Dendritic cell internalization of foot-and-mouth disease virus: influence of heparan sulfate binding on virus uptake and induction of the immune response - PubMed (original) (raw)
Comparative Study
. 2008 Jul;82(13):6379-94.
doi: 10.1128/JVI.00021-08. Epub 2008 Apr 30.
Affiliations
- PMID: 18448534
- PMCID: PMC2447102
- DOI: 10.1128/JVI.00021-08
Comparative Study
Dendritic cell internalization of foot-and-mouth disease virus: influence of heparan sulfate binding on virus uptake and induction of the immune response
Lisa J Harwood et al. J Virol. 2008 Jul.
Abstract
Dendritic cells (DC), which are essential for inducing and regulating immune defenses and responses, represent the critical target for vaccines against pathogens such as foot-and-mouth disease virus (FMDV). Although it is clear that FMDV enters epithelial cells via integrins, little is known about FMDV interaction with DC. Accordingly, DC internalization of FMDV antigen was analyzed by comparing vaccine virus dominated by heparan sulfate (HS)-binding variants with FMDV lacking HS-binding capacity. The internalization was most efficient with the HS-binding virus, employing diverse endocytic pathways. Moreover, internalization relied primarily on HS binding. Uptake of non-HS-binding virus by DC was considerably less efficient, so much so that it was often difficult to detect virus interacting with the DC. The HS-binding FMDV replicated in DC, albeit transiently, which was demonstrable by its sensitivity to cycloheximide treatment and the short duration of infectious virus production. There was no evidence that the non-HS-binding virus replicated in the DC. These observations on virus replication may be explained by the activities of viral RNA in the DC. When DC were transfected with infectious RNA, only 1% of the translated viral proteins were detected. Nevertheless, the transfected cells, and DC which had internalized live virus, did present antigen to lymphocytes, inducing an FMDV-specific immunoglobulin G response. These results demonstrate that DC internalization of FMDV is most efficient for vaccine virus with HS-binding capacity, but HS binding is not an exclusive requirement. Both non-HS-binding virus and infectious RNA interacting with DC induce specific immune responses, albeit less efficiently than HS-binding virus.
Figures
FIG. 1.
HS-variant FMDV interacts, internalizes, and transiently replicates in MoDC. Four-day-old MoDC were infected with FMDV O1 (green) and incubated for (A) 10 min or (B) 4 h. Costaining of FMDV (red) with an internal anti-caveolin marker (green) at 10 min p.i. (C) and major histocompatibility complex class II surface-staining (green) at 4 h p.i (D). (E) FMDV (green) uptake after 24 h at an MOI of 60 TCID50/cell. (F) Mock-infected cells. At the different times p.i., the cells were fixed and analyzed by confocal microscopy using either a type O- or type C-specific MAb (4C9 or 5C4, respectively). (G) pH resistance of DC-associated FMDV was measured by infecting 4-day-old MoDC with FMDV O1 at an MOI of 1 TCID50/cell for 1 h at 4°C. Cells were then washed eight times to remove unbound virus followed by the addition of prewarmed medium, and the temperature was then shifted to 39°C. At the indicated time points, half the cells were treated with 0.05 M sodium hydrogen phosphate (pH 6) to destroy virus on the cell surface. Cells were then lysed to measure the titers of cell-associated virus by titration on BHK-21 cells. (H) Replication of FMDV O1 was determined by pretreating cells with cycloheximide and measuring infectious cell-associated virus by titration on BHK-21 cells. The results shown are representative of results of three independent experiments.
FIG. 2.
Influence of metabolic inhibitors on HS-variant FMDV binding and internalization by MoDC. Four-day-old MoDC were pretreated with metabolic inhibitors for 30 min at 39°C and then infected for 4 h with FMDV O1 at an MOI of 60 TCID50/cell (also at 39°C) in the presence or absence of metabolic inhibitors. Cells were then fixed and FMDV antigen was detected using MAb 4C9 and goat anti-mouse IgG conjugated with Alexa-488. Arrows indicate the presence of virus.
FIG. 3.
Assessment of metabolic inhibitors in porcine MoDC. (A to F) Four-day-old MoDC were pretreated with metabolic inhibitors, or left untreated, for 30 min at 39°C and then 1 mg/ml DQ ovalbumin was applied. After 4 h incubation, cells were fixed, and the processing of DQ ovalbumin was assessed by confocal microscopy. The effect of cytochalasin (G) was visualized by pretreating the cells for 30 min and then staining for phalloidin and comparing the result with that seen in the absence of cytochalasin (H). Chlorpromazine activity (I) was demonstrated by inhibition of transferrin uptake compared to that for control cells (J).
