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The Journal of Immunology, 2007, 178: 2440-2447.
Copyright © 2007 by The American Association of Immunologists, Inc.

PU.1 Redirects Adenovirus to Lysosomes in Alveolar Macrophages, Uncoupling Internalization from Infection1

Brenna Carey*, Margaret K. Staudt2,*, Dana Bonaminio*, Johannes C. M. van der Loo{ddagger} and Bruce C. Trapnell3,*,{dagger},§

Divisions of * Pulmonary Biology, {dagger} Pulmonary Medicine, and {ddagger} Experimental Hematology, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH 45229; and § Division of Pulmonary, Critical Care and Sleep Medicine, University of Cincinnati College of Medicine, Cincinnati, OH 45267


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Adenovirus is endocytosed and efficiently destroyed by human and murine alveolar macrophages (AMs) and rapidly cleared from the lungs of wild-type but not GM-CSF–/– mice. We hypothesized that GM-CSF may regulate adenovirus clearance in AMs via the transcription factor PU.1 by redirecting virion trafficking from the nucleus to lysosomes. This hypothesis was tested in murine AM cell lines with altered GM-CSF and/or PU.1 expression including MH-S (GM-CSF+/+PU.1Pos), mAM (GM-CSF–/–/PU.1Neg), and mAMPU.1+ (GM-CSF–/–/PU.1Pos; PU.1-transduced mAM cells) and A549 (an epithelial-like cell line) using a human adenovirus expressing a beta-galactosidase reporter. In PU.1Neg mAM and A549 cells, adenovirus efficiently escaped from endosomes, translocated to the nucleus, and expressed the viral reporter in most cells. In marked contrast, in PU.1Pos mAMPU.1+ and MH-S cells, adenovirus failed to escape from endosomes, colocalized exclusively with endosome/lysosome markers (Rab5, Rab7, and Lamp1), and rarely expressed the reporter. Retroviral expression of PU.1 in A549 cells blocked endosomal escape, nuclear translocation and reporter expression. Inhibition of endosome acidification also blocked escape, nuclear translocation, and reporter expression in PU.1Neg cells. The effect of PU.1 on viral trafficking and transduction could not be explained by an effect on endosome acidification or on differences in viral load. PU.1 reduced expression of integrin beta5, a host factor important for endosomal escape of adenovirus, suggesting that PU.1 redirects adenoviral trafficking by modulating integrin signaling. These results demonstrate that PU.1 uncouples infection from internalization in AMs, providing a mechanism for AMs to avoid infection by adenovirus during clearance.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Alveolar macrophages (AM)4 play a central role in lung defense through their ability to clear various microbial pathogens including viruses (1, 2). Clearance is initiated by internalization of pathogens into plasmalemma-derived vesicles, which acquire microbicidal properties through a maturation process involving fusion with endocytic pathway components and culminating in formation of phagolysosomes (3, 4, 5). The process is directed by Rab GTPases, which mediate interactions between endocytic vesicles and cellular constituents (6). For example, Rab5, present on early endosomes, mediates vesicular coalescence (7), whereas Rab7, present on late endosomes, mediates fusion with lysosomes (8). Lysosome-associated membrane protein-1 (Lamp1) is a marker of lysosomal/late endosomal vesicles and may facilitate vesicular transport toward the microtubule organizing center (9). Adenovirus and other pathogens co-opt endocytic pathways during infection of target cells (10), providing both the means to explore pathways of infection and identify potential therapeutic targets.

Human adenoviruses are nonenveloped DNA viruses, comprising nearly 50 serotypes, responsible for 7–10% of all respiratory illness in infants and children and between 5 and 10 million infections annually in the United States (11). In mice, adenovirus is cleared from the lung biphasically; 60–90% is cleared rapidly in the first 24 h, and the remainder is cleared slowly over several weeks (2, 12). AMs mediate early phase clearance of adenovirus because: 1) AMs rapidly internalize adenovirus from the lungs in mice (13); 2) human, rat, and murine AMs degrade adenovirus similarly with a half-life of ~6 h (2); and 3) rapid clearance is similar in athymic and wild-type mice (14).

