Cell penetration and trafficking of polyomavirus

Abstract

The murine polyomavirus (Py) enters mouse fibroblasts and kidney epithelial cells via an endocytic pathway that is caveola-independent (as well as clathrin-independent). In contrast, uptake of simian virus 40 into the same cells is dependent on caveola. Following the initial uptake of Py, both microtubules and microfilaments play roles in trafficking of the virus to the nucleus. Colcemid, which disrupts microtubules, inhibits the ability of Py to reach the nucleus and replicate. Paclitaxel, which stabilizes microtubules and prevents microtubule turnover, has no effect, indicating that intact but not dynamic microtubules are required for Py infectivity. Compounds that disrupt actin filaments enhance Py uptake while stabilization of actin filaments impedes Py infection. Virus particles are seen in association with actin in cells treated with microfilament-disrupting or filament-stabilizing agents at levels comparable to those in untreated cells, suggesting that a dynamic state of the microfilament system is important for Py infectivity.

FIG. 1.

Effect of Colcemid addition on infectivity of BMK and NIH 3T3 cells. Cells were infected with Py, and control medium or medium containing Colcemid was added at the indicated time points postinfection. Cells were examined for Py LTAg staining at either 24 h for BMK cells or 36 h for NIH 3T3 cells. Approximately 500 nuclei were counted per sample. Results are presented as the percentages of nuclei that were Py LTAg positive in the treated sample relative to the percent positive in the untreated control. Black bars represent Colcemid-treated BMK cells, and white bars represent Colcemid-treated NIH 3T3 cells.

FIG. 2.

Colocalization of OGPy with microtubules. BMK cells were mock treated or pretreated with Colcemid. Cells were infected with OGPy in the absence (A) or presence (B) of Colcemid and then incubated further with or without Colcemid. Cells were fixed, processed for immunodetection of tubulin, imaged, and then subjected to deconvolution. Z sections (0.2 μm thick) were examined, and representative sections showing cells that were incubated for 3 h at 37°C prior to fixation are presented. Colocalized OGPy particles are indicated with white arrowheads.

FIG. 3.

Quantitation of colocalization of microtubules and OGPy. BMK cells were mock treated (▴) or pretreated with Colcemid (•) and then infected with OGPy in the presence or absence of Colcemid. Samples were fixed and processed for immunodetection of tubulin. After deconvolution, each individual 0.2-μm-thick Z section was examined for total virus particles and particles colocalized with tubulin. The averages of three randomly selected fields from three experiments were taken for each time point.

FIG. 4.

Effect of actin binding compounds on BMK cells. BMK cells were pretreated with media alone or media containing either lat A (black bars), cyto B (white bars), or jas (dotted bars). Cells were infected with Py (at an MOI of 10) for 1 h in media alone or media containing the above compounds. The compounds were washed out, and neutralizing antibody was added at the indicated time points. Cells were examined for Py LTAg staining at 24 h. Approximately 500 nuclei were counted per sample, and the data are presented as described in the legend to Fig. 1.

FIG. 5.

Effect of actin binding compounds on NIH 3T3 cells. NIH 3T3 cells were pretreated with media alone or media containing either lat A (white bars), cyto B (black bars), or jas (dotted bars). Cells were infected with Py (at an MOI of 10) for 1 h in media alone or media containing the above compounds. The compounds were washed out, and neutralizing antibody was added at the indicated time points. Cells were examined for Py LTAg staining at 32 h. Approximately 500 nuclei were counted per sample, and the data are presented as described in the legend to Fig. 1.

FIG. 6.

Colocalization of OGPy with actin. BMK cells were mock treated or pretreated with lat A or jas. Cells were infected with OGPy in the presence or absence of compounds and then further incubated in media alone (A) or with lat A (B) or jas (C). Cells were fixed, processed for immunofluorescence, imaged, and then subjected to deconvolution. Z sections (0.2 μm thick) were examined, and representative ones showing cells that were incubated for 30 min at 37°C prior to fixation are presented. Colocalized OGPy particles are indicated with white arrowheads.

FIG. 7.

Quantitation of colocalization of actin and OGPy. BMK cells were mock treated (▴) or pretreated with lat A (▪) or jas (•) and then infected with OGPy in the presence or absence of these compounds. Samples were fixed and processed for the detection of actin. After deconvolution, each individual 0.2-μm-thick Z section was examined for number of total virus particles and number of particles colocalized with actin. The averages of three randomly selected fields from three experiments were taken for each time point.