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. 2003 Dec 9;100(25):15143-8.
doi: 10.1073/pnas.2434327100. Epub 2003 Nov 20.

Apical recycling systems regulate directional budding of respiratory syncytial virus from polarized epithelial cells

Sean C Brock  1 , James R Goldenring, James E Crowe Jr
Affiliations

Apical recycling systems regulate directional budding of respiratory syncytial virus from polarized epithelial cells

Sean C Brock et al. Proc Natl Acad Sci U S A. .

Abstract

Respiratory syncytial virus (RSV) is the major viral cause of serious lower respiratory tract illness in infants and young children worldwide. RSV infection is limited to the superficial layers of the respiratory epithelium in immunocompetent individuals. Consistent with this in vivo observation, we and others have found that RSV buds preferentially from the apical surface of infected polarized epithelial cells. In contrast, directional budding is not observed in nonpolarized human epithelial cells. These findings suggest that RSV uses specific cellular trafficking pathways to accomplish viral replication. The host cell proteins that regulate directional budding of RSV are undefined. Apical sorting of cellular proteins in polarized epithelial cells involves the apical recycling endosome (ARE). To investigate whether ARE-mediated protein sorting plays a role during RSV replication, we expressed a fragment of the myosin Vb tail that functions as a dominant negative inhibitor of ARE-mediated protein sorting in polarized Madin-Darby canine kidney cells. When these cells were infected with RSV, a >9,000-fold reduction in viral yield was observed. A similar effect on virus replication was observed when a carboxyl-terminal fragment of another ARE-associated protein, the Rab11 family interacting protein 1, was expressed in Madin-Darby canine kidney cells. These data suggest that RSV requires proper ARE-mediated protein sorting for efficient egress from the apical surface of polarized epithelial cells.

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Figures

Fig. 1.
Fig. 1.
Directional release of RSV from polarized or nonpolarized cell culture monolayers. Polarized MDCK cell monolayers were infected with RSV from the apical (a) or basolateral surface (c). (b) Nonpolarized HEp-2 cell monolayers were infected with RSV from the apical surface. Apical (•) and basolateral (○) tissue culture supernatants were collected separately at the indicated times postinfection, and virus was quantified by plaque assay. The dashed line denotes the limit of detection for this assay. Titers at each time point are the average value of triplicate samples; the data are representative of at least two experiments.
Fig. 2.
Fig. 2.
GFP-myosin Vb tail expression inhibits RSV replication. MDCK cell culture monolayers with (solid bars) or without (open bars) doxycycline were plated on 48-well tissue culture plates and infected with RSV, vaccinia virus, or influenza virus. MDCK cell culture monolayers expressing the mpIgR were plated on 48-well tissue culture plates and infected with RSV. Virus was recovered from infected cell culture supernatants (a) or cell monolayers (b) separately at 120, 48, and 72 h for RSV-, vaccinia virus-, or influenza virus-infected cells, respectively, and quantified as described in Materials and Methods. Results shown are the mean results of triplicate samples (±SD) and are representative of at least two experiments.
Fig. 3.
Fig. 3.
Kinetic aspects of virus infection of GFP-myosin Vb tail-expressing MDCK cells. MDCK cell culture monolayers with (•) or without (○) doxycycline were plated on 48-well tissue culture plates and infected with RSV (a) or vaccinia virus (b). Virus was recovered from infected cell culture monolayers at 24- or 12-h intervals for periods of 120 or 48 h, for RSV- or vaccinia virus-infected cells, respectively. Data shown are the average value of duplicate titration on samples from two experiments.
Fig. 4.
Fig. 4.
Confocal microscopy analysis of the cellular distribution of RSV F protein in the presence of GFP-myosin Vb tail. Polarized MDCK cell culture monolayers expressing GFP-myosin Vb tail were infected from the apical (a and c) or basolateral (b and d) surface with RSV and examined for the cellular distribution of RSV F protein. The cellular distribution of the F protein is depicted as a single X-Z section (a and b) or a X-Z projection of the cell monolayer (c and d). It should be noted that the apparent colocalization of GFP-myosin Vb tail and RSV F protein shown in the projection images are a consequence of the projection image rather than actual colocalization, which was rarely observed in individual X-Z sections. (Magnification: ×63.)
Fig. 5.
Fig. 5.
GFP-myosin Vb tail reduces local spread of RSV. (a) Polarized MDCK cell culture monolayers with (solid bars) or without (open bars) doxycycline were infected from the apical surface with RSV and examined for the presence of RSV-infected cells by fluorescent focus assay. Data shown are the mean of four replicate samples (±SD) and are representative of two experiments. (b) Representative photomicrographs of RSV-infected MDCK cell monolayers +/- doxycycline (Dox) at 20 or 120 h postinfection. (Magnification: ×10.)
Fig. 6.
Fig. 6.
Rab11-FIP1 (576–651) expression inhibits RSV replication. MDCK cell culture monolayers with (solid bars) or without (open bars) Rab11-FIP1 (576–651) expression were plated on 48-well tissue culture plates and infected with RSV or vaccinia virus. Virus was recovered from infected cell culture supernatants at 120 or 48 h for RSV- or vaccinia virus-infected cells, respectively. Virus was quantified as described in Materials and Methods. Results shown are the mean of triplicate samples (±SD) and are representative of two experiments.
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