Cellular entry and uncoating of naked and quasi-enveloped human hepatoviruses

Figures

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  Figure 1.. Naked and quasi-enveloped HAV virions undergo clathrin-dependent endocytosis facilitated by β1 integrin. (A) Effect of endocytic inhibitors on HAV and eHAV entry quantified by RT-PCR (mean ± SD, n = 3 independent experiments). (B) Effect of siRNA-mediated depletion of endocytic regulators on HAV and eHAV entry (mean ± SD, n = 3 independent experiments). See Figure 1—figure supplement 2A for knockdown efficiencies. (C) Confocal micrographs of Huh-7.5 cells immunolabeled with anti-HAV capsid (K24F2) and anti-clathrin or anti-caveolin-1 at one hpi. Scale bar, 10 µm. (D) Effect of siRNA-mediated depletion of integrins and adhesion molecules on HAV and eHAV entry (mean ± SD, n = 3 independent experiments). See Figure 1—figure supplement 2B for knockdown efficiencies. (E) Effect of β1 integrin knockdown on HAV/eHAV attachment to cells 2 hpi at 4˚C (mean ± SD, n = 6 biological replicates from two independent experiments) or on HAV and eHAV uptake 6 hpi at 37˚C (mean ± SD, n = 3 independent experiments). (F) Levels of HAV RNA at 20 hpi in H1-HeLa engineered by CRISPR/Cas9 using a control or a specific ITGB1-targeting sgRNA (mean ± SD, n = 3 biological replicates of a representative…

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  Figure 1—figure supplement 1.. Huh-7.5 cells were incubated with DMEM supplemented with the indicated inhibitors for 1 hr. Cells were then rinsed, placed on ice for 10 min, and incubated with 10–25 µg/ml Alexa 594-conjugated Transferrin or cholera toxin subunit B diluted in supplemented DMEM for 15–20 min at 37˚C prior to fixation and imaging.

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  Figure 1—figure supplement 2.. Immunoblots showing siRNA-mediated knockdown efficiencies corresponding to data of Figure 1. (A) Immunoblots showing knockdown efficiency of siRNA transfections related to Figure 1B. (B) Immunoblots showing knockdown efficiency of siRNA transfections related to Figure 1D.

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  Figure 1—figure supplement 3.. Levels of HAV RNA at 24 hpi in H1-HeLa cells engineered by CRISPR/Cas9 to be knocked-out for the indicated integrins inoculated with naked HAV (mean ± SD). Immunoblots of whole-cell lysates harvested from the indicated cell lines.

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  Figure 1—figure supplement 4.. Huh-7.5 cells were inoculated with HAV or eHAV and subjected to immunostaining one hpi using antibodies against HAV capsid (K24F2) and α5 or αV integrins. Scale bar, 10 µm.

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  Figure 2.. Distinct endocytic sorting of naked and quasi-enveloped HAV. (A) Confocal micrographs of Huh-7.5 cells immunolabeled with anti-HAV capsid (K24F2) and anti-Rab5A, Rab7a, or Rab11A at two hpi. Scale bar, 10 µm. (B) Effect of siRNA-mediated depletion of Rab GTPases on HAV and eHAV entry (mean ± SD, n = 3 independent experiments). See Figure 2—figure supplement 1 for knockdown efficiencies. (C) Confocal micrographs of Huh-7.5 cells immunolabeled with anti-HAV capsid (K24F2) and anti-LAMP1 at six hpi. Scale bar, 10 µm. (D) Confocal micrographs of Huh-7.5 cells adsorbed with naked HAV or eHAV and immunolabeled with antibodies against HAV capsid (K24F2), β1 integrin, and either Rab7 or LAMP1. Scale bar, 10 µm. (E) Confocal micrographs of Huh-7.5 cells previously transfected with a control or ALIX-specific siRNAs and immunolabeled with anti-HAV capsid (K24F2) and anti-LAMP1 at 12 hpi with eHAV. Scale bar, 10 µm. See Figure 2—figure supplement 5 for knockdown efficiencies. (F) Effect of ALIX depletion by siRNA on eHAV and HAV entry and replication at 22 hpi (mean ± SD, n = 2 biological replicates for a representative experiment). For numeric data plotted in graphs associated with this…

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  Figure 2—figure supplement 1.. Immunoblots showing knockdown efficiency of siRNA transfections related to Figure 2B.

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  Figure 2—figure supplement 2.. Huh-7.5 cells were inoculated with gradient-purified HAV or eHAV, fixed 6 hr post-adsorption, and subjected to immunostaining using antibodies against HAV capsid (K24F2) and the lysosomal protein VAMP8. Scale bar, 10 µm.

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  Figure 2—figure supplement 3.. Huh-7.5 cells were inoculated with eHAV, fixed two hpi, and subjected to immunostaining using antibodies against HAV capsid (K24F2), β1 integrin, and Rab7. Scale bar, 10 µm.

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  Figure 2—figure supplement 4.. Huh-7.5 cells were pre-treated with the Alexa594-conjugated activating anti-β1 integrin mAb -TS2/16 or IgG control for 20 min on ice to label cell surface β1 integrin prior to virus adsorption. Inoculum was removed after 1 hr and cells were fixed and immunostained 5 hr later with anti-HAV (JC polyclonal human sera) and anti-LAMP1. Scale bar, 10 µm.

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  Figure 2—figure supplement 5.. Huh-7.5 cells were transfected with the indicated siRNAs for 72 hr prior to adsorption with eHAV. Cells were immunostained at eight hpi using anti-HAV capsid (K24F2) and anti-LAMP1. Scale bar, 10 µm. Knockdown efficiency was determined by immunoblotting or by RT-qPCR (table) using gene-specific primers to obtain calculate expression levels relative to control siRNA-transfected cells.

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  Figure 3.. Degradation of the eHAV quasi-envelope occurs within the lysosome. (A) Effect of siRNA-mediated depletion of lysosomal-associated proteins on HAV and eHAV entry (mean ± SD, n = 3 independent experiments). See Figure 3—figure supplement 1 for knockdown efficiencies. (B) Effect of inhibition of NPC1 or LAL by U18666A or Lalistat-2, respectively, on HAV and eHAV entry (mean ± SD, n = 3 independent experiments). (C) Strategy for membrane labeling of exosomes and eHAV with PKH26 dye. (D) Confocal micrographs of Huh-7.5 cells inoculated with PKH26-labeled eHAV and immunostained with anti-HAV capsid (K24F2) and anti-LAMP1. Histograms depict pixel intensities for the structures indicated with a yellow arrow. Scale bar, 10 µm. (E) Confocal micrographs of Huh-7.5 cells inoculated with PKH26-labeled eHAV in presence of Lalistat-2 and immunostained with anti-HAV capsid (K24F2) and anti-LAMP1. Graph displays the ratio of structures corresponding to PKH26-associated HAV capsid (green + red signal) over HAV capsids devoid of PKH26 fluorescence (green alone) within lysosomes per cell. Scale bar, 10 µm (F) Effect of Lalistat-2 on post-endocytic antibody-mediated neutralization of eHAV.…