Adenovirus infection has been well studied in epithelial cells and epithelial-like cell lines (e.g., A549; reviewed in Refs. 10 and 15) and albeit less extensively, also in monocytes (16), macrophages (13, 17), and macrophage cell lines (12). In A549 cells, infection begins by binding of the adenovirus fiber to high affinity Coxsackie and adenovirus receptor (18, 19) followed by binding of the penton base to low affinity integrin receptors (20). In mononuclear phagocytes, which do not express Coxsackie and adenovirus receptor, attachment is mediated by integrin receptors. Integrin {alpha}m mediates attachment but not internalization (21) whereas integrin {alpha}v mediates internalization of the virion via clathrin-dependent endocytosis (20, 21). Functional redundancy exists among integrin receptors because integrins {alpha}vbeta5, {alpha}vbeta3, and {alpha}5beta1 mediate internalization of group C adenoviruses (20, 22, 23). In permissive cells, adenovirus disrupts the endosomal membrane, escapes into the cytoplasm, and translocates along microtubules to the nucleus into which viral DNA is injected and expressed (10). Adenovirus-mediated endosome lysis and escape is critically dependent on both viral and host cell factors. Specifically, acidification of the endosome by vacuolar ATPase (24) destabilizes the adenoviral capsid causing partial disassembly (25), and release of an amphipathic capsid protein (VI) with membrane lytic properties (26). Adenovirus-mediated endosomal escape also requires sequences in the cytoplasmic tail of integrin beta5 (27).

GM-CSF promotes the survival, proliferation, and differentiation of myeloid cells including AMs (28, 29, 30, 31, 32, 33, 34). Mice deficient in GM-CSF due to targeted gene ablation (GM-CSF–/– mice) (35, 36) have increased mortality from infections (37) and impaired pulmonary clearance of microbial pathogens (38, 39, 40) including adenovirus (12). Rapid clearance of adenovirus is absent in GM-CSF–/– mice (12), suggesting that GM-CSF may be necessary for AM-mediated clearance of adenovirus. Expression of the macrophage differentiation-inducing transcription factor, PU.1, is reduced in AMs in GM-CSF–/– mice (34), and restoration of pulmonary GM-CSF restores PU.1 levels in AMs, rescues AM functions (34, 41, 42), and restores lung host defense (12, 38, 39, 40, 43). A cultured AM cell line derived from GM-CSF–/– mice (mAM cells) that does not express PU.1 (denoted PU.1Neg) has functional defects similar to those of primary AMs, which can be rescued by retroviral expression of PU.1 (34). In this study, we evaluated the role of GM-CSF and PU.1 in adenovirus trafficking and transduction in AM cell lines with altered expression of GM-CSF and PU.1. Results show that PU.1 blocks endosomal escape, prevents infection, and redirects virions to the lysosomal compartment for destruction.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Adenovirus

The adenovirus used in this study was a human serotype 5 adenovirus, deleted in E1 and E3 regions, expressing a beta-galactosidase (beta-gal) reporter from the Rous sarcoma virus promoter. Called adenovirus hereafter, its structure, production, purification, quantification, storage, and handling has been previously described (13, 44, 45, 46).

Cells

MH-S (CRL-2019; American Type Culture Collection) is a murine AM cell line (47) with a mature AM phenotype. mAMGFP+ and mAMPU.1+/GFP+ are cell lines derived from the same murine GM-CSF–/–/PU.1Neg AM cell line by retroviral transduction and have been previously described (34). The A549 (CCL-185; American Type Culture Collection), is a human epithelial-like carcinoma cell line that is readily infected by adenovirus. A549PU.1+GFP+ and A549GFP+ are A549-derived cell lines created with the retroviral vectors (34) used to create mAMGFP+ and mAMPU.1+/GFP+. For readability, the GFP subscript is omitted from the names of all transduced cell lines. Cell lines were maintained as described previously (13).

Viral infection and transduction assays

Adenoviral infections were done as described (12). Briefly, cells were seeded in 48-well plates (2 x 105 cells/well; Costar) and incubated overnight in culture medium. Medium was then replaced with infection medium containing adenovirus (100 PFU/cell for A549, or 2500 PFU/cell for AM lines), and cultures were continued for 8 h. Infection medium was then aspirated and replaced with culture medium, and plates were incubated for 72 h. Expression of the adenoviral beta-gal reporter was assessed by 5-bromo-4-chloro-3-indolyl beta-D-galactoside staining (46) and both visual inspection and microscopic examination (Axiovert 25 light microscope; Carl Zeiss). The adenoviral transduction rate was calculated as the number of blue cells divided by the total number of cells per 10x field averaged for five randomly selected fields per well in three wells per cell line or condition.

