Induction of autophagy does not affect human rhinovirus type 2 production

Abstract

Induction of autophagy has been shown to be beneficial for the replication of poliovirus, a phenomenon that might also apply for other picornaviruses. We demonstrate that de novo synthesis of human rhinovirus type 2 (HRV2), an HRV of the minor receptor group, is unaffected by tamoxifen, rapamycin, and 3-methyladenine (3-MA), drugs either stimulating (tamoxifen and rapamycin) or inhibiting (3-MA) autophagic processes. Furthermore, LC3-positive vesicles (i.e., autophagosomes) are not induced upon infection. Therefore, multiplication of this particular picornavirus is not dependent on autophagy.

FIG. 1.

Visualization of autophagosomes by MDC and LAMP-2. (A) Cells were incubated with (+) or without (−) 1 μM tamoxifen for 48 h and stained with MDC and anti-LAMP-2 antibody. A few LAMP-2-positive vesicles (arrows) were also stained with MDC in the absence of tamoxifen. However, large MDC- and LAMP-2-positive compartments are induced in all cells by the drug (arrows). (B) Control cells with or without tamoxifen and cells infected with HRV2 at 300 TCID₅₀/cell were stained with MDC. Infected cells lacked the typical tamoxifen-induced MDC staining (right). (C) Infected cells were stained with anti-HRV2 and anti-LAMP-2 antibodies followed by Alexa 568 and Alexa 488-conjugated secondary antibodies. No alteration in LAMP-2-positive compartments was observed in cells actively replicating virus (arrows). Arrowheads, noninfected cells. Bars, 10 μm (A, C) and 30 μm (B).

FIG. 2.

HRV2 infection does not induce LC3-positive vesicles. (A) Mock- or rapamycin-treated cells (100 nM, 13 h) (A) and HRV2-infected cells (1-h challenge with 300 TCID₅₀/cell) (B, C) were stained at 7.5 h p.i. for LC3 and LAMP-2 or for LC3 and HRV2. Arrows, HRV2-positive cells; arrowheads, noninfected cells. Bars, 50 μm (A) and 10 μm (B, C). Panels A and C are confocal images. Compared to that in rapamycin-treated cells (A), LC3 in infected cells is not increased (B) and does not colocalize with HRV2 (C).

FIG. 3.

Modulation of autophagy does not affect HRV2 synthesis. Cells were incubated with (+) or without 1 μM tamoxifen for 48 h, with 100 nM rapamycin for 13 h, or with 5 mM 3-MA for 3 h. (A) HRV2 (300 TCID₅₀/cell) was internalized for 1 h, and cells were further incubated for 6.5 h in the presence of the respective drug. Virus was visualized with monoclonal antibody 8F5 and nuclei with Hoechst dye. Bars, 50 μm. (B) Cells replicating virus were quantified with respect to the total number of cells per group (∼10,000) by using TissueQuest software. Data are means ± standard deviations for 15 images. (C) Cells were pretreated with tamoxifen and infected as described for panel A but with 100 TCID₅₀/cell, and the amount of infectious virus cell associated or released into the supernatant was determined at 1 and 7.5 h p.i. Data are means ± standard deviations for four parallel samples.