Influenza Infection Potentiates PLD Catalytic Activity, and That Activity Is Attenuated by VU0364739 Treatment To determine the effect of influenza infection on PLD activity, human adenocarcinomic alveolar basal epithelial cells (A549) were infected with 1 m.o.i. human influenza strain A/California/04/2009 (H1N1) for 6 h in the presence of 0.6% n-butyl alcohol. Instead of the typical biological nucleophile water, in the presence of a primary alcohol, PLD will utilize the alcohol to produce a metabolically stable transphosphatidylation product, phosphatidylalcohol, as an alternative to hydrolysis producing PA. Fig. 1A illustrates that influenza infection markedly stimulated PLD activity as measured by the increase in phosphatidylbutanol production compared with cells that were not exposed to influenza virus (mock-infected). Treatment with a PLD2-preferring inhibitor, VU0364739, was sufficient to block the influenza-mediated PLD activity increase. As shown in Fig. 1B, both PLD1 and PLD2 were active during the viral entry phase, and complete ablation of catalytic activity required knockdown of both PLD1 and -2. This result suggests independent roles for both isoenzymes in entry. View larger version: Download as PowerPoint Slide FIGURE 1. Influenza infection stimulates PLD activity. A, A549 cells treated for 1 h with or without 10 μm PLD inhibitor VU0364739 were then infected for 1 h with 1 m.o.i. influenza A/California/04/2009 (or mock-infected) after which the inoculum was removed. The cells were cultured for 6 h in the presence of 0.6% n-butyl alcohol. PLD activity as a measure of phosphatidylbutanol (PtdBuOH) was determined in cell lysates by mass spectrometric analysis. B, RNAi of PLD1, PLD2, or PLD1 and -2. A549 cells were transfected with isoform-specific siRNA 1 day before a 5 m.o.i. infection of influenza A/Brisbane/59/2007 (H1N1). 30 min after infection, PLD catalytic activity was measured in the same manner as in A. C, D, and E, A549 cells were infected with 1 m.o.i. influenza A/California/04/2009 (H1N1), and samples were fixed and probed for influenza NP and PLD2 at 0, 30, 90, and 360 min after infection. Z-stacks of the infected cultures were collected using confocal microscopy, and ImageJ was used to determine PLD and influenza NP colocalization (C) during infection as well as protein accumulation (D). Quantification of the degree of correlation between NP and PLD shows that they significantly colocalize as early as 30 min postinfection, and the colocalization has the highest correlation coefficient 360 min postinfection. PLD2 signal begins to intensify 30 min after infection and continues to increase in both size and intensity throughout the duration of the infection. NP signal lags behind that of PLD2 but begins to intensify 90 and 360 min postinfection. E, representative images from a 1 m.o.i. influenza A/California/04/2009 (H1N1) infection taken 0, 30, 90, and 360 min postinfection. The green signal is influenza (NP), the magenta signal is PLD2, and the blue signal is DAPI staining. Scale bars, 10 μm. As the infection progresses, PLD2 staining begins to accumulate at the cell periphery, then intensifies, and finally moves to a perinuclear region. NP staining first appears at the cell periphery and then intensifies in the nucleus. F, representative images from PLD2 monoclonal antibody validation. A549 cells were electroporated with 100 nm scrambled control or PLD2 siRNA and left to rest for 24 h. Cells were fixed 8 h after a 1 m.o.i. A/Brisbane/59/2007 (H1N1) infection. Green signal is influenza NP, red signal is PLD2, and blue signal is nuclei. All data are mean ± S.E. (error bars). *, p < 0.05; ***, p < 0.001. KD, knockdown. Confocal microscopy-based experiments were conducted to assess changes in the accumulation and localization of PLD during an influenza infection. A549 cells were grown on chamber slides and infected with 1 m.o.i. influenza A/California/04/2009 (H1N1). Samples were fixed at 0, 30, 90, and 360 min postinfection and probed for influenza NP and PLD2. PLD2 began to accumulate at the periphery of the cell as early as 30 min postinfection (Fig. 1, C, D, and E). At the same time point, very low levels of NP, also occurring at the outer reaches of the cell (Fig. 1E), were detected. PLD2 staining continued to intensify 90 and 360 min postinfection (Fig. 1D), and the observed PLD2 signal was increasing as well as moving from the cytoplasm to a perinuclear region (Fig. 1E). Influenza NP signal also intensified 360 min postinfection, and NP was moving from the cytoplasm (30 and 90 min postinfection) toward the nucleus (Fig. 1, D and E). During the infection, PLD2 and NP were trafficking to similar subcellular locations. The extent of this colocalization was quantified. PLD2 and NP were increasingly colocalized at 30, 90, and 360 min postinfection (Fig. 1C) as measured by both Pearson's coefficient and rank correlation (Spearman). To further confirm that siRNA treatment was knocking down levels of PLD2 and that the PLD2 antibody was specific, we probed for PLD2 expression after infection of cells electroporated with control or PLD2 siRNA. We imaged more than 75 cells for each condition and found a consistent loss of 50% or more of stainable PLD2 in the siRNA-treated cells (1F). Together these data suggest that PLD activity is stimulated by influenza infection, endogenous PLD is redistributed during the infection, and the accumulation of PLD is occurring in the same subcellular location as influenza NP.