Confocal microscopy

Cells were cultured on poly-D-lysine-coated coverslips (BD Biosciences) for 16 h before adenoviral infection. In some experiments, bafilomycin A1 (BAF; 20 µg/ml) was added 30 min before adding adenovirus and maintained throughout infection, and/or Lysotracker (1 µM; Molecular Probes) was included during the last 30 min of infection. Cells were then fixed in 4% paraformaldehyde (Electron Microscopy Sciences), incubated in 150 mM glycine to quench autofluorescence, permeabilized with 0.01% Triton X-100 (Sigma-Aldrich), and incubated in blocking buffer (2% BSA in PBS at room temperature for 30 min). Cells were then incubated (room temperature, 60 min) with primary Ab diluted 1/100 in PBS containing 2% BSA. Primary Abs included goat anti-mouse Rab5C, goat anti-mouse Rab7, rat anti-mouse Lamp1 (all from Santa Cruz Biotechnology), mouse anti-human Lamp1 (BD Pharmingen), or rabbit anti-adenovirus Ab (isolated from serum of rabbits immunized with intact serotype 5 human adenovirus using the Montage Ab purification kit (Millipore)). After washing, cells were incubated (room temperature, 60 min) with secondary Ab diluted 1/500 in PBS containing 2% BSA. Secondary Abs included Cy3-labeled goat anti-rabbit IgG, Cy5-labeled donkey anti-goat IgG, Cy5- labeled goat anti-rat IgG, and/or Cy5-labeled donkey anti-mouse IgG (Jackson ImmunoResearch Laboratories). Cells were washed, stained with Sytox green (diluted 1/10,000; Molecular Probes), washed, mounted onto slides with Vectashield mounting medium (Vector Laboratories), and examined with a LSM510 triple beam confocal laser scanning microscope equipped with a 63x 1.4 PlanApo objective (Carl Zeiss) focused to 488, 543, and 633 nm to detect FITC, Cy3, Cy5-labeled Abs, respectively. Images were analyzed with AxioVision Viewer 3.0 software (Carl Zeiss), and immunostaining in some photomicrographs was pseudocolored using Adobe Photoshop (version 7.0) software. Colocalization of adenovirus with Lamp1 was calculated by enumerating all intracellular pixels associated both with green and red fluorescence (representing Lamp1 and adenoviral load, respectively), dividing by the total number of intracellular red pixels and multiplying by 100. Fifty cells were evaluated per cell line and each experiment was done twice. Adenoviral load was quantitated by enumerating intracellular pixels associated with red fluorescence in photomicrographs of MH-S, mAM, mAMPU.1+, A549, and A549PU.1+ cells. At least 20 cells were evaluated per cell line.

Virus-mediated endosome lysis assay

Cells were seeded into 48-well plates (2 x 105 cells/well), cultured for 16 h, washed once in PBS, and cultured in infection medium for 2 h. Luciferase reporter plasmid DNA (pRSVL, 1.5 µg; a gift from Dr. P. Seth, Northwestern University, Chicago, IL) was added, and the cells were cultured for 30 min before adding adenovirus (2 x 104 PFU/cell for A549; 2 x 105 PFU/cell for AMs). After 24 h, infection medium was aspirated and replaced with culture medium. After 24 h, cell lysates were prepared, and luciferase activity was measured using a kit (Promega) as directed by the manufacturer.

Real time PCR amplification

Total RNA was isolated from cells using TRIzol (Invitrogen Life Technologies), and RNA was converted to cDNA using the Superscript First Strand Synthesis System (Invitrogen Life Technologies) per manufacturer’s protocols. Aliquots of cDNA (100 ng, quantified by optical density) were used to quantify transcript levels using quantitative real-time PCR amplification using a SmartCycler (Cepheid) and transcript-specific oligonucleotide primers (mouse integrin beta5, 5'-ttt gct gtg acg aag aac cac-3', 5'-cag gtg gca gtg aag aag aga-3'; human integrin beta5, 5'-agc cct gat acc tgg aac aac-3' and 5'-cca atc ttc aga ccc tcg cac-3'; GAPDH, 5'-ctt cac cac cat gga gaa ggc-3', 5'-ggc atg gac tgt ggt cat gag-3'). The PCR amplification algorithm for murine integrin beta5 transcripts was: 95°C, 10 s; 62°C, 15 s, 72°C, 20 s, 82°C, 6 s (detection optics on) for 45 cycles. Conditions for human integrin beta5 and GAPDH transcripts were similar except optics were turned on for 6 s at 80°C or 84°C, respectively. Expression levels were normalized to GAPDH transcript levels. At least three determinations were made for each gene evaluated in a given experiment.

