body as well as saliva. Our results suggest a novel mechanism adopted by WNV to overcome mosquito immune response and increase viral replication.</p></div
<i>Culex</i> cullin (Cul4) blocks the Jak-STAT pathway.
<p><b>A</b>. Hsu cells were transfected with plasmid overexpressing <i>Culex</i> cullin (<i>Cx</i>Cul4). Empty vector (Control) was used as transfection control. At 24 h post-transfection, the cells were infected with WNV and total RNA was collected at 48 hpi. Real-time RT-qPCR was performed using <i>Culex</i> Vir1-specific and <i>Culex</i> Vago-specific primers. RpL32 primers were used as an internal control. Error bars represent standard error from three separate experiments with assays performed in triplicate (Student’s t-test *p < 0.05, comparing between control and Cul4-overexpressing cells). <b>B</b>. Hsu cells were transfected with plasmid overexpressing <i>Culex</i> cullin (CxCul4). Empty vector (Control) was used as transfection control. At 24 h post-transfection, the cells were treated with <i>Cx</i>Vago-containing medium and total RNA was collected 48 h later. Real-time RT-qPCR was performed using <i>Culex</i> Vir1-specific primers. RpL32 primers were used as an internal control. Error bars represent standard error from three separate experiments with assays performed in triplicate (Student’s t-test *p < 0.05, comparing between control and Cul4-overexpressing cells). <b>C</b>. Hsu cells were transfected with p6x2DRAF-Luc (STAT reporter) and pAct-Renilla (control) plasmids along with dsRNA against <i>Culex</i> Cul4 (Cul4 dsRNA) or GFP (GFP dsRNA). Cells were treated with heat-inactivated <i>E</i>. <i>coli</i>, WNV or PBS for 1 h. Luciferase activity was measured 16 h post-stimulation. Fold increase over untreated control were plotted after normalising with Renilla transfection control. Error bars represent standard error from six separate samples (Student’s t-test *p < 0.05, comparing between GFP-dsRNA and Cul4-dsRNA cells for each stimulation). <b>D</b>. Hsu cells were transfected with plasmid overexpressing <i>Culex</i> cullin (CxCul4). Empty vector (Control) was used as a silencing control. At 24 h post-transfection, the cells were infected with WNV and treated with MG132 (1 μM) or PYR-41 (1 μM) or treated with drugs alone. Total RNA was collected at 48 hpi and real time RT-qPCR was performed using <i>Culex</i> Vir1 and WNV NS1-specific primers. RpL32 primers were used as internal control. Error bars represent standard error from three separate experiments with assays performed in triplicate (Student’s t-test *p < 0.05, comparing between control and Cul4-overexpressing cells treated with or without inhibitors). <b>E.</b> Hsu cells were transfected with dsRNA against <i>Culex</i> cullin (Cul4 dsRNA). No dsRNA or GFP dsRNA was used as a silencing control. At 24 h post-transfection, the cells were infected with WNV and total RNA was collected 48 hpi. Real-time RT-qPCR was performed using <i>Cx</i>Vir1 (Vir1) and <i>Cx</i>Vago (Vago)-specific primers. RpL32 primers were used as internal control. Error bars represent standard error from three separate experiments with assays performed in triplicate (*p < 0.05, comparing between No dsRNA, GFP dsRNA and Cul4-knock-down cells). <b>F.</b> Hsu cells were transfected with dsRNA against <i>Culex</i> cullin (Cul4 dsRNA) or <i>Culex</i> STAT (STAT dsRNA) or both. GFP dsRNA was used as a silencing control (Control). At 24 h post-transfection, the cells were infected with WNV and total RNA was collected 48 hpi. Real-time RT-qPCR was performed using WNV NS1-specific primers. RpL32 primers were used as internal control. Error bars represent standard error from three separate experiments with assays performed in triplicate (Student’s t-test *p < 0.05, comparing between Control and Cul4 dsRNA or STAT dsRNA cells).</p
WNV NS1 and NS5 are responsible for <i>Cx</i>Cul4 regulation.
