Involvement of Cytoskeletal Components in BK Virus
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病菌学杂志 2005年第18期
Graduate Program in Pathobiology
Department of Molecular Microbiology and Immunology, Brown University, Providence, Rhode Island 02912
ABSTRACT
Posttransplant reactivation of BK virus (BKV) in the renal allograft progresses to polyomavirus-associated nephropathy in 1% to 8% of kidney recipients. Graft dysfunction and loss in 30% to 45% of polyomavirus-associated nephropathy-affected patients are secondary to extensive tubular epithelial cell injury induced by the lytic replication of BKV. The early events in productive BKV infection are not thoroughly understood. We have previously shown that BKV enters cells by caveola-mediated endocytosis. In this report we examine the role of microfilaments and microtubules during early viral infection. Our results show that BKV infection of Vero cells is sensitive to nocodazole-induced disassembly of the microtubule network for the initial 8 hours following virus binding. In contrast, suppression of microtubule turnover with the stabilizing agent paclitaxel has no effect on BKV infectivity. Selective disassembly of the actin filaments with latrunculin A does not impede BKV infection, while inhibition of microfilament dynamics with jasplakinolide results in reduced numbers of viral antigen-positive cells. These data demonstrate that BKV, like other polyomaviruses, relies on an intact microtubule network during early infection. BKV, however, does not share the requirement with the closely related JC virus for an intact actin cytoskeleton during intracellular transport.
INTRODUCTION
First discovered in 1970, BK virus (BKV) has recently drawn renewed interest as the causative agent of an infectious complication termed polyomavirus-associated nephropathy in renal transplant recipients (16, 52). Asymptomatic BKV infection is ubiquitous, occurring in 70% to 90% of healthy adults worldwide (41). Surgical injury and potent immunosuppressive therapy following renal transplantation lead to BKV reactivation and progression to polyomavirus-associated nephropathy in 1% to 10% of kidney recipients (24, 51). The clinical concern stems from the extensive virally induced damage to the kidney, which in turn results in severe allograft dysfunction and ultimate graft loss in 45% to 60% of polyomavirus-associated nephropathy-affected patients (32, 52).
BKV belongs to the family Polyomaviridae, which are small, nonenveloped, double-stranded DNA viruses. Other well-studied polyomaviruses include simian virus 40 (SV40), mouse polyomavirus (PyV), and JC virus (JCV). Despite established differences in receptor specificity (3-5, 9, 13, 14, 27, 28, 47, 48), mode of internalization (1, 12, 17, 33, 36, 37, 39, 40, 43), and intracellular trafficking mechanisms (2, 19, 36, 37), all the members of the polyomavirus family must deliver their genome to the nucleus of the host cell in order to execute a productive viral life cycle. The nucleus is the site of viral gene transcription, viral DNA replication, and progeny assembly (45).
Efforts in our laboratory are focused on understanding the early events during BKV infection, namely host cell entry and subsequent intracellular trafficking to the nucleus. We and others have previously demonstrated that BKV enters cells through a caveola-mediated internalization mechanism (11, 12, 38). Images from ultrastructural analysis of renal tubular cells during various stages of infection consistently reveal BK virions inside monopinocytotic vesicles and/or tubuloreticular network as the viral aggregates traverse the cytoplasm en route to the host cell nucleus (11). These observations are indicative of deliberate and directional movement of the virus through the cytoplasm of the target cell.
Having successfully penetrated the interior of the host, BKV, like other DNA viruses, is faced with the challenging task of reaching the nucleus—the most critical destination in the viral life cycle. Passive random diffusion through the cytoplasm is hardly the most efficient means for vectoral movement. Endocytic entry provides the virus with the advantage of active membrane transport (49). The cytoskeleton, an elaborate meshwork of fibrous proteins, provides the tracks along which membrane-bound vesicles and organelles move in the cytosol. The three classes of fibers that comprise the cytoskeleton include microtubules, polymers of globular tubulin subunits; microfilaments, polymers of the protein actin; and intermediate filaments, polymers of one or more rod-shaped protein subunits.
In this report we investigate the involvement of microfilaments and microtubules in cytoplasmic transport of BKV by selectively perturbing their architecture, and therefore function, with well-characterized pharmacological agents. Perturbing the integrity of the microfilament network by disruption of the actin polymers did not reduce BKV infection levels in Vero cells, while stabilization of the actin filaments interfered with BKV infectivity. Arrest in microtubule (MT) dynamics, however was inconsequential to virus trafficking during early infection. In contrast, selective induction of MT disassembly exhibited an inhibitory effect on BKV infection. The results from these experiments provide insight in BKV interaction with its host cell. Also, the data reveal similarities and differences during a productive infection between BKV and the other polyomaviruses.
MATERIALS AND METHODS
Cell line and virus. Vero cells were purchased from the American Type Culture Collection (Manassas, Va.) and were maintained in a humidified 37°C incubator in Eagle's minimal essential medium (Mediatech Inc., Herdon, Va.) supplemented with 5% heat-inactivated fetal bovine serum (Mediatech Inc.). BKV Gardener strain was obtained from the American Type Culture Collection and propagated in Vero cells as previously described (42).
Antibodies and reagents. The monoclonal antibody PAb 416 (Ab2) purchased from Oncogene Research Products (Cambridge, Mass.) detects the N-terminal portion of SV40, JCV, and BKV large T antigen (T-Ag). The mouse monoclonal anti--tubulin antibody, clone DM1A was purchased from Sigma-Aldrich (St. Louis, Mo.). Alexa Fluor 488-conjugated Phalloidin, a high-affinity probe for F-actin, was obtained from Molecular Probes. Rabbit anti-BKV serum was produced by injecting a New Zealand White rabbit with highly purified preparation of BK virus in complete Freund's adjuvant (23). The rabbit received two additional BKV boosts in incomplete Freund's adjuvant. The anti-BKV serum was titered by a Western blot assay and the immunoglobulin G portion was purified and isolated on a protein G column (Pierce Biotechnology, Rockford, Ill.). Latrunculin A (Lat A), nocodazole and paclitaxel (Taxol) were purchased from Sigma Aldrich and jasplakinolide was obtained from Calbiochem (San Diego, Calif.).