PLD Is a Targetable Host Factor That Facilitates Efficient Influenza Infection To determine the role of PLD-mediated PA production in influenza infection, we used a spatial infection model in the presence of 0.6% n-butyl alcohol with tert-butyl alcohol used as a negative control. This assay utilizes the use of primary alcohols as preferred nucleophiles in a PLD transphosphatidylation reaction as an assessment of exclusively PLD-produced PA. Twenty-four hours postinfection, infected cells were counted by anti-influenza NP staining (Fig. 2A). Infection with influenza strain A/Brisbane/59/2007 (H1N1), A/California/04/2009 (H1N1), or A/Brisbane/10/2007 (H3N2) resulted in fewer infected cells in the presence of n-butyl alcohol, indicating that blocking PLD production of PA substantially reduces the rate of cell to cell transmission of infection. Notably, averting the production of PLD-generated PA to PLD-generated phosphatidylbutanol by use of primary alcohol did not entirely prevent influenza infection but did significantly decrease the rate of infectious spread within the cultures. View larger version: Download as PowerPoint Slide FIGURE 2. PLD2 is required for efficient influenza infection, and inhibition of PLD-generated PA by primary alcohol, RNAi, or small molecule VU0364739 dramatically hinders cell to cell spread of influenza. A, number of influenza-infected cells in cultures of A549 cells treated with 0.6% tert-butyl alcohol or n-butyl alcohol for 1 h prior to spatial infection with the indicated influenza virus strain at 1 m.o.i. for 24 h. Differences were assessed by t test. B, upper panel, RNAi of PLD isoforms. A549 cells were transfected with siRNA targeting PLD1 or PLD2 24 h prior to infection with 5 m.o.i. influenza A/Brisbane/59/2007 (H1N1); 8 h postinfection, influenza replication was measured by TCID 50 assay. Lower panel, immunoblot of human PLD1 and PLD2 from A549 cells following RNAi treatment. Lysates were separated on a 10% polyacrylamide gel, transferred to nitrocellulose overnight, and incubated with primary antibodies for phosphatidylcholine-PLD1 (Santa Cruz Biotechnology sc-25512) or PLD2 (Abgent AT3337a). Immunoblots were developed with ECL Western blotting substrate (Pierce catalog number 32106). C, A549 cells were transiently transfected with PLD2-specific siRNA or scrambled control siRNA 24 h before infection. An hour before infection, the cells were treated with DMSO or VU0364739 (10 μm). The cells were infected with 5 m.o.i. A/Brisbane/59/2007 (H1N1), and influenza replication was measured by TCID 50 assay 24 h after infection. Differences between amounts of infected cells as well as influenza replication were compared using a one-way ANOVA and Dunnett's post-test. D, influenza spatial infection (following 1-h 10 μm PLD2-preferring inhibitor VU0364739 pretreatment) with clinically relevant strains of influenza at 1 m.o.i. At both 6 and 24 h postinfection, significantly fewer numbers of influenza-infected cells were counted in VU0364739-pretreated samples. Data were analyzed by t test. E, representative fluorescent photomicrograph mosaics following a 24-h spatial infection of A549 cells with influenza A/Brisbane/59/2007 (H1N1). Green signal is influenza-infected cells, and blue signal is DAPI staining. Scale bars, 10 μm. All data are mean ± S.E. (error bars). *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001. To determine whether PLD1 or PLD2 was preferentially required for efficient influenza infection, RNAi was used to selectively knock down individual PLD isoforms. A549 cells were transiently transfected with siRNA that targeted PLD1 or PLD2 24 h prior to a 5 m.o.i. influenza infection of A/Brisbane/59/2007 (H1N1). Twenty-four hours postinfection, a TCID 50 assay was used to measure influenza virus titers in culture supernatants (Fig. 2B). RNAi of either PLD1 or PLD2 inhibited influenza replication. However, the magnitude of the effect was greater with PLD2 knockdown. Although each isoform appears to contribute to efficient influenza replication, the stronger inhibitory effects seen after PLD2 knockdown led us to focus on PLD2 inhibition