Western blotting

Western blotting was performed as previously described (34). Primary Abs included rabbit anti-mouse integrin beta5 (1/100) or rat anti-mouse actin (Santa Cruz Biotechnology; 1/200). Immunoreactive proteins were visualized by ECL (Amersham Biosciences) and exposed to Kodak X-Omat AR film. Films were subjected to densitometric analysis on an Alphaimager 2000 with the supplied software.

Statistical analysis

All numerical data are presented as the mean ± SEM. Statistical comparisons were made with Sigma Stat (version 3) software. Normality was evaluated by the Kolmogrov-Smirnov method and equal variance by Levene median test. Except where noted, statistical comparisons were made using Kruskal-Wallis one-way ANOVA on ranks with post hoc analysis by the Student-Newman-Keuls method for multiple group comparisons.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
PU.1 blocks adenoviral transduction in AMs

The role of GM-CSF and PU.1 in adenoviral infection of AMs was first evaluated by quantifying expression of the viral beta-gal reporter gene in virus-exposed cells. A549 and mAM cells were readily transduced by adenovirus whereas MH-S and mAMPU.1+ cells were not, as judged by visual inspection of cell staining (Fig. 1A). beta-gal activity was not seen in uninfected cells. Microscopic inspection revealed blue staining in 80–90% of A549 and mAM cells, only 1% of MH-S and 5% of mAMPU.1+ cells (p < 0.001; Fig. 1B). These results indicate that PU.1 markedly reduces adenoviral gene expression in AMs.


Figure 1
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  		FIGURE 1. PU.1 blocks adenovirus-mediated gene transfer in AMs. Cells were infected with adenovirus expressing a beta-gal reporter, cultured for 72 h, and then stained with 5-bromo-4-chloro-3-indolyl beta-D-galactoside to detect beta-gal activity by visual inspection (A) or microscopic examination to determine the percent of beta-gal+ cells (B). Bars represent the mean percentage of stained cells in five random fields per well in three wells per cell line. Data are representative of three separate experiments.

 
Adenovirus is confined to endosomes and lysosomes in PU.1-expressing AMs

To determine the effect of PU.1 on adenovirus trafficking in AMs, we colocalized adenovirus with markers of early and late endosomes and with a lysosomal marker (Lamp1) by confocal microscopy. In the human epithelial-like cell line, A549, adenovirus was primarily found at the nuclear membrane, consistent with the high rate of viral gene expression in these cells, and only a small proportion colocalized with Lamp1 (Fig. 2, A–C) or with Rab5 or Rab7 (data not shown). In contrast, in the phenotypically mature AM cell line, MH-S, adenovirus was confined to large paranuclear aggregates that colocalized entirely with Lamp1 (Fig. 2, D–F) and also with Rab5 and Rab7 (data not shown). In the phenotypically immature AM cell line, mAM, adenoviral trafficking was similar to that seen in A549 cells (Fig. 2, G–I). Retrovirus-mediated PU.1 expression in mAM cells substantially altered trafficking, redirecting nearly all adenovirus to a Lamp1+, paranuclear aggregate as seen in MH-S cells (Fig. 2, J–L). Morphometric analysis of digital fluorescent micrographs revealed that 98% of adenovirus in MH-S and mAMPU.1+ cells colocalized with Lamp1, compared with <10% in A549 and mAM cells (p < 0.001; Fig. 2M). Thus, in PU.1Neg epithelial cells and immature AMs, adenovirus primarily translocates to the nucleus; whereas in PU.1Pos, mature AMs, adenovirus remains associated with endosomal/lysosomal markers, with few virions reaching the nucleus.


Figure 2
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  		FIGURE 2. PU.1 redirects intracellular trafficking of adenovirus to the lysosome in AMs. Cells were infected with adenovirus for 8 h and then immunostained to visualize adenovirus and Lamp1. Shown are confocal photomicrographs of cells illustrating the intracellular localization of adenovirus (in red; A, D, G, and J), Lamp1 (in green; B, E, H, and K), or the colocalization of adenovirus in Lamp1+ organelles (in yellow; C, F, I, and L). Nuclei are counterstained (in blue). Scale bar, 10 µm. Photomicrographs are representative of at least five cells examined in detail per cell line in each experiment in six separate experiments. Digital image files were also analyzed morphometrically to quantify the percentage of adenovirus colocalizing within Lamp1+ organelles, as described in Materials and Methods (M).