Indirect immunofluorescent analysis of BKV infection. To detect expression of early T-Ag, BKV-infected Vero cells were fixed in 2% paraformaldehyde at the end of a 48-hour incubation period after BKV infection. Following three washes in phosphate-buffered saline (PBS) (137 mM NaCl, 2.682 mM KCl, 8.1 mM Na2HPO4, 1.47 mM KH2PO4, pH 7.2), the cells were incubated with a 1:20 dilution of PAb 416 for 1 h at 37°C. The cells were then washed three times in PBS followed by a 45-minute incubation at 37°C with a 1:150 goat anti-mouse immunoglobulin G, F(ab')2 fragments conjugated to Alexa Fluor 488 (Molecular Probes, Eugene, Oreg.). Next the cells were extensively rinsed in PBS and counterstained with Evan's Blue solution (red cytoplasmic dye). The coverslips were mounted on glass slides with Vectashield mounting medium for fluorescence with 4',6'-diamidino-2-phenylindole (DAPI) (Vector Laboratories, Inc., Burlingame, Calif.). Cells expressing T-Ag were visualized with a Nikon epifluorescence microscope (Eclipse E800; Nikon, Inc.). A minimum of four fields were counted for each sample from three or more independent experiments.
Pharmacological treatment of Vero cell with cytoskeleton-binding agents. Collapse of the actin filaments was achieved by treating Vero cells with 0.1 μM Lat A for 1 h at 37°C. The microtubule network was disrupted by treating the cells with 30 μM nocodazole for 1 h at 37°C. A 24-hour treatment with 20 μM paclitaxel was employed to prevent microtubule disassembly. The actin cytoskeleton was stabilized by incubating the cells in 200 nM jasplakinolide for 24 h. Following either a mock or a given cytoskeleton treatment the cells were fixed and stained to evaluate the action of the respective drug.
To visualize the microtubule network, treated Vero cells were washed three times in 1X PBS, fixed and permeabilized in microtubule-stabilizing buffer (1% Triton X-100, 2% formaldehyde, 4% PEG-6000, 1 M EGTA, 1 M MgSO4, 1 M PIPES, pH 6.8) for 30 min at room temperature. The cells were then incubated with 1:100 dilution of mouse anti--tubulin antibody for 1 h at 37°C. After three washes in PBS the primary antibody was recognized by a secondary goat anti-mouse Alexa Fluor 488 antibody used at 1:150 dilution for 1 h at 37°C. The cells were washed and mounted on glass slides with anti-fade medium containing DAPI (Vector Laboratories). Staining was analyzed at a magnification of 63x on a laser scanning confocal microscope (TCS SP2 AOBS; Leica Microsystems, Exton, Pa.). All images were processed with Adobe Photoshop version 7.0.
To visualize the actin filaments, treated Vero cells were washed three times in PBS and fixed in 2% paraformaldehyde for 30 min. The 20-minute permeabilization in 0.2% saponin was followed by a 30-minute blocking step in 1% bovine serum albumen to reduce nonspecific binding. Alexa Fluor 488-conjugated phalloidin was used at dilution 1:40 for 1 h at 37°C as a probe for actin filaments. The cells were rinsed in PBS and mounted with medium containing DAPI for nuclear localization. Staining was analyzed as described above.
In the case of BKV infection, pretreated Vero cells were challenged with the virus in the continued presence of the drug for either the entire duration of the incubation (48 h) or only for the first 4 h of the virus life cycle. In the latter case, after removal of the pharmacological agent, the cells were incubated in fresh medium containing neutralizing concentrations of anti-BKV antisera. Cells were fixed at 48 h postinfection (h.p.i.) and stained for T-Ag expression as described above.
RESULTS
Effect of actin disassembly on BKV infection. As one of the major structural proteins responsible for cell shape, motility, and adhesion, actin is highly conserved and greatly abundant in eukaryotic cells (6, 50). Inside the cell actin filaments are arranged in bundles and networks that provide the supporting framework of the plasma membrane (15). To establish a role for intact actin cytoskeleton in BKV productive infection, Vero cells were treated with the chemical inhibitor of actin filament elongation latrunculin A. This natural marine product binds rapidly and exclusively to monomeric actin in a 1:1 complex, altering the subunit interface, which in turn prevents its polymerization (30).
As shown in panel 2 of Fig. 1A, incubation of Vero cells with a low dose of Lat A resulted in a complete disassembly of the actin filaments in contrast to the untreated cells, where intact bundles of actin stress fibers span the entire cytosol (Fig. 1A, panel 1). The phase-contrast images of untreated and Lat A-treated Vero cells (Fig. 1A, panels 3 and 4) depict the collapse of the cytoplasm following actin cytoskeleton disruption.
Lat A-treated Vero cells were infected with BKV and left in the continuous presence of the drug for the entire duration of the 48-hour incubation period (Fig. 1B, panel 3). Alternatively, the drug was removed after the initial four hours of virus internalization and the cells were refed with medium supplemented with anti-BKV neutralizing serum (Fig. 1B, panel 6). The latter experimental condition ensures that in the case of Lat A inactivation due to cell metabolism, no reinfection by extracellular free virus will take place. At 48 h.p.i. all cells were fixed and examined for BKV T-Ag expression by immunofluorescence. Untreated and uninfected cells in the absence or presence of anti-BKV anti-serum (Fig. 1B, panels 1 and 4) served as a negative control. Positive control cells were mock-treated and infected with BKV followed by the addition of control medium (Fig. 1B, panel 2) or medium containing anti-BKV neutralizing sera (Fig. 1B, panel 5).
No inhibition of BKV infection was seen upon disruption of the actin filaments with Lat A, suggesting that internalization and subsequent events in the BKV life cycle occur independent of an intact actin cytoskeleton.
Effect of actin stabilization on BKV infection. Previous studies on the involvement of the actin cytoskeleton in polyomavirus trafficking have reported decreased infection efficiency upon arrest in actin filament depolymerization (19, 37). To assess the requirement for dynamic and constant rearrangement of the microfilament meshwork in BKV infectious entry, Vero cells were treated with the F-actin probe jasplakinolide (Jas) prior to virus inoculation. Jas is readily cell-permeable and specifically binds to and stabilizes actin polymers by locking adjacent subunits together (7, 8). Based on previously published data describing Jas kinetics and mechanism of action (8) and on other reports utilizing this compound as a tool to manipulate microfilament assembly dynamics (8, 19, 37), we subjected Vero cells to a 24-hour Jas treatment with the nontoxic dose of 200 nM. Analysis of the accumulated changes in actin distribution reveals, as expected, large perinuclear actin aggregates and thick F-actin bundles at the cell margins when compared to untreated cells (Fig. 2A, panels 1 and 2). We then proceeded to challenge mock treated and Jas pretreated Vero cells with BKV. Stabilization of the actin cytoskeleton by the addition of Jas lead to a reduced efficiency of infection in Vero cells by BKV (Fig. 2B). The results from this experiment demonstrate that perturbation of the microfilament assembly and disassembly interferes with BKV trafficking and infection.