and its effect on the host and viral replication for subsequent studies. The development and optimization of the PLD inhibitors have been described (12) with the PLD2-preferring inhibitor VU0364739 having a 75-fold selectivity for PLD2 over PLD1 (13). Based on previous studies, VU0364739 was used at 10 μm in all cell-based assays unless otherwise indicated (21). To demonstrate the importance of PLD2 during influenza replication, another RNAi experiment was conducted while treating the PLD2-deficient cells with PLD2 inhibitor VU0364739. Viral titer was measured by a TCID 50 assay after a 24-h, 5 m.o.i. influenza A/Brisbane/59/2007 (H1N1) infection. Pretreatment with VU0364739 for 1 h caused a dramatic decrease in viral replication (Fig. 2C). Similar reductions in titer were also observed when PLD2 was knocked down by RNAi in vehicle-treated samples. Finally, the specificity of this compound was confirmed by combining the apparent marginal, non-statistically significant additive nature of RNAi with VU0364739 treatment. These observations were extended to establish the importance of PLD2 as a host factor required for infection by other clinically relevant strains of influenza. Pretreating cells for 1 h with VU0364739 resulted in a significant decrease in the number of infected cells measured in the spatial infection assay with multiple viral strains at both 6 and 24 h postinfection (Fig. 2D). Representative images of the spatial infection model assay (Fig. 2E) illustrate the reduction in influenza spread observed after VU0364739 treatment. PLD2 inhibition by VU0364739 effectively protected A549 cells from cell to cell spread of influenza, further demonstrating that PLD2 is required for efficient influenza infection and spread. A time-of-addition study was conducted where inhibitor was added over a time course spanning 2 h before infection to 4 h postinfection. Protection from infection was observed at all times of PLD inhibitor addition (data not shown).

Inhibition of PLD Activity Dramatically Reduces Influenza Replication The spatial infection model assay data were validated using the more traditional TCID 50 assay to assess viral reproduction in vitro. A549 cells were treated with 10 μm VU0364739 for 1 h before infection, and the cells were then infected with the indicated doses and strains of influenza. At the indicated time points, infectious supernatant was removed from the A549 cells and titrated on Madin-Darby canine kidney cells to measure viral reproduction. Using either a low m.o.i. (Fig. 3A) or a high m.o.i. (Fig. 3B) of influenza A/Brisbane/10/2007 (H3N2), a lower viral reproduction was observed when PLD2 was inhibited. In the case of the low m.o.i. infection, viral titers were first noted to be lower at 16 h postinfection, and the effect was sustained 24 h postinfection. Using the high m.o.i. model, when A549 cells were treated with VU0364739, a significant reduction in viral reproduction was noted 12 h postinfection and lasted through at least 24 h. View larger version: Download as PowerPoint Slide FIGURE 3. Influenza replication is severely reduced when PLD2 is inhibited by VU034739. A549 cells were pretreated with 10 μm VU0364739 or DMSO for 1 h and then infected with either low 0.01 m.o.i. H3N2 influenza (A), high 5.0 m.o.i. H3N2 influenza (B), 0.01 m.o.i. H5N1 influenza (C), or 0.01 m.o.i. H7N9 influenza (D) for an hour at 4 °C, and then the infectious supernatant containing the virus was removed and titrated on Madin-Darby canine kidney cells to assess viral production at the indicated times postinfection. Under PLD2 inhibitor treatment, poor viral replication in all influenza strains tested by 24 h postinfection was observed, and the viral output defect was noted as early as 12 h postinfection in the case of H3N2 and H7N9. Differences were assessed using a two-way ANOVA and Bonferroni's post-test where * represents p < 0.05, *** represents p < 0.001, and **** represents p < 0.0001. E and F, dose-response curves to determine the IC 50 of VU0364739 on influenza titer after a 24-h infection with rg-A/Vietnam/1204/2004 (H5N1) (E) or A/Anhui/1/2013 (H7N9) (F). A549 