 
Adenovirus-mediated endosome lysis is blocked in PU.1-expressing AMs

Because adenovirus efficiently escapes from endocytic vesicles in epithelial cells by disrupting its membrane (48, 49, 50, 51), we asked whether PU.1 interfered with this step during infection of AMs using an endosome lysis assay (51). Adenovirus caused a >3-log higher expression levels of a cointernalizing luciferase reporter plasmid in A549 cells and a 5-log increase in mAM cells, indicating that adenovirus-mediated endosome lysis was efficient in these cells (p = 0.015; Fig. 3). In contrast, adenovirus infection had little effect on cointernalizing reporter plasmid expression in MH-S and mAMPU.1+ cells, indicating that PU.1 reduces adenovirus-mediated endosome lysis.


Figure 3
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  		FIGURE 3. PU.1 blocks adenovirus-mediated endosome lysis and reporter plasmid transfection of AMs. Cells were incubated with a luciferase reporter plasmid, coinfected with adenovirus, and cultured for 48 h; then, luciferase activity was quantified in cell lysates. Bars represent the mean luciferase activity in three separate infections per cell line. In the absence of adenovirus coinfection, luciferase activity was similar in the absence or presence of reporter plasmid exposure. Data are representative of three separate experiments.

 
Endosome acidification is required for adenoviral infection of AMs and unaffected by PU.1

Because adenoviral escape from endosomes depends on acidification (10, 50, 51, 52), we determined whether PU.1 alters endosome acidification. In the presence of BAF to block the endosomal ATP-dependent H+ pump (24), adenoviral transduction was markedly reduced in A549 and completely blocked in mAM cells (p < 0.001; Fig. 4). Evaluation of similarly exposed cells by confocal microscopy revealed that BAF blocked adenoviral trafficking to the nuclear membrane, redirecting adenovirus to a Rab7+ paranuclear compartment in both A549 cells (Fig. 5, A–F) and mAM cells (Fig. 5, G–L). PU.1 did not affect endosome acidification, which occurred normally in mAMPU.1+ cells (Fig. 5, M–O) and MH-S cells (data not shown), as revealed by confocal microscopy and use of a pH-sensitive molecular probe. BAF did not alter adenoviral trafficking in mAMPU.1+ cells (Fig. 5, P–R) or MH-S cells (data not shown). Thus, PU.1 blocks adenoviral escape from endosomes in AMs, but not by altering endosome acidification.


Figure 4
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  		FIGURE 4. BAF reduces adenoviral reporter gene expression. Cells were infected with adenovirus in the absence and presence of BAF as indicated and cultured for 72 h; then, reporter gene expression was quantified as described in the legend to Fig. 1B. Bars represent the mean percentage of stained cells in five random fields per well in three wells per cell line. Data are representative of two separate experiments.

 

Figure 5
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  		FIGURE 5. Blocking endosome acidification alters adenoviral trafficking in PU.1Neg AMs, but not PU.1Pos AMs. Cells were infected in the absence (A–C, G–I, and M–O) or presence (D–F, J–L, and P–R) of BAF to block endosome acidification, cultured for 8 h, and then immunostained to visualize adenovirus and Rab7 (A–L). In some (M–R), Lysotracker was added during the last 30 min of infection and Rab7 staining was omitted. Shown are confocal photomicrographs illustrating the intracellular localization of adenovirus (in red; A, D, G, J, M, and P), Rab7 (in green; B, E, H, and K), colocalization of adenovirus in Rab7+ organelles (in yellow; C, F, I, and L), endosome acidification (in green N and Q) or colocalization of adenovirus in acidified endosomes (in yellow (O and R). Nuclei are counterstained (in blue). Scale bar, 10 µm. Photomicrographs are representative of at least five cells examined in detail per cell line in each experiment in two separate experiments.