Effect of microtubule disassembly on BKV infection. The microtubule (MT) network is assembled in bundles of noncovalent tubulin polymers that originate from the microtubule-organizing center and radiate outward to the cell membrane (10), as seen on Fig. 3A, panel 1. MTs provide cytoplasmic order by positioning the nucleus and other organelles within the cell; they also serve as tracks for directional intracellular transport of membrane-bound vesicles (20). To examine the requirement for intact MTs during BKV infection, Vero cells were incubated with 30 μM of the MT poison nocodazole for 2 h at 37°C. Disassembly of the tubulin polymers was visualized by immunofluorescence staining with a monoclonal antibody directed against -tubulin (Fig. 3A, panel 2).
We then evaluated the ability of BKV to infect nocodazole-treated Vero cells (Fig. 3B, panels 3 and 4). Positive control cells were incubated with the virus in the absence of the drug (Fig. 3B, panels 1 and 2). Pharmacological disruption of the microtubule network resulted in decreased levels of BKV infection, pointing to an important role for these filaments in BKV trafficking. The inhibitory effect of nocodazole treatment on BKV infectivity was reversible, which indicated that the drug had not damaged the structural integrity of the virions, but had indeed impeded proper virus trafficking. To this end, Vero cells incubated with BKV in the presence of nocodazole were washed free of the drug and infection was allowed to proceed for 48 h in regular medium. Removal of the inhibitor rescued viral infectivity as shown by the number of BK T-Ag-positive cells in panels 1 and 3 of Fig. 3C. Nocodazole treatment of the cells 24 h after initiation of infection did not interfere with the expression of early viral proteins (Fig. 3C, panels 2 and 4). These data suggest that intact cellular MT network is necessary for BKV early infection.
Kinetics of microtubule disassembly and BKV infection. We next set out to establish the time frame of MT-dependent traffic in BKV early infection. Vero cells were inoculated with BKV at 4°C to synchronize virus entry. After shifting the cells to 37°C to initiate infection, nocodazole was added at the indicated time points and remained present for the duration of the 48-hour incubation period. Infection was then scored by staining for expression of early viral T-Ag. The inhibitory effect of nocodazole-induced disassembly of the MT fibers on BKV infection persisted until 8 h postinitiation of viral entry. Introducing the MT poison into the infectious medium at or after 8 h.p.i. did not result in the reduced infection efficiency that was recorded in the prior time points (Fig. 4).
Effect of microtubule stabilization on BKV infection. Having recognized a role for the MT network in BKV early infection, we next asked whether the intrinsic mechanical properties of these fibers such as dynamic instability and treadmilling were exploited by the virus for directional movement. To suppress MT subunit turnover Vero cells were exposed to a nontoxic dose of paclitaxel (20 μM) for 24 h at 37°C. In a highly specific manner this pharmacological agent interacts with tubulin to enhance both the rate and the yield of MT formation by increasing the number of MT nucleation events and by stabilizing assembled MTs to depolymerization (29, 34). The characteristic effect of paclitaxel treatment, namely the formation of distinct ordered bundles of MTs, can be seen in panel 2 of Fig. 5A. We then evaluated the ability of BKV to traffic and express early viral protein T-Ag in paclitaxel-treated cells. Negative control cells were either untreated or treated with paclitaxel and left uninfected (Fig. 5B, panels 1 and 4). Vero cells infected with BKV in the absence of paclitaxel served as positive control (Fig. 5B, panels 2 and 3). Paclitaxel treatment did not exhibit an inhibitory effect on BKV early infection (Fig. 5B, panels 5 and 6). The result from this experiment indicates that infection by BKV occurs independent of intrinsic MT dynamics such as the poleward flow of tubulin subunits within the polymers or lengthening and shortening fluctuations in the MT arrays.
DISCUSSION
Having successfully crossed the plasma membrane, BK virus now has to traverse the cytoplasm and deliver its genome to the host cell nucleus for replication. The parasitic nature and simplicity of viruses account for their exploitation of the cellular machinery throughout their infectious life cycle (35). Directed traffic of BKV between compartments in the cytosol is most likely aided by the cellular transport network. We report the results from a set of experiments designed to better understand the mechanism and key elements in BKV trafficking from the plasma to the nucleus. By selectively disrupting the architecture and dynamics of the two major cytoskeleton components, microfilaments and microtubules, we have begun to expose the participants in BKV transport during early infection.
We first turned our attention to the role of microfilaments during BKV productive infection. We used two actin-binding compounds with exactly opposite modes of action to manipulate the integrity and turnover rate of actin subunits. The results from our experiments demonstrate that disruption of the actin filaments with Lat A did not impede BKV infectivity, while prevention of actin polymerization dynamics with Jas exerted an inhibitory effect on BKV infection.
Collapse of the actin network has proven nondetrimental to the early stages in the life cycle of other polyomaviruses (2, 19, 46). SV40 T-Ag expression in CV-1 or SVG-A cells was insensitive to cytochalasin B- or cytochalasin D-induced disassembly of the actin cytoskeleton (2, 46). In addition, disruption of the microfilaments with four different treatment agents not only failed to inhibit PyV infection in either epithelial or fibroblast cells but also resulted in an enhanced, albeit saturable, PyV infectivity (19). The authors of the study attribute the increase in PyV infectivity to a probable redistribution leading to a better accessibility of the viral receptor or to a facilitated viral penetration upon elimination of dense network of cortical actin (19). A two-to threefold reduction in PyV infectivity was observed when the dynamic state of actin filaments was arrested by Jas prior to or up to 4 h.p.i., indicating that the need for microfilament turnover most likely maps to an early step in PyV virion uptake rather than intracellular trafficking (19).
Conversely, infection of SVG-A cells by JCV was severely impaired in the absence of intact actin filaments (2). As sequestering of actin monomers has been shown to disrupt clathrin-dependent endocytosis (26), the internalization mechanism of JCV (39), the authors speculate that the observed inhibition of JCV infection was occurring early at the stage of virus entry (2). It is worth noting that a study aimed at deciphering the fate of SV40 virions during caveola-mediated uptake reported a transient rearrangement of actin stress fibers following the binding of the virus to caveolae. In addition, the use of either actin monomer-sequestering or actin polymer-stabilizing agents led to a reduction in SV40 infectivity. Nonetheless, a fraction of the SV40 virions successfully trafficked through and infected the target cell. This suggests that actin filaments were not necessary for transport of virus-containing vesicles but rather enhanced a step in viral uptake (37). Our findings regarding the progression of BKV early infection in cells with a disassembled microfilament network are in agreement with previous reports stating the lack of requirement for intact actin cytoskeleton during cellular trafficking of other polyomaviruses internalized by clathrin-independent endocytosis (2, 19, 37, 44, 46).
