The Spike Protein VP4 Defines the Endocytic Pathway Used by Rotavirus To Enter MA104 Cells
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Fig 1Effect of hypertonic medium on the infectivities of the UK × RRV reassortants. MA104 cells were incubated with sucrose at 250 mM in MEM for 10 min at 37°C before they were infected with the indicated viruses at an MOI of 0.02 for 1 h at 37°C in the presence of the same hypertonic medium. After the adsorption period, the virus inoculum was removed, the cells were washed twice with 5 mM EGTA, and the infection was left to proceed for 14 h at 37°C. The infected cells were fixed and immunostained as indicated in Materials and Methods. Data are expressed as the percent infectivity of each virus observed in nontreated cells, which represent 100% infectivity. Black bars, RRV and reassortants bearing RRV VP4; white bars, UK and reassortants bearing UK VP4. The arithmetic means ± standard deviation of three independent experiments performed in duplicate are shown. The asterisks indicate significant differences between the infectivity of each virus in mock- and sucrose-treated cells (P ≤ 0.001).
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Fig 2Infectivities of UK × RRV reassortants in CHC knocked-down cells. (A) MA104 cells transfected with a SMARTpool siRNA against the clathrin heavy chain (CHC) were infected with the indicated viruses at an MOI of 0.02. At 14 hours postinfection (hpi), the cells were fixed and immunostained as described in Materials and Methods. Data are expressed as the percent infectivity of each virus compared to infection of cells transfected with an irrelevant siRNA (luciferase siRNA), which represents 100% infectivity. Black bars, RRV and reassortants bearing RRV VP4; white bars, UK and reassortants bearing UK VP4. The arithmetic means ± standard deviation of three independent experiments performed in duplicate are shown. The asterisks indicate significant differences between the infectivities of each virus in irrelevant and CHC siRNA-transfected cells (P ≤ 0.001). (B) Representative immunoblot for determination of the abundance of CHC in MA104 cells transfected with either irrelevant or CHC siRNAs (Irr and CHC, respectively). Vimentin (Vim) was used as a loading control. The antibodies used are indicated.
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Fig 3Effects of cell treatments that affect clathrin-dependent endocytosis on the infectivities of RRV variants. MA104 cells treated with sucrose or transfected with the siRNA against CHC were infected with the indicated viruses at an MOI of 0.02. Cells were fixed and immunostained at 14 hpi as described in Materials and Methods. Data are expressed as the percent infectivity of each virus compared to their infectivity under mock sucrose conditions or in cells transfected with an irrelevant siRNA. The arithmetic means ± standard deviation of three independent experiments performed in duplicate are shown. **, P < 0.01, and ****, P ≤ 0.0001, for statistically significant differences between the infectivities of each virus under experimental and control conditions. Revertant viruses rNar3-14 and rNar3-18 are denoted as rN3-14 and rN3-18.
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Fig 4Effects of NA treatment and GphA on the infectivities of RRV variants in CHC-silenced cells. (A) MA104 cells transfected with an irrelevant siRNA or an siRNA against CHC were treated or not with NA (80 mU/ml for 1 h) and infected with the indicated RRV variants. Cells were fixed and immunostained at 14 hpi as described in Materials and Methods. Data are expressed as the percent infectivity of each virus compared to its infectivity in cells transfected with the irrelevant siRNA but not treated with NA. (B) MA104 cells transfected with an irrelevant siRNA or an siRNA against CHC were infected with the indicated viruses that had been incubated with GphA. Cells were fixed and immunostained at 14 hpi as described in Materials and Methods. Data are expressed as the percent infectivity of each virus compared to its infectivity in the absence of GphA in cells transfected with an irrelevant siRNA. The arithmetic means ± standard deviation of three independent experiments performed in duplicate are shown. Revertant viruses rNar3-14 and rNar3-18 are denoted as rN3-14 and rN3-18.
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Fig 5Infectivities of different rotavirus strains in cells with treatments that inhibited clathrin-mediated endocytosis. MA104 cells treated with sucrose or transfected with an siRNA against CHC were infected with the indicated rotavirus strains at an MOI of 0.02. At 14 hpi the cells were fixed and immunostained as described in Materials and Methods. Data are expressed as the percent infectivity of each virus compared to their infectivities under mock sucrose conditions or in cells transfected with an irrelevant siRNA. The arithmetic means ± standard deviation of three independent experiments performed in duplicate are shown. **, P < 0.01, ***, P ≤ 0.001, and ****, P ≤ 0.0001, for statistically significant differences between the infectivities of each virus under experimental and control conditions.
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