Role of autophagy during the replication and pathogenesis of common mosquito-borne flavi- and alphaviruses

Arboviruses that are transmitted to humans by mosquitoes represent one of the most important causes of febrile illness worldwide. In recent decades, we have witnessed a dramatic re-emergence of several mosquito-borne arboviruses, including dengue virus (DENV), West Nile virus (WNV), chikungunya virus (CHIKV) and Zika virus (ZIKV). DENV is currently the most common mosquito-borne arbovirus, with an estimated 390 million infections worldwide annually. Despite a global effort, no specific therapeutic strategies are available to combat the diseases caused by these viruses. Multiple cellular pathways modulate the outcome of infection by either promoting or hampering viral replication and/or pathogenesis, and autophagy appears to be one of them. Autophagy is a degradative pathway generally induced to counteract viral infection. Viruses, however, have evolved strategies to subvert this pathway and to hijack autophagy components for their own benefit. In this review, we will focus on the role of autophagy in mosquito-borne arboviruses with emphasis on DENV, CHIKV, WNV and ZIKV, due to their epidemiological importance and high disease burden

Mitochondrial protein BNIP3 regulates Chikungunya virus replication in the early stages of infection

Chikungunya virus (CHIKV) is a human pathogen causing outbreaks of febrile illness for which vaccines and specific treatments remain unavailable. Autophagy-related (ATG) proteins and autophagy receptors are a set of host factors that participate in autophagy, but have also shown to function in other unrelated cellular pathways. Although autophagy is reported to both inhibit and enhance CHIKV replication, the specific role of individual ATG proteins remains largely unknown. Here, a siRNA screen was performed to evaluate the importance of the ATG proteome and autophagy receptors in controlling CHIKV infection. We observed that 7 out of 50 ATG proteins impact the replication of CHIKV. Among those, depletion of the mitochondrial protein and autophagy receptor BCL2 Interacting Protein 3 (BNIP3) increased CHIKV infection. Interestingly, BNIP3 controls CHIKV independently of autophagy and cell death. Detailed analysis of the CHIKV viral cycle revealed that BNIP3 interferes with the early stages of infection. Moreover, the antiviral role of BNIP3 was found conserved across two distinct CHIKV genotypes and the closely related Semliki Forest virus. Altogether, this study describes a novel and previously unknown function of the mitochondrial protein BNIP3 in the control of the early stages of the alphavirus viral cycle.</p

Mitochondrial protein BNIP3 regulates Chikungunya virus replication in the early stages of infection.

Chikungunya virus (CHIKV) is a human pathogen causing outbreaks of febrile illness for which vaccines and specific treatments remain unavailable. Autophagy-related (ATG) proteins and autophagy receptors are a set of host factors that participate in autophagy, but have also shown to function in other unrelated cellular pathways. Although autophagy is reported to both inhibit and enhance CHIKV replication, the specific role of individual ATG proteins remains largely unknown. Here, a siRNA screen was performed to evaluate the importance of the ATG proteome and autophagy receptors in controlling CHIKV infection. We observed that 7 out of 50 ATG proteins impact the replication of CHIKV. Among those, depletion of the mitochondrial protein and autophagy receptor BCL2 Interacting Protein 3 (BNIP3) increased CHIKV infection. Interestingly, BNIP3 controls CHIKV independently of autophagy and cell death. Detailed analysis of the CHIKV viral cycle revealed that BNIP3 interferes with the early stages of infection. Moreover, the antiviral role of BNIP3 was found conserved across two distinct CHIKV genotypes and the closely related Semliki Forest virus. Altogether, this study describes a novel and previously unknown function of the mitochondrial protein BNIP3 in the control of the early stages of the alphavirus viral cycle

BNIP3 involvement in CHIKV infectivity is independent of cellular death pathways.

U2OS cells were reverse-transfected with siBNIP3 or siScramble for 48 h. Cells were infected with 5’GFP-CHIKV-LR at the indicated MOI for 10 and 16 h. (A) Gating strategy and (B) bar plots showing the frequencies of Annexin V-, FVD- and double positive cells measured by flow cytometry in GFP+ (infected) cells. Data represents mean ± SEM of at least three independent experiments. NT denotes for non-transfected, siScram for siScramble. Student’s test: no symbol implies non-statistically significant.</p

Genes targeted by the customised siRNA library and gene accession numbers.

Genes targeted by the customised siRNA library and gene accession numbers.</p

BNIP3 controls CHIKV replication early in infection.