We next turned our attention to the role of the microtubule network in BKV early infection of Vero cells. For this purpose we took advantage of two well-characterized MT poisons, nocodazole and paclitaxel, which exert opposing activities on MT stability. Reversible inhibition of MT polymerization by nocodazole resulted in substantial decrease in BKV T-Ag expression levels. Viral infectivity was restored once the drug was removed and the cells were allowed to recover indicating that nocodazole did not permanently damage the cellular or virion structure. The degree of infection inhibition was similar under both experimental conditions: removing of the drug at 4 h.p.i. followed by neutralization of extracellular virus (Fig. 3C, panels 2 and 4) or leaving the drug in continuously for the entire duration of the incubation (Fig. 3C, panels 1 and 3).
There are reports of accumulation and immobilization of invaginated caveolae at the surface of CHO cells upon microtubule disruption with nocodazole (31). The reduced infection levels seen upon nocodazole removal followed by anti-BKV serum addition would be explained by the neutralization of extracellular/noninternalized virus. The general notion, however, is that microtubules are involved largely in the shuttling of newly formed endocytic vesicles to various intracellular locations, and not during the budding process (25, 53). To this end, we followed the intracellular fate of cholera toxin subunit B, a caveola-dependent ligand, in untreated and nocodazole-treated Vero cells. Our results demonstrate that although cholera toxin subunit B was successfully taken up by cells with depolymerized microtubules, the toxin was unable to move directionally and reach the Golgi, as did the cholera toxin subunit B internalized in untreated control Vero cells (data not shown). The misguided pattern of intracellular trafficking of cholera toxin subunit B seen in cells treated with nocodazole suggests that in the absence of polymerized microtubules caveolar cargo is successfully endocytosed but its subsequent intracellular trajectory is altered.
We speculate that BK virions taken up by nocodazole-treated cells might be subjected to a similar misdirected course of intracellular transport that diverts them to a degradation pathway, thereby precluding their progression through the optimal route for productive infection. Presumably in nocodazole-treated cells the prolonged initial arrest of virions in an early vesicular intermediate, due to the lack of polymerized microtubules, leads to their sequestration into endocytic compartments such as the lysosome that proves detrimental for the viral life cycle. Addition of neutralizing anti-BKV serum binds to extracellular virus, and in turn prevents subsequent reinfection of cells even after the drug is removed and microtubules repolymerize. The recovery of infection observed when nocodazole was washed out and the cells were incubated in drug-free medium (Fig. 3C, panels 1 and 3) would result from the invasion of cells by newly internalized virions following the restoration of microtubule structures. The inhibitory effect of MT disassembly on BKV infection levels was lost when the nocodazole was added at or after 8 h.p.i.. Therefore, the results from the experiment on the kinetics of nocodazole action lead us to conclude that by 8 h.p.i. BKV has entered an MT-independent stage of its infectious life cycle.
The requirement for intact microtubule cytoskeleton appears to be a common theme in the productive infection of all polyomaviruses that are studied, regardless of their mode of plasma membrane penetration (2, 18, 19, 36, 44, 46). Reduced efficiency in PyV infectivity upon MT disruption is observed until 8 h.p.i. or in some cases as late as 12 h.p.i., depending on the host cell type. In the case of SV40, the need for intact preassembled MTs arises between 3 and 6 h.p.i. (36). Another study also reports that addition of the MT poison colcemid within 4 h.p.i. resulted in maximal inhibition of SV40 T-Ag expression (46). The microtubule network is also essential for efficient trafficking of JCV, as viral infectivity is severely reduced in the presence of nocodazole (2).
Arrest in the dynamic state of the MT filaments, however, did not interfere with BKV infectivity. Previous studies demonstrating that paclitaxel-induced stabilization of MTs resulted in significant inhibition of the frequency and velocity of small MT-dependent vesicle movements (21, 22) prompted us to examine the effect of selective MT stabilization during BKV early infection. Pretreatment of Vero cells with paclitaxel leads to the formation of the characteristic thick and extensive parallel arrays of MTs due to enhanced polymerization and block in depolymerization of the fibers. Our observations demonstrate that the level of BKV infection remained unaltered in cells with suppressed MT dynamics. It appears that during cellular invasion the virus takes advantage of the preexisting MT tracks and does not rely on the intrinsic mechanical properties such as treadmilling and dynamic instability of these fibers. Similar findings were reported for PyV as viral infectivity was sharply reduced upon MT disassembly, but was unaffected by a block in tubulin subunit turnover (18, 19).
In summary, we examined the role of microfilaments and microtubules in BKV early infection by exposing Vero cells to a selection of pharmacological agents that specifically target the structure and properties of the above cytoskeletal elements. Our findings demonstrate that BKV infection proceeded successfully under the circumstances of induced disassembly of actin filaments. Arrest in microfilament dynamics, however, interfered with BKV infectivity. In contrast, selective depolymerization of MT filaments resulted in sharp reduction in BKV infectivity. The inhibitory effect of microtubule disruption on viral infectivity was observed up until 8 h.p.i.. We speculate that MTs are needed to transport vesicles and tubulovesicular structures containing BKV (11) from the site of entry, the plasma membrane, to the site of viral replication, the nucleus. BKV infection remained unaffected under the conditions of inhibited turnover of cellular MTs.
The exact nature of the interactions between BKV and the participating cytoskeletal elements as well as the identity of the various organelles involved in the BKV early infection remain to defined. Current and future studies in our laboratory are aimed at elucidating the cellular receptor complex for BKV and the subsequent intracellular fate of BK virions during productive infection.
ACKNOWLEDGMENTS
We thank all the members of the Atwood laboratory for critical discussions during the course of this work. We thank Robbert Creton for critical help with confocal microscopy, Bethany O'Hara for technical assistance, and Amanda Robinson, Amy Bozek, and Lorie St. Pierre for administrative assistance.
Work in our laboratory was supported by a grant from the National Cancer Institute, R01 CA71878, and by a grant from the National Institute of Neurologic Disorders and Stroke, R01 NS43097.