<p><b>A.</b> Hsu cells were transfected with WNV genes (C-Capsid; NS1, NS3, NS4A or NS5). Total RNA was collected at 24 hours post-transfection and real time RT-qPCR was performed using <i>Culex</i> Cul4-specific primers. RpL32 primers were used as internal control. Error bars represent standard error from three separate experiments with assays performed in triplicate (Student’s t-test *p < 0.05, significant from empty vector control). <b>B.</b> Real time Rt-qPCr was performed on cells treated as in <b>A</b>, using <i>Culex</i> Cullin3-specific primers. <b>C</b>. Hsu cells were transfected with dsRNA against <i>Culex</i> cullin (Cul4 dsRNA). GFP dsRNA was used as a silencing control (Control). At 24 h post-transfection, the cells were infected with WNV and cell lysates were collected 24 hours post-infection. Western blot was performed using anti-<i>Culex</i> STAT and anti-beta actin antibodies, with marker representing proteins with known molecular weight (in kDa). The experiment was repeated 3 times and representative blots shown here. <b>D</b>. Hsu cells were transfected with WNV genes (NS1, NS5 or NS3) along with dsRNA against <i>Culex</i> cullin (+). GFP dsRNA was used as a silencing control (-). Control indicates empty vector transfection. Cell lysates were collected 24 hours post-infection. Western blot was performed using anti-<i>Culex</i> STAT and anti-beta actin antibodies. The experiment was repeated and representative blots shown here. <b>E.</b> Hsu cells were transfected with dsRNA against <i>Culex</i> cullin (Cul4 dsRNA). GFP dsRNA was used as a silencing control (GFP dsRNA). At 24 h post-transfection, the cells were infected with WNV and treated with MG132 (1 μM) and cell lysates were collected 24 hours post-infection. Immunoprecipitation was performed using anti-ubiquitin antibody, followed by Western blot using anti-ubiquitin (Upper) and anti-<i>Cx</i>STAT (lower) antibodies. WCL represents whole cells lysate (pre-IP), FT represents flow-through and IP represents immunoprecipitate (eluate). The experiment was repeated twice and representative blots shown here. <b>F.</b> Female <i>Culex annulirostris</i> mosquitoes were microinjected with dsRNA against Culex Cullin (Cul4). GFP dsRNA was used as a silencing control. At 24 h post-injection, mosquitoes were blood-fed with WNV for 1h. At 2 days and 10 days post-infection, mosquitoes were collected and processed for Western blot using anti-<i>Cx</i>STAT and anti-actin antibodies. The experiment was repeated and representative blots shown here.</p
<i>Culex</i> cullin (Cul4) is pro-viral.
<p><b>A</b>. Female <i>Culex annulirostris</i> mosquitoes (n = 40) were infected with WNV by blood feeding. Mosquitoes were micro-dissected at 24 hpi and total RNA was collected from the midgut and the remaining carcass. Real-time RT-qPCR was performed using <i>Culex</i> cullin (Cul4) primers. RpL32 primers were used as an internal control. Error bars represent standard error from four pooled mosquitoes with assays performed in triplicate (Student’s t-test *p < 0.05, comparing between control and WNV-infected carcasses and midguts). <b>B</b>. Hsu cells were transfected with dsRNA against <i>Culex</i> cullin (Cul4 dsRNA). No dsRNA or GFP dsRNA was used as a silencing control. At 24 h post-transfection, the cells were infected with WNV and total RNA was collected 48 hpi. Real-time RT-qPCR was performed using WNV NS1-specific primers. RpL32 primers were used as internal control. Error bars represent standard error from three separate experiments with assays performed in triplicate (*p < 0.05, comparing between No dsRNA, GFP dsRNA and Cul4-knock-down cells). <b>C</b>. Viral titer estimation by plaque assays conducted on the supernatant media collected from cells treated as in <b>B</b>. <b>D</b>. Western blot using anti-WNV NS5 and anti-β-actin antibodies conducted on lysates collected from cells treated as in <b>B</b>. Molecular weight markers represented as kDa. The band at around 95 kDa is expected size of WNV-NS5; while band at 191 kDa is non-specific. <b>E</b>. Hsu cells were transfected with <i>Culex</i> Cul4 overexpression plasmid. Empty vector (Control) was used as a transfection control. At 24 h post-transfection, the cells were infected with WNV and cell lysates were collected at 48 hpi. Total RNA was collected from cells and real-time RT-qPCR was performed using WNV NS1-specific primers. RpL32 primers were used as an internal control. Error bars represent standard error from three separate experiments with assays performed in triplicate (Student’s t-test *p < 0.05, comparing between control and Cul4-transfected cells). <b>F</b>. Viral titer estimation by plaque assays conducted on the supernatant media collected from cells treated as in <b>E</b>. <b>G</b>. Hsu cells were transfected with <i>Culex</i> Cul4 overexpression plasmid. Empty vector (Empty) was used as a transfection control. At 24 h post-transfection, the cells were infected with WNV and cell lysates were collected at 48 hpi. Western blot was performed using anti-V5 (detecting <i>Cx</i>Cul4 overexpression) and anti-β-actin antibodies. Molecular weight markers represented as kDa.</p
Pathway analysis and validation.