 
PU.1 reduces integrin beta5 expression in AMs

Because integrin beta5 is important in adenovirus-mediated endosome lysis (27), we evaluated the role of PU.1 in integrin beta5 expression in AMs. Integrin beta5 mRNA was readily detected in A549 and mAM cells and was markedly reduced in MH-S cells and mAMPU.1+ cells as determined by quantitative real time PCR amplification (p < 0.001; Fig. 6A). Integrin beta5 protein was also readily detected in A549 cells and mAM cells and markedly reduced in MH-S cells and mAMPU.1+ cells (Fig. 6B). Thus, PU.1 reduces expression of integrin beta5 in AMs, suggesting that PU.1 may block endosomal escape of adenovirus by repressing expression of integrin beta5.


Figure 6
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  		FIGURE 6. PU.1 reduces integrin beta5 expression in AMs. Integrin beta5 gene expression was examined in cell lines by quantifying mRNA transcript levels after conversion to cDNA by quantitative real time PCR (A) or by quantifying protein levels by Western blotting analysis (B). In A, bars represent the mean of three determinations of integrin beta5 transcript levels (normalized to GAPDH mRNA levels) for each cell line evaluated. Data are representative of three separate experiments. In B, aliquots of cell lysates representing equal amounts of cellular protein (10 µg/lane) were subjected to electrophoresis on 4–20% polyacrylamide gradient gels containing SDS and then immunoblotted to detect integrin beta5 or actin as described in Materials and Methods. Results are representative of three separate experiments.

 
PU.1 redirected adenovirus trafficking and reduced adenoviral transduction in A549 cells

To further explore the mechanism by which PU.1 mediates antiadenoviral effects, we evaluated adenovirus transduction rate and trafficking in A549 cells ectopically expressing PU.1 from a retroviral vector (A549PU.1+ cells). Adenovirus transduced nearly 100% of unmodified A549 cells; however, transduction was markedly reduced in A549PU.1+ cells (p < 0.001; Fig. 7). Confocal microscopy revealed that PU.1 substantially altered adenoviral trafficking in A549PU.1+ cells, completely blocking detectable nuclear translocation and redirecting virions to paranuclear aggregates that colocalized with Rab5 (Fig. 8, A–F) and Rab7 (Fig. 8, G–L). Because integrin beta5 was previously demonstrated to play a critical role in adenoviral-mediated endosome lysis (27) and PU.1 repressed its expression in AMs, integrin beta5 mRNA and protein levels were quantified in A549 and A549PU.1+ cells. PU.1 markedly reduced but did not eliminate integrin beta5 mRNA (p < 0.01; Fig. 9A) and protein (Fig. 9, B and C) in parallel with the marked reduction (but not elimination) of adenoviral transduction (Fig. 7). These data demonstrate that PU.1 expression alters trafficking of adenovirus in both AMs and epithelial-like cells.


Figure 7
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  		FIGURE 7. Ectopic expression of PU.1 in A549 cells impairs adenovirus-mediated gene transduction. Cells were infected with adenovirus, cultured, and evaluated for adenoviral reporter gene expression as described in the legend to Fig. 1B. Bars represent the mean percentage of stained cells in five random fields per well in three wells per cell line. Data are representative of two separate experiments.

 

Figure 8
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  		FIGURE 8. Ectopic expression of PU.1 in A549 cells confines adenovirus to endosomes. Cells were infected with adenovirus for 8 h and then immunostained to visualize adenovirus, Rab5, or Rab7. Shown are confocal photomicrographs of cells illustrating the intracellular localization of adenovirus (in red; A, D, G and J), Rab5 (in green; B and E), Rab7 (in green; H and K), colocalization of adenovirus in Rab5+ organelles (in yellow; C and F) or colocalization of adenovirus in Rab7+ organelles (in yellow; I and L). Nuclei are counterstained (in blue). Scale bar, 10 µm. Photomicrographs are representative of at least five cells examined in detail per cell line in each experiment in three separate experiments.

 

Figure 9
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  		FIGURE 9. Ectopic expression of PU.1 in A549 cells reduces integrin beta5 expression. Integrin beta5 gene expression was examined in A549 cells and A549 cells expressing PU.1 from a retroviral vector as described in the legend to Fig. 6. Bars in A represent the mean of three determinations of integrin beta5 transcript levels (normalized to GAPDH mRNA levels) for each cell line evaluated. Data are representative of two separate experiments. B, Aliquots of cell lysates representing equal amounts of cellular protein (10 µg/lane) were subjected to electrophoresis on 4–20% polyacrylamide gradient gels containing SDS and then immunoblotted to detect integrin beta5 or actin as described in Materials and Methods. C, The immunoblot films were evaluated by densitometry to quantify the relative expression of integrin beta5 level in each cell line. Results are representative of three separate experiments.