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Department of Molecular Microbiology and Immunology, Brown University, Providence, Rhode Island 02912
ABSTRACT
Posttransplant reactivation of BK virus (BKV) in the renal allograft progresses to polyomavirus-associated nephropathy in 1% to 8% of kidney recipients. Graft dysfunction and loss in 30% to 45% of polyomavirus-associated nephropathy-affected patients are secondary to extensive tubular epithelial cell injury induced by the lytic replication of BKV. The early events in productive BKV infection are not thoroughly understood. We have previously shown that BKV enters cells by caveola-mediated endocytosis. In this report we examine the role of microfilaments and microtubules during early viral infection. Our results show that BKV infection of Vero cells is sensitive to nocodazole-induced disassembly of the microtubule network for the initial 8 hours following virus binding. In contrast, suppression of microtubule turnover with the stabilizing agent paclitaxel has no effect on BKV infectivity. Selective disassembly of the actin filaments with latrunculin A does not impede BKV infection, while inhibition of microfilament dynamics with jasplakinolide results in reduced numbers of viral antigen-positive cells. These data demonstrate that BKV, like other polyomaviruses, relies on an intact microtubule network during early infection. BKV, however, does not share the requirement with the closely related JC virus for an intact actin cytoskeleton during intracellular transport.
INTRODUCTION
First discovered in 1970, BK virus (BKV) has recently drawn renewed interest as the causative agent of an infectious complication termed polyomavirus-associated nephropathy in renal transplant recipients (16, 52). Asymptomatic BKV infection is ubiquitous, occurring in 70% to 90% of healthy adults worldwide (41). Surgical injury and potent immunosuppressive therapy following renal transplantation lead to BKV reactivation and progression to polyomavirus-associated nephropathy in 1% to 10% of kidney recipients (24, 51). The clinical concern stems from the extensive virally induced damage to the kidney, which in turn results in severe allograft dysfunction and ultimate graft loss in 45% to 60% of polyomavirus-associated nephropathy-affected patients (32, 52).
BKV belongs to the family Polyomaviridae, which are small, nonenveloped, double-stranded DNA viruses. Other well-studied polyomaviruses include simian virus 40 (SV40), mouse polyomavirus (PyV), and JC virus (JCV). Despite established differences in receptor specificity (3-5, 9, 13, 14, 27, 28, 47, 48), mode of internalization (1, 12, 17, 33, 36, 37, 39, 40, 43), and intracellular trafficking mechanisms (2, 19, 36, 37), all the members of the polyomavirus family must deliver their genome to the nucleus of the host cell in order to execute a productive viral life cycle. The nucleus is the site of viral gene transcription, viral DNA replication, and progeny assembly (45).
Efforts in our laboratory are focused on understanding the early events during BKV infection, namely host cell entry and subsequent intracellular trafficking to the nucleus. We and others have previously demonstrated that BKV enters cells through a caveola-mediated internalization mechanism (11, 12, 38). Images from ultrastructural analysis of renal tubular cells during various stages of infection consistently reveal BK virions inside monopinocytotic vesicles and/or tubuloreticular network as the viral aggregates traverse the cytoplasm en route to the host cell nucleus (11). These observations are indicative of deliberate and directional movement of the virus through the cytoplasm of the target cell.
Having successfully penetrated the interior of the host, BKV, like other DNA viruses, is faced with the challenging task of reaching the nucleus—the most critical destination in the viral life cycle. Passive random diffusion through the cytoplasm is hardly the most efficient means for vectoral movement. Endocytic entry provides the virus with the advantage of active membrane transport (49). The cytoskeleton, an elaborate meshwork of fibrous proteins, provides the tracks along which membrane-bound vesicles and organelles move in the cytosol. The three classes of fibers that comprise the cytoskeleton include microtubules, polymers of globular tubulin subunits; microfilaments, polymers of the protein actin; and intermediate filaments, polymers of one or more rod-shaped protein subunits.
In this report we investigate the involvement of microfilaments and microtubules in cytoplasmic transport of BKV by selectively perturbing their architecture, and therefore function, with well-characterized pharmacological agents. Perturbing the integrity of the microfilament network by disruption of the actin polymers did not reduce BKV infection levels in Vero cells, while stabilization of the actin filaments interfered with BKV infectivity. Arrest in microtubule (MT) dynamics, however was inconsequential to virus trafficking during early infection. In contrast, selective induction of MT disassembly exhibited an inhibitory effect on BKV infection. The results from these experiments provide insight in BKV interaction with its host cell. Also, the data reveal similarities and differences during a productive infection between BKV and the other polyomaviruses.
MATERIALS AND METHODS
Cell line and virus. Vero cells were purchased from the American Type Culture Collection (Manassas, Va.) and were maintained in a humidified 37°C incubator in Eagle's minimal essential medium (Mediatech Inc., Herdon, Va.) supplemented with 5% heat-inactivated fetal bovine serum (Mediatech Inc.). BKV Gardener strain was obtained from the American Type Culture Collection and propagated in Vero cells as previously described (42).
Antibodies and reagents. The monoclonal antibody PAb 416 (Ab2) purchased from Oncogene Research Products (Cambridge, Mass.) detects the N-terminal portion of SV40, JCV, and BKV large T antigen (T-Ag). The mouse monoclonal anti--tubulin antibody, clone DM1A was purchased from Sigma-Aldrich (St. Louis, Mo.). Alexa Fluor 488-conjugated Phalloidin, a high-affinity probe for F-actin, was obtained from Molecular Probes. Rabbit anti-BKV serum was produced by injecting a New Zealand White rabbit with highly purified preparation of BK virus in complete Freund's adjuvant (23). The rabbit received two additional BKV boosts in incomplete Freund's adjuvant. The anti-BKV serum was titered by a Western blot assay and the immunoglobulin G portion was purified and isolated on a protein G column (Pierce Biotechnology, Rockford, Ill.). Latrunculin A (Lat A), nocodazole and paclitaxel (Taxol) were purchased from Sigma Aldrich and jasplakinolide was obtained from Calbiochem (San Diego, Calif.).
