ACCEPTED INDUCTION OF AUTOPHAGY DOES NOT AFFECT HUMAN RHINOVIRUS 2 PRODUCTION. Marianne Brabec-Zaruba, Ursula Berka, Dieter Blaas 1, and Renate Fuchs*

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1 JVI Accepts, published online ahead of print on 1 August 00 J. Virol. doi:./jvi Copyright 00, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved. 1 INDUCTION OF AUTOPHAGY DOES NOT AFFECT HUMAN RHINOVIRUS PRODUCTION Marianne Brabec-Zaruba, Ursula Berka, Dieter Blaas 1, and Renate Fuchs* Department of Pathophysiology and 1 Max F. Perutz Laboratories, University Departments at the Vienna Biocenter, Department of Medical Biochemistry, Medical University of Vienna, Austria 1 1 Running title: Autophagy and HRV production 1 1 *Corresponding author: Department of Pathophysiology, Medical University of Vienna, 1 Währinger Gürtel 1-0, A-0 Vienna, Austria. Phone: () Fax: () renate.fuchs@meduniwien.ac.at. 1 1

2 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 HRV, a human rhinovirus of the minor receptor group, is unaffected by tamoxifen, rapamycin, and -methyladenine, drugs respectively stimulating and inhibiting autophagic processes. Furthermore, LC-positive vesicles (i.e. autophagosomes) are not induced upon infection. Therefore, multiplication of this particular picornavirus is not dependent on autophagy. Human rhinoviruses (HRVs) are responsible for roughly 0% of respiratory tract infections. As picornaviruses they are non-enveloped with a positive-sense RNA genome (1). Of the HRV serotypes 1 (the minor group) use members of the low-density lipoprotein receptor family and (the major group) use intercellular adhesion molecule-1 for cell entry (1). The prototype minor group virus HRV is internalized and routed to late endosomes where RNA uncoating occurs (, ). Subsequently, viral protein synthesis is initiated and followed by RNA replication. Finally, infective viruses are assembled and released from the host cell. Replication has been most extensively studied for the closely related poliovirus and Kirkegaard and coworkers found double-membraned vesicles in infected cells. These compartments could be immunoisolated with an antibody against viral protein C and contained markers of the secretory pathway and lysosomal membrane protein-1 (LAMP-1), indicative for an autophagic origin (). Furthermore, induction of autophagy by tamoxifen and rapamycin increased poliovirus yield (). Based on immunofluorescence co-localization of GFP-LC (an autophagosome marker) and LAMP-1 as well as staining with mono-dansylcadaverin (MDC), autophagosomes were also observed after infection with HRV and the major group virus

3 HRV1. Focusing on HRV, we therefore investigated whether replication of this rhinovirus is also affected by modulators of autophagy. Autophagosomes are induced by treatment with tamoxifen in cells expressing the estrogen receptor. In accordance with Bursch (), 1 µm tamoxifen was sufficient to induce autophagy in all HeLa-H1 cells after h. MDC and LAMP- staining was used to reveal autophagic vacuoles (, ). Following incubation with tamoxifen, MDC ( µm in DMSO/ethanol) was added for 1 h prior to fixation with % paraformaldehyde (Sigma) in PBS for 1 min at room temperature (). The cells were viewed under a Zeiss Axioscop microscope. Subsequently, cells were permeabilized with 0.0 % Triton-X 0 in PBS for min, quenched with 0 mm NH Cl in PBS and incubated with monoclonal anti-lamp- antibody (1:00; HB; BD Pharmingen) followed by Alexa -labeled goat anti-mouse IgG (1:00; Invitrogen). The samples were washed, mounted in glycerol / PBS (:1) and the areas previously photographed for MDC staining were relocated for digital imaging. As depicted in Fig. 1A tamoxifen treatment induced large MDC-positive compartments that also contained LAMP-. Then, the effect of viral infection on MDC (Fig. 1B) and LAMP- staining (Fig. 1C) was studied. Cells were challenged for 1 h with HRV (00 TCID 0 / cell; C), washed, and further incubated. MDC was added at. h post infection (p.i.), and the cells were fixed and immediately photographed as above. For comparison, a tamoxifen control was included (Fig. 1B) and showed the expected increase of vesicular MDC-staining (compare to Fig. 1A). No such increase was seen in the infected cells (right panel). Next, cells infected as above were fixed and permeabilized. h p.i. and stained for viral protein with rabbit anti-hrv antiserum (1:0) followed by Alexa -conjugated goat anti-rabbit IgG (1:00, Invitrogen; Fig. 1C, upper panel) and for LAMP- (as in Fig. 1A; Fig. 1C, lower panel). No increase in the intensity or number of LAMP--positive compartments was seen in cells replicating virus (arrows) as