<p><b>A</b>. Pathway (molecular and cellular function) analysis was performed for all differentially expressed genes using Ingenuity software. The results are represented here as a pie-chart to indicate the contribution of major cellular pathways during WNV infection. <b>B</b>. Hsu cells were infected with WNV and total RNA was collected at 48 hpi. Cells were mock-infected as controls. Real-time RT-qPCR was performed using primers for ubiquitin-proteasomal pathway genes found to be differentially regulated in the RNA-Seq screen. RpL32 (ribosomal protein L32) primers were used as an internal control. The graph was plotted as fold-increase over control (set arbitrarily at 1, not included). Error bars represent standard error from three separate experiments with assays performed in triplicate. The results from the RNA-Seq experiment were also plotted on the graph for comparison.</p
PO activity in U4.4 cell-conditioned medium.
<p>(<b>A</b>) PO activity in conditioned medium without immune challenge (Control) or after the addition of <i>E. coli</i> or purified SFV virions. One unit (U) of PO activity was defined as <b>Δ</b>A<sub>490</sub> = 0.001 after 30 minutes incubation (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002977#s4" target="_blank">Materials and Methods</a>). Each bar represents the mean from 10 reactions; error bars show standard deviation. This experiment was repeated three times with similar results. (<b>B</b>) SFV virion viability after a 1 h incubation at 28°C in unconditioned culture medium or medium conditioned by U4.4 cells for 48 h. Viability was then determined by titration of SFV on BHK-21 cells. PFU: plaque forming units. Each bar represents the mean from triplicate incubations; error bars show standard deviation. This experiment was repeated three times with similar results. (<b>C</b>) Staining for intracellular PO activity in U4.4 cells. Arrow indicates a U4.4 cell that melanised after fixation and incubation with the PO substrate dopamine. Note the larger size of this cell and its rounded morphology relative to surrounding cells that have not melanised.</p
Spread of SFV in mosquito and vertebrate cells.
<p>(<b>A</b>) The addition of glutathione (GSH) to medium enhances the spread of SFV. U4.4 cells were infected with SFV4(3H)-<i>FFLuc</i>-Egf1.0F (Egf1.0F) or SFV4(3H)-<i>FFLuc</i>-Egf1.0R (Egf1.0R) at an MOI of 0.005 followed by determination of <i>FFLuc</i> activity at 48 h p.i. + GSH: 0.5 mM GSH; − GSH: negative control. Each bar represents the mean from triplicate cultures; error bars show standard deviation. This experiment was repeated three times with similar results. (<b>B</b>) Egf1.0 has no effect on SFV spread in BHK-21 cells. Cells were infected with SFV4(3H)-<i>FFLuc</i>-Egf1.0F (Egf1.0F) or SFV4(3H)-<i>FFLuc</i>-Egf1.0R (Egf1.0R) at an MOI of 0.005 followed by determination of <i>FFLuc</i> activity at 24 h and 48 h p.i. Each bar represents the mean from triplicate cultures; error bars show standard deviation. This experiment was repeated three times with similar results.</p
Expression of Egf1.0 increases mortality of <i>Ae. aegypti</i> and replication of SFV <i>in vivo</i>.