U2OS cells were reverse-transfected with siBNIP3 or siScramble for 48 h. (A) Transfected cells were incubated with DiD-labelled CHIKV-LR and the number of fusion-positive cells was quantified by TIRF microscopy. (B-G) siRNA-transfected cells were infected with 5’GFP-CHIKV-LR at the indicated MOI. (B) Representative blots and (C) bar plot showing the expression levels of CHIKV nsP2, E2, E1 and capsid proteins in BNIP3-depleted cells compared to siScramble-transfected cells after 10 hpi. GAPDH and vinculin were both used as loading controls. (D) Bar plot showing the levels of intracellular CHIKV E1 and nsP1 RNA in BNIP3-knockdown cells compared to siScramble-transfected cells. (E) Bar plot showing the quantification by RT-qPCR of genome equivalent copies per ml of supernatant (GECs/ml) after 10 hpi, relative to NT cells. (F) Bar plot showing the titration of infectious CHIKV particles per ml of supernatant (PFU/ml, determined by plaque assay) after 10 hpi and expressed as relative to NT cells. (G) Bar plot showing the specific infectivity (GECs/ml divided by the PFU/ml) compared to NT cells. FC denotes for fold change. Data represents mean ± SEM of at least three independent experiments. Student’s test: *** p p p < 0.05, no symbol implies non-statistically significant. NT denotes for non-transfected, siScram for siScramble.</p

siRNA-based screen to determine the relevance of ATG proteins and autophagy receptors in CHIKV-infected U2OS cells and validation of BNIP3 as a factor controlling infection.

(A) Heat-map showing the results of the siRNA screen (n = 4). Data represent the ratio of GFP-positive cells (%GFP+) and the average GFP intensity per well (GFP int/well), for each siRNA knockdown as compared to cells transfected with a siRNA control (siScramble, bottom). The significant hits (p (B) Distribution of the Z-score values of the siRNA screen as compared to the siScramble based on the percentage of GFP-positive cells. Z-scores between -1.96 and +1.96 represent significant hits with p (C-D) U2OS cells were reverse-transfected in 24-well plates with siRNAs targeting BNIP3 (siBNIP3) or with siScramble (siScram) for 48 h. (C) Bar plot showing BNIP3 transcript levels relative to the siScramble as measured by RT-qPCR. (D) Transfected cells were infected with the 5’GFP-CHIKV-LR strain at the indicated MOI. Cells were collected and fixed at 10 hpi, and infection (i.e., GFP-positive cells) was determined by flow cytometry. Bar plot shows the percentage of infection relative to non-transfected (NT) cells. (E) MRC-5, HeLa or HuH-7 cells were reverse-transfected with siBNIP3 or siScram for 48 h and infected at MOI 5 (HeLa/HuH-7 cells) or MOI 1 (MRC-5 cells). After 9–10 hpi, infection was evaluated as stated in 1D. Data represents mean ± SEM of at least three independent experiments. Student’s test: *** p p < 0.05.</p

Raw data belonging to Fig 1E.

Chikungunya virus (CHIKV) is a human pathogen causing outbreaks of febrile illness for which vaccines and specific treatments remain unavailable. Autophagy-related (ATG) proteins and autophagy receptors are a set of host factors that participate in autophagy, but have also shown to function in other unrelated cellular pathways. Although autophagy is reported to both inhibit and enhance CHIKV replication, the specific role of individual ATG proteins remains largely unknown. Here, a siRNA screen was performed to evaluate the importance of the ATG proteome and autophagy receptors in controlling CHIKV infection. We observed that 7 out of 50 ATG proteins impact the replication of CHIKV. Among those, depletion of the mitochondrial protein and autophagy receptor BCL2 Interacting Protein 3 (BNIP3) increased CHIKV infection. Interestingly, BNIP3 controls CHIKV independently of autophagy and cell death. Detailed analysis of the CHIKV viral cycle revealed that BNIP3 interferes with the early stages of infection. Moreover, the antiviral role of BNIP3 was found conserved across two distinct CHIKV genotypes and the closely related Semliki Forest virus. Altogether, this study describes a novel and previously unknown function of the mitochondrial protein BNIP3 in the control of the early stages of the alphavirus viral cycle.</div

CHIKV does not trigger an autophagic response in U2OS cells.

(A) Representative LC3 western blots and (B) determination of the autophagic flux index (LC3-II/LC3-I ratio) in protein lysates from U2OS cells either mock-treated or infected with CHIKV-LR at MOI 10 at the indicated time-points. (C) Representative fluorescence microscopy images and (D) quantification of GFP-WIPI2 puncta in U2OS cells stably expressing GFP-WIPI2 and infected with CHIKV-LR at MOI 10. Cells were analysed at the indicated time-points. (E) Data presented in Fig 2A and 2B was quantified and shown as LC3-II normalised to the housekeeping gene (GAPDH). In all cases, starvation-induced autophagy is used as a positive control (HBSS/EBSS). Baf, Bafilomycin A1. Data shown represent mean ± SEM of at least three independent experiments. Student’s test: *** p p  (DOCX)</p

Schematic representation of CHIKV replication cycle and the involvement of BNIP3 in the early stages of replication.

Schematic representation of CHIKV replication cycle and the involvement of BNIP3 in the early stages of replication.</p