 
PU.1 expression and not viral load regulates adenoviral trafficking and infection in AMs and epithelial-like cells

To determine whether viral load had an effect on intracellular trafficking of adenovirus, the amount of internalized adenovirus was measured using confocal microscopy. In the approach used, at 8 h of infection, the viral load reflects net differences between virus uptake and degradation. A549 cells had the highest viral load and translocated nearly all virions to the nuclear envelope, resulting in transduction of the vast majority of cells (Table I). Expression of PU.1 in A549 cells reduced viral load by only 39% but reversed the trafficking pattern, redirecting nearly all virions to the endosomal/lysosomal compartment. In mAM cells, the viral load was only 35% lower than in A549PU.1+ cells, but the trafficking pattern was reversed with nearly all virions directed to the nuclear envelope. In PU.1-expressing mAMPU.1+ and MH-S cells, the viral load was reduced a further 46 and 53%, respectively, and again the trafficking pattern was reversed with nearly all virions redirected to the endosomal/lysosomal compartment. Thus, as the viral load goes from highest to lowest, the virion trafficking pattern toggles from nuclear to lysosomal in parallel with the absence or presence of PU.1. Thus, PU.1 and not viral load correlates with the pattern of nuclear trafficking and adenoviral infection in AMs and epithelial-like cells.


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  		Table I. Internalization and trafficking of adenovirus in expressing or not expressing PU.1

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In this report, we show that GM-CSF, via PU.1, uncouples adenovirus internalization from infection by confining adenovirus to the endosome/lysosomal pathway, thus providing the AM with a mechanism to avoid being infected by adenovirus during clearance. This finding is supported by several lines of evidence: 1) PU.1 blocked adenovirus-mediated endosome lysis; 2) adenovirus colocalized entirely with endosomal markers in PU.1-expressing cells (MH-S, mAMPU.1+, and A549PU.1+) and poorly with these markers in cells not expressing PU.1 (mAM and A549); 3) PU.1 caused accumulation of adenovirus in Lamp1+ paranuclear aggregates in AMs and A549 cells, consistent with vesicular fusion with tubular lysosomes at the microtubule organizing center (53); 4) PU.1 blocked expression of the adenoviral reporter gene in AMs and markedly reduced its expression in A549 cells. Finally, PU.1 redirected adenoviral trafficking in AMs and A549 cells in a manner similar to that caused by BAF but did not alter endosome acidification. Rather, PU.1 reduced expression of integrin beta5, a molecule previously reported to play an important role in adenovirus-mediated endosome lysis (27).

Adenoviral gene transfer and expression were efficient in GM-CSF–/–/PU.1Neg AMs, similar to epithelial cells (10, 15, 54). Therefore, neither GM-CSF nor PU.1 is required for expression of host cell factors necessary for adenoviral infection, including virion internalization (20, 55, 56, 57), endosome lysis (24, 27, 58), virion transport along microtubules (59, 60), capsid disassembly (10), transfer of the viral genome into the nucleus (61), and expression of adenoviral genes (15). Our results are consistent with a model (Fig. 10) in which adenovirus enters AMs by endocytosis and, in the absence of GM-CSF/PU.1, rapidly escapes from endosomes and translocates to the nucleus. In PU.1Pos AMs, endosomal escape is blocked, thereby preventing the subsequent steps of adenoviral infection. This model is consistent with reports that GM-CSF is required for pulmonary clearance of adenovirus (12) and that adenovirus is rapidly degraded by AMs (2) and provides a mechanism explaining the low transduction efficiency of adenoviral vectors in AMs (13). The net result is redirection of adenovirus trafficking to lysosomes. These findings may have further implications because a number of viruses co-opt endocytic mechanisms to infect target cells including rhinovirus (62), papillomavirus (63), Hantaan virus (64), and vesicular stomatitis virus (65). Because productive infection is dependent on escape from the endosome/lysosome pathway, understanding these mechanisms may facilitate development of novel antiviral therapeutic strategies.