cells were treated with the indicated concentration of VU0364739 for 1 h before infection, and a TCID 50 assay was used to measure viral load. All data are mean ± S.E. (error bars). The H3N2 strain used in our previous experiments is considered a low pathogenicity strain of influenza. To determine whether host PLD is required for high pathogenicity and quickly replicating strains of influenza, VU0364739-treated A549 cells were infected with 0.01 m.o.i. influenza rg-A/Vietnam/1203/2004 (H5N1), and viral reproduction was assessed during a more severe infection. Twenty-four hours postinfection, a massive decrease in viral titer was observed when PLD2 was inhibited during H5N1 infection (Fig. 3C). Subsequent investigation to determine whether host PLD2 activity is required for infection was conducted using a recently emergent virus with pandemic potential, influenza strain A/Anhui/01/2013 (H7N9). After 1 h of 10 μm VU0364739 pretreatment, viral reproduction was effectively blocked 24 h postinfection (Fig. 3D). Viral titer was near the limit of detection when PLD catalytic activity was inhibited, consistent with PLD2 being a host factor required for low and high pathogenicity influenza infections. Using the same model, in vitro dose-response experiments were performed using VU0364739 to determine the efficacy of the PLD2 inhibitor during H5N1 or H7N9 infection. A549 cells were treated for 1 h with varying concentrations of VU0364739 before a 0.01 m.o.i. infection of either influenza rg-A/Vietnam/1204/2004 or A/Anhui/1/2013. Supernatant was then used to measure viral reproduction in a TCID 50 assay 24 h postinfection. In the case of the H5N1 infection, the IC 50 of VU0364739 was calculated by non-linear regression analysis to be 2.1 μm (Fig. 3E). Similarly, the IC 50 of VU0364739 was found to be 3.4 μm when cells were infected with an H7N9 influenza strain (Fig. 3F). These IC 50 values are consistent with in vitro dose-response experiments using influenza A/Brisbane/59/2007 (H1N1), A/Brisbane/10/2007 (H3N2), and A/California/04/2009 (H1N1) as well (data not shown). Based on these results, it was determined that inhibition of PLD2 can significantly lower influenza reproduction in vitro and that the decrease in viral titer occurs in a dose-dependent fashion.

PLD2-preferring Inhibitor VU0364739 Reduced Viral Titer in Mouse Lungs and Delayed Mortality during Lethal H7N9 Influenza Infection Having shown that abrogation of PLD2 activity leads to a decrease in viral spread and reproduction in vitro, we wanted to determine whether the loss of PLD2 could reduce viral titer in vivo. Female C57BL/6 (B6) mice were treated intraperitoneally with dilutions of PLD2-preferring inhibitor VU0364739 every 8 h from day −1 to 8 h after infection with 1 LD 50 (4000 EID 50 ) influenza A/Puerto Rico/8/1934 (H1N1) (PR8) administered intranasally on day 0. Because of solubility issues and observed acute vehicle toxicity, a long term survival study with optimal dosing to achieve the therapeutic in vivo concentration continuously was not feasible at this time, and viral titer was used as a readout to determine the role of PLD2 in a mouse model of influenza infection. Viral titer in lungs decreased significantly with PLD2 inhibitor VU0364739 treatment (Fig. 4A), and these protective effects were dose-dependent. Additionally, to determine whether PLD2 inhibition could lead to lower viral titers in a more chronic situation, mice were given 13 mg/kg VU0364739 every 8 h intraperitoneally from day −1 to day 3 after infection. Animals treated with VU0364739 had significantly less viral replication in their lungs (Fig. 4B) on day 3. Concomitant with the decrease in viral titers, 8 h after PR8 influenza infection, significant up-regulation of the innate immune proteins Mx1, OASL (2′-5′-oligoadenylate synthetase-like protein), and IFITM3 was observed when PLD2 was inhibited (Fig. 4C), indicating that the early immune response may be an important part of the mechanism of protection when mice are treated with VU0364739. IFITM3 has recently been described as a human restriction factor for influenza infection (22). View larger version: Download as PowerPoint Slide FIGURE 4. PLD2 inhibitor decreased early viral titer in a dose-dependent manner and delayed mortality in a lethal H7N9 model. A, mice treated with VU0364739 displayed lung viral titers that were drastically reduced 8 h after 4000 EID 50 PR8 influenza infection, and the reduction occurred in a dose-dependent manner. Data were compared by ANOVA and Bonferroni's post-test with n ≥ 5 mice used for each dose. **, p < 0.01. B, viral replication was similarly inhibited 3 days after 4000 EID 50 PR8 influenza infection in mice treated with 13 mg/kg VU0364739 three times a day from day −1 to day 3 postinfluenza infection. Data were analyzed by t test. **, p < 0.01. C, RNA from PR8 influenza-infected lungs was isolated, and gene expression was measured using TaqMan-based quantitative PCR 8 h after infection. Innate immune proteins Mx1, OASL (2′-5′-oligoadenylate synthetase-like protein), and IFITM3 were significantly up-regulated as early as 8 h after infection as measured by t test. *, p < 0.05. D, mice receiving 13 mg/kg VU0364739 twice a day from day −1 to day 5 postinfection were inoculated with 103.5 TCID 50 (1 LD 50 ) influenza A/Anhui/1/2013 (H7N9). The PLD2 inhibitor conferred a substantial delay in mortality and a 20% survival advantage, a significant benefit as measured by either log rank test (p = 0.0194) or Gehan-Breslow-Wilcoxon test (p = 0.0165). *, p < 0.05. E, plasma, brain, lung, and liver exposures following a single 10 mg/kg intraperitoneal dose of the PLD inhibitor VU0364739 in mouse. Samples were stored at −80 °C until extraction and LC-MS/MS analysis (n = 2 samples per time point). Data are means ± S.E. (error bars). To identify survival benefits conferred by the observed reduction in viral titers, a study was conducted dosing mice with 13 mg/kg VU0364739 every 12 h from day −1 to day 3 of a lethal influenza A/Anhui/1/2013 (H7N9) infection (Fig. 4D). Relative concentrations of the PLD inhibitor in various tissues of interest over time are presented in Fig. 4E. Administration of the PLD2-preferring inhibitor resulted in a modest yet significant increase in survival and in addition delayed mortality (Fig. 4D). Although 80% of the mice administered VU0364739 eventually succumbed, death occurred considerably later after infection (compared with influenza-infected mice administered vehicle), clearly providing an extended window for further supportive therapy in a clinical setting. This demonstrates that by inhibiting PLD activity in the mouse viral replication is decreased and that antiviral responses are up-regulated, leading to some protective benefits during a lethal influenza infection. No significant toxicity or neurological impairment was noted in rats receiving identical treatment (Tables 1 and 2). This inhibitor is a preclinical compound that has not been optimized for pharmacokinetic or pharmacodynamic properties, and yet it demonstrates robust effects on viral spread and reproduction. View this table: TABLE 1 Rat liver toxicity panel shows little effect of PLD inhibitor VU0364739 Only globulin and total protein showed higher levels with inhibitor treatment (n = 5–6 rats per treatment; p < 0.05 by Mann-Whitney test), and both were within normal clinical ranges for both the vehicle control and PLD inhibitor treatment groups. Principal component analysis of the z-score standardized values across the screening panel displayed no clustering of the multivariate results by treatment, and none of the principal components differed significantly (p > 0.05 by t test). View this table: TABLE 2 Modified Irwin Neurological Battery with VU0364739 PLD inhibitor VU0364739 is without effect in a Modified Irwin Neurological Battery in rats. Changes in the Modified Irwin Neurological Battery were evaluated using a rating scale from 0 to 2: 0, no effect; 1, modest effects; 2, robust effect. Male Harlan Sprague-Dawley rats (n = 6; approximately 250 g) were pretreated with vehicle alone or a 13 mg/kg intraperitoneal dose of VU0364739 and then tested in the Irwin battery at 30 min after treatment and subsequently monitored for 8 h.