Indirect immunofluorescent analysis of BKV infection. To detect expression of early T-Ag, BKV-infected Vero cells were fixed in 2% paraformaldehyde at the end of a 48-hour incubation period after BKV infection. Following three washes in phosphate-buffered saline (PBS) (137 mM NaCl, 2.682 mM KCl, 8.1 mM Na2HPO4, 1.47 mM KH2PO4, pH 7.2), the cells were incubated with a 1:20 dilution of PAb 416 for 1 h at 37°C. The cells were then washed three times in PBS followed by a 45-minute incubation at 37°C with a 1:150 goat anti-mouse immunoglobulin G, F(ab')2 fragments conjugated to Alexa Fluor 488 (Molecular Probes, Eugene, Oreg.). Next the cells were extensively rinsed in PBS and counterstained with Evan's Blue solution (red cytoplasmic dye). The coverslips were mounted on glass slides with Vectashield mounting medium for fluorescence with 4',6'-diamidino-2-phenylindole (DAPI) (Vector Laboratories, Inc., Burlingame, Calif.). Cells expressing T-Ag were visualized with a Nikon epifluorescence microscope (Eclipse E800; Nikon, Inc.). A minimum of four fields were counted for each sample from three or more independent experiments.
Pharmacological treatment of Vero cell with cytoskeleton-binding agents. Collapse of the actin filaments was achieved by treating Vero cells with 0.1 μM Lat A for 1 h at 37°C. The microtubule network was disrupted by treating the cells with 30 μM nocodazole for 1 h at 37°C. A 24-hour treatment with 20 μM paclitaxel was employed to prevent microtubule disassembly. The actin cytoskeleton was stabilized by incubating the cells in 200 nM jasplakinolide for 24 h. Following either a mock or a given cytoskeleton treatment the cells were fixed and stained to evaluate the action of the respective drug.
To visualize the microtubule network, treated Vero cells were washed three times in 1X PBS, fixed and permeabilized in microtubule-stabilizing buffer (1% Triton X-100, 2% formaldehyde, 4% PEG-6000, 1 M EGTA, 1 M MgSO4, 1 M PIPES, pH 6.8) for 30 min at room temperature. The cells were then incubated with 1:100 dilution of mouse anti--tubulin antibody for 1 h at 37°C. After three washes in PBS the primary antibody was recognized by a secondary goat anti-mouse Alexa Fluor 488 antibody used at 1:150 dilution for 1 h at 37°C. The cells were washed and mounted on glass slides with anti-fade medium containing DAPI (Vector Laboratories). Staining was analyzed at a magnification of 63x on a laser scanning confocal microscope (TCS SP2 AOBS; Leica Microsystems, Exton, Pa.). All images were processed with Adobe Photoshop version 7.0.
To visualize the actin filaments, treated Vero cells were washed three times in PBS and fixed in 2% paraformaldehyde for 30 min. The 20-minute permeabilization in 0.2% saponin was followed by a 30-minute blocking step in 1% bovine serum albumen to reduce nonspecific binding. Alexa Fluor 488-conjugated phalloidin was used at dilution 1:40 for 1 h at 37°C as a probe for actin filaments. The cells were rinsed in PBS and mounted with medium containing DAPI for nuclear localization. Staining was analyzed as described above.
In the case of BKV infection, pretreated Vero cells were challenged with the virus in the continued presence of the drug for either the entire duration of the incubation (48 h) or only for the first 4 h of the virus life cycle. In the latter case, after removal of the pharmacological agent, the cells were incubated in fresh medium containing neutralizing concentrations of anti-BKV antisera. Cells were fixed at 48 h postinfection (h.p.i.) and stained for T-Ag expression as described above.
RESULTS
Effect of actin disassembly on BKV infection. As one of the major structural proteins responsible for cell shape, motility, and adhesion, actin is highly conserved and greatly abundant in eukaryotic cells (6, 50). Inside the cell actin filaments are arranged in bundles and networks that provide the supporting framework of the plasma membrane (15). To establish a role for intact actin cytoskeleton in BKV productive infection, Vero cells were treated with the chemical inhibitor of actin filament elongation latrunculin A. This natural marine product binds rapidly and exclusively to monomeric actin in a 1:1 complex, altering the subunit interface, which in turn prevents its polymerization (30).
As shown in panel 2 of Fig. 1A, incubation of Vero cells with a low dose of Lat A resulted in a complete disassembly of the actin filaments in contrast to the untreated cells, where intact bundles of actin stress fibers span the entire cytosol (Fig. 1A, panel 1). The phase-contrast images of untreated and Lat A-treated Vero cells (Fig. 1A, panels 3 and 4) depict the collapse of the cytoplasm following actin cytoskeleton disruption.
Lat A-treated Vero cells were infected with BKV and left in the continuous presence of the drug for the entire duration of the 48-hour incubation period (Fig. 1B, panel 3). Alternatively, the drug was removed after the initial four hours of virus internalization and the cells were refed with medium supplemented with anti-BKV neutralizing serum (Fig. 1B, panel 6). The latter experimental condition ensures that in the case of Lat A inactivation due to cell metabolism, no reinfection by extracellular free virus will take place. At 48 h.p.i. all cells were fixed and examined for BKV T-Ag expression by immunofluorescence. Untreated and uninfected cells in the absence or presence of anti-BKV anti-serum (Fig. 1B, panels 1 and 4) served as a negative control. Positive control cells were mock-treated and infected with BKV followed by the addition of control medium (Fig. 1B, panel 2) or medium containing anti-BKV neutralizing sera (Fig. 1B, panel 5).
No inhibition of BKV infection was seen upon disruption of the actin filaments with Lat A, suggesting that internalization and subsequent events in the BKV life cycle occur independent of an intact actin cytoskeleton.
Effect of actin stabilization on BKV infection. Previous studies on the involvement of the actin cytoskeleton in polyomavirus trafficking have reported decreased infection efficiency upon arrest in actin filament depolymerization (19, 37). To assess the requirement for dynamic and constant rearrangement of the microfilament meshwork in BKV infectious entry, Vero cells were treated with the F-actin probe jasplakinolide (Jas) prior to virus inoculation. Jas is readily cell-permeable and specifically binds to and stabilizes actin polymers by locking adjacent subunits together (7, 8). Based on previously published data describing Jas kinetics and mechanism of action (8) and on other reports utilizing this compound as a tool to manipulate microfilament assembly dynamics (8, 19, 37), we subjected Vero cells to a 24-hour Jas treatment with the nontoxic dose of 200 nM. Analysis of the accumulated changes in actin distribution reveals, as expected, large perinuclear actin aggregates and thick F-actin bundles at the cell margins when compared to untreated cells (Fig. 2A, panels 1 and 2). We then proceeded to challenge mock treated and Jas pretreated Vero cells with BKV. Stabilization of the actin cytoskeleton by the addition of Jas lead to a reduced efficiency of infection in Vero cells by BKV (Fig. 2B). The results from this experiment demonstrate that perturbation of the microfilament assembly and disassembly interferes with BKV trafficking and infection.