<p>(<b>A</b>) <i>Ae. aegypti</i> were fed blood containing SFV4(3H)-<i>FFLuc</i>-Egf1.0F or SFV4(3H)-<i>FFLuc</i>-Egf1.0R. Uninfected blood meals served as a control. Mosquito mortality was then monitored daily post-bloodmeal. Combined survival data from three independent experiments (cohorts of 22–25 infected mosquitoes per virus or control mosquitoes in each experiment) are shown. Error bars show standard deviation. (<b>B</b>) SFV genome copy number as determined by real time qPCR. Total RNA was extracted 3 days post-bloodmeal from mosquitoes infected with SFV4(3H)-<i>FFLuc</i>-Egf1.0F or SFV4(3H)-<i>FFLuc</i>-Egf1.0R. Viral genome RNA levels from 10 mosquitoes for each virus are shown. Values at 0 represent uninfected mosquitoes. Horizontal bar indicates average genome copy number from infected mosquitoes. This experiment was repeated three times with similar results.</p
Recombinant SFV expresses Egf1.0 and inhibits PO activity in U4.4 cell-conditioned medium.
<p>(<b>A</b>) Immunoblots showing Egf1.0 expression and secretion from mosquito cells. U4.4 cells were infected with SFV4(3F)-<i>ZsGreen</i>-Egf1.0F or SFV4(3F)-<i>ZsGreen</i>-Egf1.0R at an MOI of 10 followed by preparation of cell lysate and medium samples at 48 h p.i. as indicated in the <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002977#s4" target="_blank">Materials and Methods</a>. The left blot was probed with an anti-SFV nsP3 antibody with individual lanes labeled as follows: U4.4 cells infected with SFV4(3F)-<i>ZsGreen</i>-Egf1.0R (R = cell lysate, Rm = conditioned medium), U4.4 cells infected with SFV4(3F)-<i>ZsGreen</i>-Egf1.0F (F = cell lysate, Fm = conditioned medium), or uninfected cells (U = cell lysate, Um = conditioned medium). Black star identifies the nsP3-ZsGreen protein, only detected in lysates from SFV-infected cells. Black diamond indicates bovine serum albumin (non-specifically detected because of high abundance). The right blot shows the same samples probed with an anti-Egf1.0 antibody. A control lane (C) was added to this blot (purified, recombinant Egf1.0). Note that Egf1.0 is only detected in the control lane, and F and Fm lanes. Black arrow indicates uncut Egf1.0; open arrow identifies a band corresponding to the predicted C-terminal domain of Egf1.0 after PAP cleavage. Molecular mass markers indicated to the left. (<b>B</b>) PO activity in conditioned medium from uninfected U4.4 cells (Control), cells infected with SFV4(3H)-<i>FFLuc</i>-Egf1.0F (Egf1.0F), SFV4(3H)-<i>FFLuc</i>-Egf1.0R (Egf1.0R), cells infected with SFV4(3H)-<i>FFLuc</i>-Egf1.0F with <i>E. coli</i> added to the medium (Egf1.0F+<i>E. coli</i>), cells infected with SFV4(3H)-<i>FFLuc</i>-Egf1.0R with <i>E. coli</i> added to the medium (Egf1.0R+<i>E. coli</i>), or medium from uninfected cells with <i>E. coli</i> added (<i>E. coli</i>). PO activity was measured as outlined in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002977#ppat-1002977-g002" target="_blank">Fig. 2A</a>; 1 ml of conditioned medium was taken at 48 h p.i. from 2.6×10<sup>5</sup> U4.4 cells infected at an MOI of 10, or uninfected (Control). Each bar represents the mean from 10 reactions; error bars show standard deviation. This experiment was repeated three times with similar results.</p