PLD2 Inhibition Alters Endocytosis Kinetics and Aggregation of Endocytic Proteins Inhibition of PLD function has been shown to decrease uptake of ligands in various systems (9, 23, 24). Given that perturbation of influenza virus trafficking during the early infection process can lead to degradation of the entering virus (11), it was hypothesized that PLD inhibition during influenza infection led to alterations of entry events that occur in the first minutes of infection. To support this hypothesis, the effect of PLD2 inhibition on normal endocytosis rates was assessed using the established transferrin uptake model, a classic demonstration of a clathrin-dependent trafficking process. A549 cells pretreated with VU0364739 were labeled with fluorescent transferrin at 4 °C for 1 h and then placed into a heated microscope for live cell imaging. Images were recorded for 1 h, and the frame and time of the transferrin fluorescence disappearance was noted (Fig. 5, A and B). Cells treated with vehicle control were able to take up, traffic, and degrade transferrin by the 14th frame, corresponding to an average time of 26 min after warming. In contrast, cells treated with VU0364739 took ∼56 min to process the fluorescent ligand. These data indicate that inhibiting PLD2 activity with VU0364739 alters trafficking kinetics by extending the endocytosis process rather than acting as a strict blockade. View larger version: Download as PowerPoint Slide FIGURE 5. PLD inhibition alters ligand trafficking kinetics and accumulation of endocytosis regulatory proteins. A and B, A549 cells were treated for 1 h with DMSO or VU0364739 (10 μm) prior to being labeled for 1 h at 4 °C with 100 nm Alexa Fluor 647-transferrin. Live cell imaging was used to assess the kinetics of transferrin uptake. To quantify this assay, both the frame (A) and the time (B) were used to determine when the fluorescent signal disappeared, signaling recycling of transferrin. Live cell imaging was performed using Slidebook imaging software, and frame time measurements were compared using t tests. Data are mean ± S.E. (error bars). C–E, A549 cells were treated for 1 h with DMSO or VU0364739 (10 μm) and then infected with 0.05 m.o.i. influenza A/California/04/2009 (H1N1). Cells were stained and examined by confocal microscopy for the accumulation of clathrin (C), Rab5 (early endosome) (D), and CD63 (late endosome) (E). Less clathrin is recruited at 50 and 80 min postinfection under inhibitor treatment, and less Rab5 (D) and CD63 (E) accumulate after 10 and 90 min postinfection. In these experiments, protein accumulation was defined by gating signal intensity as well as size such that higher y axis values represent bigger and brighter foci of signal. A two-way ANOVA with Bonferroni's post-test was used to examine data from C. t tests were used to analyze data from D and E with p values indicated. Data are mean ± S.E. (error bars). *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001. Influenza entry is not entirely dependent on clathrin-mediated endocytosis, but it is widely accepted that the majority of incoming viruses enter via clathrin-dependent events primarily by the de novo formation of clathrin-coated pits on the cell surface after exposure to influenza virus (25). After entry, influenza virus needs to be properly trafficked from the membrane to the nucleus. Along the way, the endosome is acidified (26), and the hemagglutinin protein undergoes a conformational change that creates a fusion pore between the viral and vesicle membranes through which viral ribonucleoproteins gain access to the cytoplasm, eventually entering the nucleus to initiate new virus production. The accumulation of trafficking-associated proteins was visualized by confocal microscopy in A549 cells during a 0.05 m.o.i. influenza A/California/04/2009 (H1N1) infection. Signal intensity and particle size were gated to determine protein accumulation. As proteins accumulate, the brightness and size of the fluorescence increases on a per cell basis. Clathrin recruitment was inhibited 50 min postinfection and remained significantly lower (up to 80 min postinfection) with VU0364739 administration (Fig. 5C). During the first phase of viral entry, Rab5 accumulated on the early endosomes; however, in the presence of VU0364739, much less Rab5 accumulated 10 min postinfection (Fig. 5D). Similarly, VU0364739 treatment led to a significant reduction in recruitment of the late endosome marker CD63 90 min postinfection (Fig. 5E). Vesicular trafficking is also important for key elements of the late stages of viral replication, and defects in Rab11 accumulation associated with viral protein trafficking to the membrane were consistently observed (data not shown). These data indicate that when PLD2 is inhibited during an infection the normal cascade of protein accumulation required for proper endosomal maturation is disrupted, leading to inefficient trafficking of incoming virus particles, indicating that PLD2 is a host factor required for the efficient trafficking of influenza virus once within the cell.