Figure 10
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  		FIGURE 10. Proposed mechanism by which GM-CSF/PU.1 uncouples adenovirus internalization from infection in AMs. Binding of the adenoviral penton protein to integrin {alpha}V triggers internalization of virions into endosomes. Escape of adenovirus from endosomes is dependent on their acidification, which can be blocked by BAF, and on the presence of integrin beta5 and possibly integrin signaling (10 16 21 27 ). The drop in endosomal pH promotes capsid destabilization, resulting in dissociation of viral proteins including the internal hidden amphipathic capsid protein VI, which mediates lysis of the endosome membrane (26 ). Once in the cytoplasm, partially disassembled virions traffic along microtubules to the nuclear membrane where viral DNA is imported into the cell nucleus followed by expression its genetic program. GM-CSF-stimulated PU.1 expression blocks the escape of adenovirus from endosomes, possibly by reducing integrin beta5 levels and/or integrin signaling. Endocytic vesicles acquire Rab7 and are translocated to the microtubule organizing center and fuse with the lysosomes. This model does not exclude potential participation of other PU.1-dependent molecules in the mechanism confining adenovirus to the endosome/lysosome pathway in AMs.

 
In contrast to endocytosis of small particles, which is reduced but not absent in both GM-CSF–/–/PU.1Neg AMs (mAM) and in GM-CSF–/–/PU.1Low AMs (i.e., primary AMs from GM-CSF–/– mice) (12), phagocytosis of large (2–10 µm) particles is severely impaired in both (12, 34, 43, 66). Because endocytosis and phagocytosis are mediated by mechanisms the molecular components of which are overlapping, mAM and mAMPU.1+ cells may be useful in studies to further define the molecular factors comprising these vital mechanisms of host defense.

Our results shed light on mechanisms of infection by adenovirus. Endosomal escape, nuclear translocation, and viral gene expression were completely blocked by BAF treatment in AMs and in A549 cells, indicating that endosome acidification is required for infection in both AMs and epithelial cells. This is consistent with some reports (10, 26) but not others (67). A recent study (26) appears to have resolved this controversy by establishing that: 1) partial disassembly of adenovirus (i.e., dissociation of the fiber) occurs before virus-mediated penetration of the endosomal membrane; 2) acidification destabilizes the viral capsid; and 3) adenoviral protein VI, an internal capsid protein exposed during viral disassembly, exhibits pH-independent membrane lytic activity. It is unlikely that PU.1 blocks endosome escape by altering capsid destabilization because acidification occurred normally in PU.1Pos cells. Nor is it likely to have directly affected protein VI-mediated membrane disruption, because the adenovirus used here was able to infect PU.1Neg cells. The effect of PU.1 on adenovirus-mediated endosome lysis was not explained by an effect of the viral load. Adenoviral infection may require integrin signaling because CS-1 cells, which express integrin {alpha}V but not integrin beta chains, are poorly infected by adenovirus and endosomal membrane lysis in CS-1 cells required specific sequences within the cytoplasmic tail of integrin beta5 (27). PU.1 reduced expression of integrin beta5 in AMs and A549 cells in parallel with blockade of endosomal escape, nuclear translocation, and transduction, suggesting that PU.1 blocks infection by altering integrin signaling. This is consistent with a report showing that GM-CSF, via PU.1, reduces integrin beta5 transcription in macrophages (68). Notwithstanding, the exact mechanism by which adenoviral protein VI, integrin beta5 and other viral and host factors participate in mediate endosomal escape of adenovirus requires further study. The cell lines developed for this study should be useful in further delineating the mechanism(s) by which PU.1 confines pathogens to the lysosomal pathway of destruction.


   Acknowledgment
 
We thank Prem Seth (Northwestern University, Chicago, IL) for the gift of the pRSVL reporter plasmid.


   Disclosures
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 Materials and Methods
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The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by National Institutes of Health Grants HL69549 and HL071823 (to B.C.T.). Back

2 Current address: Laboratory of Neuro-Oncology, Rockefeller University, 1230 York Avenue, New York, NY 10021. Back

3 Address correspondence and reprint requests to Dr. Bruce Trapnell, Cincinnati Children’s Hospital Medical Center, Division of Pulmonary Biology, 3333 Burnet Avenue, Cincinnati, OH 45229. E-mail address: Bruce.Trapnell{at}cchmc.org Back

4 Abbreviations used in this paper: AM, alveolar macrophage; Lamp1, lysosome-associated membrane protein-1; beta-gal, beta-galactosidase; BAF, bafilomycin A1. Back

Received for publication July 19, 2006. Accepted for publication November 30, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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