4 compared to cells not expressing viral proteins (arrowheads). To further verify the lack of induction of autophagic vacuoles in HRV-replicating cells, endogenous LC was monitored (, ). As control, autophagic vesicles were induced by rapamycin (0 nm, 1 h; ()) and after permeabilization with 0.1 % saponin LC was detected with rabbit anti-lc antibody PD01 (MBL; 1:00) and LAMP- with mouse anti-lamp- antibodies. As expected, rapamycin treatment resulted in increased and co-staining of the two markers (Fig. A). Upon infection (Fig. B), virus producing cells were revealed with monoclonal anti-hrv antibody (F; 1:00; ()). No increased LC-staining was seen in HRV replicating (arrows) as compared to non-infected cells (arrow heads). It is of note that LC expression levels varied to a similar extent in infected as well as in non-infected cells. Furthermore, no co-localization between HRV replication sites and LC was observed (Fig. C). Finally, we analyzed whether the autophagy inducers tamoxifen and rapamycin or the inhibitor -methyladenine (-MA; ()) affect viral yield. Cells were pretreated with tamoxifen for h, with rapamycin for 1 h or with mm -MA for h and virus was internalized for 1 h at C. After further incubation in culture medium for. h in the presence of the respective drug, viral proteins and nuclei were revealed by fluorescence microscopy (Fig. A) as above. HRV-positive cells were quantified using TissueQuest software (TissueGnostics Gmbh, Vienna) by analyzing,000 cells / treatment (Fig. B). About % of the cells were producing virus with no significant difference upon drug treatment. This is in line with the lack of influence of tamoxifen on the viral titer (Fig. C). The amount of virus taken up during 1 h as well as of de novo synthesized cell-associated virus at. h p.i. was virtually identical in the absence and presence of the drug. In addition, no influence of tamoxifen on virus release into the supernatant was observed. Taken together, our data demonstrate that autophagic vesicles are

5 not involved in replication and/or release of HRV in HeLa cells. Autophagy generates sites that may facilitate virus replication (1). For poliovirus Jackson and colleagues demonstrated that modulation of autophagy affected virus yield as well as release from the cell (). Moreover, they observed co-localization of poliovirus A with GFP- LC in infected cells. For HRV, they only took co-localization of GFP-LC and LAMP-1 to indicate autophagosome formation following infection. Using a different experimental approach, we demonstrate that i) endogenous LC and LAMP- are unchanged in cells actively replicating HRV and, ii) these markers do not co-localize of with viral proteins. On the other hand, iii) infection is neither affected by induction of autophagy by tamoxifen nor rapamycin that act via different signaling pathways, nor by its inhibition by -MA. Finally, iv) viral yield and virus release into the medium is not increased by tamoxifen treatment. These results are in line with previous data demonstrating that the phosphatidylinositol -kinase and autophagy inhibitor wortmannin () does not modify viral yield (1). Thus, at least this particular rhinovirus serotype neither induces the synthesis of LAMP- and LC-positive compartments, nor does modification of autophagy result in increased viral synthesis. Supported by Austrian Science Fund P to R.F. and Austrian Academy of Sciences DOC- FORTE to U.B. We thank Irene Goesler for TCID 0 determination and Francesca Demarchi for providing the detailed protocol for LC staining. 0

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8 FIGURE LEGENDS FIG. 1: Visualization of autophagosomes by MDC and LAMP-. (A) Cells were incubated ± 1µM tamoxifen for h and stained with MDC and anti-lamp- antibody. Some few LAMP--positive vesicles (arrows) are also stained with MDC in the absence of tamoxifen. However, large MDC- and LAMP--positive compartments are induced in all cells by the drug (arrows). (B) Control cells ± tamoxifen and cells infected with HRV at 00 TCID 0 /cell were stained with MDC. Infected cells lacked the typical tamoxifen-induced MDC staining (B, right panel). (C) Infected cells were stained with anti-hrv and anti-lamp- antibodies followed by Alexa and Alexa -conjugated secondary antibodies. No alteration in LAMP-- positive compartments was observed in cells actively replicating virus (C, arrows; arrowheads: non-infected cells). Bars: A, C) µm; B) 0 µm. FIG. : HRV infection does not induce LC-positive vesicles. (A) Mock infected ± rapamycin (0 nm, 1 h) and (B, C) HRV infected cells (1 h challenge with 00 TCID 0 / cell) were stained at. h p.i. for LC and LAMP- or for LC and HRV. Arrows, HRV-positive cells; arrowheads, non-infected cells. Bars: A) 0 µm; B, C) µm. A and C are confocal images. When compared to rapamycin treated cells (A), in infected cells LC is not increased (B) and does not co-localize with HRV (C) FIG. : Modulation of autophagy does not affect HRV synthesis. Cells were incubated ± 1µM tamoxifen for h, 0 nm rapamycin for 1 h or mm -MA for h. A) HRV (00 TCID 0 /cell) was internalized for 1 h and cells further incubated for. h in the presence of the respective drug. Virus was visualized with mab F and nuclei with Hoechst dye. Bars, 0 µm. B) Cells replicating virus were quantified with respect to the total number (~,000 cells /

9 group) using TissueQuest software. Data are mean ± S.D. from 1 images. C) Cells were pretreated with tamoxifen and infected as in A) but with 0 TCID 0 /cell and infectious virus cell associated and released into the supernatant was determined at 1 h and at. h p.i. Data are mean ± S.D. from parallel samples.

10 Fig. 1: Brabec-Zaruba et al. A mock C infected MDC LAMP- MDC B - tamoxifen + tamoxifen + HRV LAMP- HRV - tamoxifen + tamoxifen + HRV

11 igure : Brabec-Zaruba et al. infected A LC LAMP- overlay + rapamycin mock B infected LC HRV overlay C LC HRV overlay

12 Figure : Brabec-Zaruba et al. A control + tamoxifen B C % cells synthesizing HRV 0 TCID rapamycin + -methyladenine control + tamoxifen + rapamycin + -methyladenine 1 h. h cells supernatant cells supernatant control + tamoxifen