Effect of microtubule disassembly on BKV infection. The microtubule (MT) network is assembled in bundles of noncovalent tubulin polymers that originate from the microtubule-organizing center and radiate outward to the cell membrane (10), as seen on Fig. 3A, panel 1. MTs provide cytoplasmic order by positioning the nucleus and other organelles within the cell; they also serve as tracks for directional intracellular transport of membrane-bound vesicles (20). To examine the requirement for intact MTs during BKV infection, Vero cells were incubated with 30 μM of the MT poison nocodazole for 2 h at 37°C. Disassembly of the tubulin polymers was visualized by immunofluorescence staining with a monoclonal antibody directed against -tubulin (Fig. 3A, panel 2).
We then evaluated the ability of BKV to infect nocodazole-treated Vero cells (Fig. 3B, panels 3 and 4). Positive control cells were incubated with the virus in the absence of the drug (Fig. 3B, panels 1 and 2). Pharmacological disruption of the microtubule network resulted in decreased levels of BKV infection, pointing to an important role for these filaments in BKV trafficking. The inhibitory effect of nocodazole treatment on BKV infectivity was reversible, which indicated that the drug had not damaged the structural integrity of the virions, but had indeed impeded proper virus trafficking. To this end, Vero cells incubated with BKV in the presence of nocodazole were washed free of the drug and infection was allowed to proceed for 48 h in regular medium. Removal of the inhibitor rescued viral infectivity as shown by the number of BK T-Ag-positive cells in panels 1 and 3 of Fig. 3C. Nocodazole treatment of the cells 24 h after initiation of infection did not interfere with the expression of early viral proteins (Fig. 3C, panels 2 and 4). These data suggest that intact cellular MT network is necessary for BKV early infection.
Kinetics of microtubule disassembly and BKV infection. We next set out to establish the time frame of MT-dependent traffic in BKV early infection. Vero cells were inoculated with BKV at 4°C to synchronize virus entry. After shifting the cells to 37°C to initiate infection, nocodazole was added at the indicated time points and remained present for the duration of the 48-hour incubation period. Infection was then scored by staining for expression of early viral T-Ag. The inhibitory effect of nocodazole-induced disassembly of the MT fibers on BKV infection persisted until 8 h postinitiation of viral entry. Introducing the MT poison into the infectious medium at or after 8 h.p.i. did not result in the reduced infection efficiency that was recorded in the prior time points (Fig. 4).
Effect of microtubule stabilization on BKV infection. Having recognized a role for the MT network in BKV early infection, we next asked whether the intrinsic mechanical properties of these fibers such as dynamic instability and treadmilling were exploited by the virus for directional movement. To suppress MT subunit turnover Vero cells were exposed to a nontoxic dose of paclitaxel (20 μM) for 24 h at 37°C. In a highly specific manner this pharmacological agent interacts with tubulin to enhance both the rate and the yield of MT formation by increasing the number of MT nucleation events and by stabilizing assembled MTs to depolymerization (29, 34). The characteristic effect of paclitaxel treatment, namely the formation of distinct ordered bundles of MTs, can be seen in panel 2 of Fig. 5A. We then evaluated the ability of BKV to traffic and express early viral protein T-Ag in paclitaxel-treated cells. Negative control cells were either untreated or treated with paclitaxel and left uninfected (Fig. 5B, panels 1 and 4). Vero cells infected with BKV in the absence of paclitaxel served as positive control (Fig. 5B, panels 2 and 3). Paclitaxel treatment did not exhibit an inhibitory effect on BKV early infection (Fig. 5B, panels 5 and 6). The result from this experiment indicates that infection by BKV occurs independent of intrinsic MT dynamics such as the poleward flow of tubulin subunits within the polymers or lengthening and shortening fluctuations in the MT arrays.
DISCUSSION
Having successfully crossed the plasma membrane, BK virus now has to traverse the cytoplasm and deliver its genome to the host cell nucleus for replication. The parasitic nature and simplicity of viruses account for their exploitation of the cellular machinery throughout their infectious life cycle (35). Directed traffic of BKV between compartments in the cytosol is most likely aided by the cellular transport network. We report the results from a set of experiments designed to better understand the mechanism and key elements in BKV trafficking from the plasma to the nucleus. By selectively disrupting the architecture and dynamics of the two major cytoskeleton components, microfilaments and microtubules, we have begun to expose the participants in BKV transport during early infection.
We first turned our attention to the role of microfilaments during BKV productive infection. We used two actin-binding compounds with exactly opposite modes of action to manipulate the integrity and turnover rate of actin subunits. The results from our experiments demonstrate that disruption of the actin filaments with Lat A did not impede BKV infectivity, while prevention of actin polymerization dynamics with Jas exerted an inhibitory effect on BKV infection.
Collapse of the actin network has proven nondetrimental to the early stages in the life cycle of other polyomaviruses (2, 19, 46). SV40 T-Ag expression in CV-1 or SVG-A cells was insensitive to cytochalasin B- or cytochalasin D-induced disassembly of the actin cytoskeleton (2, 46). In addition, disruption of the microfilaments with four different treatment agents not only failed to inhibit PyV infection in either epithelial or fibroblast cells but also resulted in an enhanced, albeit saturable, PyV infectivity (19). The authors of the study attribute the increase in PyV infectivity to a probable redistribution leading to a better accessibility of the viral receptor or to a facilitated viral penetration upon elimination of dense network of cortical actin (19). A two-to threefold reduction in PyV infectivity was observed when the dynamic state of actin filaments was arrested by Jas prior to or up to 4 h.p.i., indicating that the need for microfilament turnover most likely maps to an early step in PyV virion uptake rather than intracellular trafficking (19).
Conversely, infection of SVG-A cells by JCV was severely impaired in the absence of intact actin filaments (2). As sequestering of actin monomers has been shown to disrupt clathrin-dependent endocytosis (26), the internalization mechanism of JCV (39), the authors speculate that the observed inhibition of JCV infection was occurring early at the stage of virus entry (2). It is worth noting that a study aimed at deciphering the fate of SV40 virions during caveola-mediated uptake reported a transient rearrangement of actin stress fibers following the binding of the virus to caveolae. In addition, the use of either actin monomer-sequestering or actin polymer-stabilizing agents led to a reduction in SV40 infectivity. Nonetheless, a fraction of the SV40 virions successfully trafficked through and infected the target cell. This suggests that actin filaments were not necessary for transport of virus-containing vesicles but rather enhanced a step in viral uptake (37). Our findings regarding the progression of BKV early infection in cells with a disassembled microfilament network are in agreement with previous reports stating the lack of requirement for intact actin cytoskeleton during cellular trafficking of other polyomaviruses internalized by clathrin-independent endocytosis (2, 19, 37, 44, 46).