FIG. 4.
Effect of metabolic inhibitors on binding and internalization of FMDV by DC with and without pronase treatment. Four-day-old MoDC were pretreated for 30 min with metabolic inhibitors at 39°C, as in Fig. 2, or were left untreated (No inhibitor). The cultures were then placed at 4°C and infected with FMDV O1 at an MOI of 1 TCID50/cell for 1 h at 4°C. Cells were washed eight times to remove any unbound virus followed by the addition of prewarmed medium and were then shifted back to 39°C. At 30 min (A) and 4 h (B) p.i., cells were harvested and treated with pronase to destroy bound virus. Cells were then lysed, and titers of cell-associated virus were assessed by titration on BHK-21 cells. (C) Percentage of bound virus internalized (remaining after pronase treatment) by the MoDC. The results shown are representative of results of three independent experiments.
FIG. 5.
DSTP inhibits the infection of DC by HS-binding virus. (A) Four-day-old MoDC were infected for 4 h with FMDV O1 at an MOI of 60 TCID50/cell in the presence of different amounts of the HS-binding inhibitor DSTP (25 μg, 100 μg, and 400 μg). This infection was performed at 39°C without a washing step, to give the virus the maximum chance for interacting with the cells. The 4-h infection was repeated with DSTP (C and E, +DSTP) and without 100 μg DSTP (B and D, −DSTP) and with the addition of bafilomycin (B and C) or chloroquine (D and E). Cells were fixed, and FMDV antigen was detected using MAb 4C9 and goat anti-mouse IgG conjugated to Alexa-488. Arrows indicate the presence of virus.
FIG. 6.
Influence of circumventing the HS structures on MoDC with respect to viral RNA translation and infectious virus association with the cells. (A) Production of FMDV protease 3A following transfection of BHK-21 cells with infectious RNA (O1K/C-S8c1). The FMDV protease 3A protein production was detected by FACS analysis at 48 h posttransfection. (B) Comparison of HS-binding and non-HS-binding virus interaction with MoDC in terms of the presence of infectious virus. Four-day-old MoDC were infected with FMDV O1 and FMDV C-S8c1 at an MOI of 1 TCID50/cell for 1 h at 4°C. Cells were then shifted to 39°C for the duration of the experiment. At each of the time points shown on the x axis, the cells were washed eight times to remove any unbound virus. The cells were then lysed after the final centrifugation to release cell-associated virus, and the infectious virus titers were measured by titration on BHK-21 cells. The results shown are representative of results of three independent experiments.
FIG. 7.
Transfection of MoDC with infectious RNA results in abortive replication. (A) Following transfection of MoDC with O1K/C-S8c1 infectious RNA, the presence of ECV and cell-associated virus (CAV) was determined at different times posttransfection by titrating on BHK-21 cells. (B) Four-day-old MoDC transfected with rhodamine-labeled O1K/C-S8c1 RNA (red). At 4 h posttransfection, the MoDC were washed eight times. Replicate cultures were either prepared immediately for confocal microscopy or incubated further at 39°C for the indicated times. The preparation for microscopy entailed fixation and permeabilization of the cells as described in Materials and Methods, followed by staining with an Alexa-labeled anti-FMDV capsid antibody (green). Arrows denote presence of virus. (C) FACS analysis of viral protease 3A production in the MoDC transfected with O1K/C-S8c1 infectious RNA, using MAb against FMDV protease 3A following fixation and permeabilization of the cells. (D) Detection of dsRNA after transfection of the MoDC with O1K/C-S8c1 RNA. The dsRNA was detected using MAb J2 and confocal microscopy at 24 h, 48 h, and 72 h posttransfection.
FIG. 8.
MoDC transfected with infectious RNA and virus induce lymphocyte recall responses in vitro. A coculture system of CD172a− cells (containing T and B lymphocytes) and O1K/C-S8c1 RNA-transfected MoDC were used to measure T- and B-lymphocyte-dependent anti-FMDV IgG production. MoDC were transfected with O1K/C-S8c1 and cocultured with CD172a− cells from a vaccinated pig (A) and a naïve pig (B). MoDC from a vaccinated pig were infected for 4 h with HS-variant FMDV (C1, MARLS, and O1K/C-S8c1/p5) and non-HS variant (C-S8c1 and O1K/C-S8c1/p2), after which the cells were extensively washed. Cocultures of CD172a− were set up, and T- and B-lymphocyte-dependent anti-FMDV IgG production was measured (C). These experiments are representative of results of three separate experiments.
References
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