The cytotoxicity of coxsackievirus B3 is associated with a blockage of autophagic flux mediated by reduced syntaxin 17 expression

Abstract

Coxsackievirus B3 (CVB3) is an important human pathogen linked to cardiac arrhythmias and acute heart failure. CVB3 infection has been reported to induce the formation of autophagosomes that support the viral replication in host cells. Interestingly, our study shows that the accumulation of autophagosomes during CVB3 infection is caused by a blockage of autophagosome-lysosome fusion rather than the induction of autophagosome biogenesis. Moreover, CVB3 decreases the transcription and translation of syntaxin 17 (STX17), a SNARE (soluble N-ethylmaleimide-sensitive factor activating protein receptor) protein involved in autophagosome-lysosome fusion. Overexpression of STX17 restored the autophagic flux, alleviated the virus-induced lysosomal dysfunction, and decreased the apoptosis induced by CVB3 infection in HeLa cells. Taken together, our results suggest that CVB3 infection impairs the autophagic flux by blocking autophagosome-lysosome fusion. These findings thus point to potential new therapeutic strategies targeting STX17 or autophagosome-lysosome fusion for treating CVB3-associated diseases.

Fig. 1. CVB3 induces LC3-II accumulation in dose-dependent manner.

a HeLa cells were infected with CVB3 at a low (CVB3-L) and high MOI (CVB-H) and incubated for 36 h. Endogenous LC3B was detected by immunofluorescence. b Western blotting was employed to detect LC3B; VP1: a CVB3 viral protein; p62: a substrate for autophagic degradation; actin: a loading control. c Statistical analysis for a vs. Control, **p < 0.01. d Statistical analysis of the ratio of LC3B-II to LC3B-I, normalized to actin for b vs. Control, **p < 0.01. d Western blotting was employed to detect Triton X-100-soluble and Triton X-insoluble ubiquitin-conjugated proteins after infection with CVB3 at low (CVB3-L) and high MOI (CVB-H), respectively, and incubation for 36 h. Actin is a loading control. e Statistical analysis of the ratio of insoluble ubiquitin conjugates to actin, normalized to actin for d. *p < 0.05, **p < 0.01

Fig. 2. Baf1 blocks accumulation of LC3-II induced by CVB3 infection.

a HeLa cells were infected with CVB3 at a high MOI and treated with 50 nM Baf1 for 12 h. Endogenous LC3B was detected by western blotting. VP1: a CVB3 viral protein. Actin: a loading control. b Statistical analysis of the ratio of LC3B-II to LC3B-I, normalized to actin vs. Control. *p < 0.01. NS, no statistical significance

Fig. 3. CVB3 infection increases autophagic vesicles by blockage of autophagosome–lysosome fusion.

a The monomeric red fluorescent protein (mRFP)-GFP-LC3 tandem reporter was used to transfect HeLa cells with or without CVB3 infection. If autophagic flux is increased, the number of both yellow and red puncta are increased; if autophagosome maturation into autolysosomes is blocked, only the number of yellow puncta is increased, without a concomitant increase of the number of red puncta. b After 24 h of transfection, yellow puncta were counted. Quantification of the percentage of yellow dots (GFP+/RFP+ dots), which contain both GFP and mRFP signal (autophagosomes), out of the total number of red dots (RFP+ dots) vs. Control. **p < 0.01. c A representative ultrastructural picture of CVB3-infected HeLa cells. Autophagosome-like vacuoles are indicated by arrows. Scale bar = 1 µm

Fig. 4. CVB3 infection inhibits transcription of STX17.

a Endogenous STX17 was detected in CVB3-infected HeLa cells by western blotting. VP1: a CVB3 viral protein. Actin: a loading control. b Statistical analysis for a vs. Control. **p < 0.01. c Transcription of STX17 and Lamp1 was measured in CVB3- infected HeLa cells by real-time PCR vs. Control. **p < 0.01

Fig. 5. Exogenous STX17 restores impaired autophagosome–lysosome fusion following CVB3 infection.

a CVB3-infected HeLa cells were transfected with STX17-myc or empty vector. LC3 was detected in CVB3-infected HeLa cells by western blotting. VP1: a CVB3 viral protein. Actin: a loading control. b Statistical analysis for a vs. CVB3. *p < 0.05. c The monomeric red fluorescent protein (mRFP)-GFP-LC3 tandem reporter and STX17-myc were used to transfect HeLa cells with CVB3 infection. d Yellow puncta were counted. Quantification of the percentage of yellow dots (GFP+/RFP+ dots), which contain both GFP and mRFP signal (autophagosomes), out of the total number of red dots (RFP+ dots). vs. CVB3. *p < 0.05. e Ultrastructural analysis of CVB3-infected HeLa cells transfected with STX17-myc or sham. Autophagosome-like vacuoles are indicated by arrows. Scale bar = 1 µm

Fig. 6. STX17 restored lysosomal function and blocked apoptosis induced by CVB3 infection.

a Western blotting was employed to detect Triton X-100-insoluble ubiquitin-conjugated proteins in cells with or without exogenous expression of STX17 after infection with CVB3. b Statistical analysis of the ratio of insoluble ubiquitin conjugates to actin, normalized to actin for a, *p < 0.05, **p < 0.01. c The activity of cathepsin B/L was measured in cells with or without exogenous expression of STX17 after infection with CVB3, *p < 0.05. d Annexin V + PI staining for the flow-cytometric measurement of apoptosis induced by CVB3 infection. e Cleaved caspase-3 was detected in CVB3-infected HeLa cells transfected with STX17-myc or sham. f Statistical analysis for a vs. CVB3. *p < 0.01