We next turned our attention to the role of the microtubule network in BKV early infection of Vero cells. For this purpose we took advantage of two well-characterized MT poisons, nocodazole and paclitaxel, which exert opposing activities on MT stability. Reversible inhibition of MT polymerization by nocodazole resulted in substantial decrease in BKV T-Ag expression levels. Viral infectivity was restored once the drug was removed and the cells were allowed to recover indicating that nocodazole did not permanently damage the cellular or virion structure. The degree of infection inhibition was similar under both experimental conditions: removing of the drug at 4 h.p.i. followed by neutralization of extracellular virus (Fig. 3C, panels 2 and 4) or leaving the drug in continuously for the entire duration of the incubation (Fig. 3C, panels 1 and 3).
There are reports of accumulation and immobilization of invaginated caveolae at the surface of CHO cells upon microtubule disruption with nocodazole (31). The reduced infection levels seen upon nocodazole removal followed by anti-BKV serum addition would be explained by the neutralization of extracellular/noninternalized virus. The general notion, however, is that microtubules are involved largely in the shuttling of newly formed endocytic vesicles to various intracellular locations, and not during the budding process (25, 53). To this end, we followed the intracellular fate of cholera toxin subunit B, a caveola-dependent ligand, in untreated and nocodazole-treated Vero cells. Our results demonstrate that although cholera toxin subunit B was successfully taken up by cells with depolymerized microtubules, the toxin was unable to move directionally and reach the Golgi, as did the cholera toxin subunit B internalized in untreated control Vero cells (data not shown). The misguided pattern of intracellular trafficking of cholera toxin subunit B seen in cells treated with nocodazole suggests that in the absence of polymerized microtubules caveolar cargo is successfully endocytosed but its subsequent intracellular trajectory is altered.
We speculate that BK virions taken up by nocodazole-treated cells might be subjected to a similar misdirected course of intracellular transport that diverts them to a degradation pathway, thereby precluding their progression through the optimal route for productive infection. Presumably in nocodazole-treated cells the prolonged initial arrest of virions in an early vesicular intermediate, due to the lack of polymerized microtubules, leads to their sequestration into endocytic compartments such as the lysosome that proves detrimental for the viral life cycle. Addition of neutralizing anti-BKV serum binds to extracellular virus, and in turn prevents subsequent reinfection of cells even after the drug is removed and microtubules repolymerize. The recovery of infection observed when nocodazole was washed out and the cells were incubated in drug-free medium (Fig. 3C, panels 1 and 3) would result from the invasion of cells by newly internalized virions following the restoration of microtubule structures. The inhibitory effect of MT disassembly on BKV infection levels was lost when the nocodazole was added at or after 8 h.p.i.. Therefore, the results from the experiment on the kinetics of nocodazole action lead us to conclude that by 8 h.p.i. BKV has entered an MT-independent stage of its infectious life cycle.
The requirement for intact microtubule cytoskeleton appears to be a common theme in the productive infection of all polyomaviruses that are studied, regardless of their mode of plasma membrane penetration (2, 18, 19, 36, 44, 46). Reduced efficiency in PyV infectivity upon MT disruption is observed until 8 h.p.i. or in some cases as late as 12 h.p.i., depending on the host cell type. In the case of SV40, the need for intact preassembled MTs arises between 3 and 6 h.p.i. (36). Another study also reports that addition of the MT poison colcemid within 4 h.p.i. resulted in maximal inhibition of SV40 T-Ag expression (46). The microtubule network is also essential for efficient trafficking of JCV, as viral infectivity is severely reduced in the presence of nocodazole (2).
Arrest in the dynamic state of the MT filaments, however, did not interfere with BKV infectivity. Previous studies demonstrating that paclitaxel-induced stabilization of MTs resulted in significant inhibition of the frequency and velocity of small MT-dependent vesicle movements (21, 22) prompted us to examine the effect of selective MT stabilization during BKV early infection. Pretreatment of Vero cells with paclitaxel leads to the formation of the characteristic thick and extensive parallel arrays of MTs due to enhanced polymerization and block in depolymerization of the fibers. Our observations demonstrate that the level of BKV infection remained unaltered in cells with suppressed MT dynamics. It appears that during cellular invasion the virus takes advantage of the preexisting MT tracks and does not rely on the intrinsic mechanical properties such as treadmilling and dynamic instability of these fibers. Similar findings were reported for PyV as viral infectivity was sharply reduced upon MT disassembly, but was unaffected by a block in tubulin subunit turnover (18, 19).
In summary, we examined the role of microfilaments and microtubules in BKV early infection by exposing Vero cells to a selection of pharmacological agents that specifically target the structure and properties of the above cytoskeletal elements. Our findings demonstrate that BKV infection proceeded successfully under the circumstances of induced disassembly of actin filaments. Arrest in microfilament dynamics, however, interfered with BKV infectivity. In contrast, selective depolymerization of MT filaments resulted in sharp reduction in BKV infectivity. The inhibitory effect of microtubule disruption on viral infectivity was observed up until 8 h.p.i.. We speculate that MTs are needed to transport vesicles and tubulovesicular structures containing BKV (11) from the site of entry, the plasma membrane, to the site of viral replication, the nucleus. BKV infection remained unaffected under the conditions of inhibited turnover of cellular MTs.
The exact nature of the interactions between BKV and the participating cytoskeletal elements as well as the identity of the various organelles involved in the BKV early infection remain to defined. Current and future studies in our laboratory are aimed at elucidating the cellular receptor complex for BKV and the subsequent intracellular fate of BK virions during productive infection.
ACKNOWLEDGMENTS
We thank all the members of the Atwood laboratory for critical discussions during the course of this work. We thank Robbert Creton for critical help with confocal microscopy, Bethany O'Hara for technical assistance, and Amanda Robinson, Amy Bozek, and Lorie St. Pierre for administrative assistance.
Work in our laboratory was supported by a grant from the National Cancer Institute, R01 CA71878, and by a grant from the National Institute of Neurologic Disorders and Stroke, R01 NS43097.
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