Early activation of MyD88-mediated autophagy sustains HSV-1 replication in human monocytic THP-1 cells

Abstract

Autophagy is a cellular degradation pathway that exerts numerous functions in vital biological processes. Among these, it contributes to both innate and adaptive immunity. On the other hand, pathogens have evolved strategies to manipulate autophagy for their own advantage. By monitoring autophagic markers, we showed that HSV-1 transiently induced autophagosome formation during early times of the infection of monocytic THP-1 cells and human monocytes. Autophagy is induced in THP-1 cells by a mechanism independent of viral gene expression or viral DNA accumulation. We found that the MyD88 signaling pathway is required for HSV-1-mediated autophagy, and it is linked to the toll-like receptor 2 (TLR2). Interestingly, autophagy inhibition by pharmacological modulators or siRNA knockdown impaired viral replication in both THP-1 cells and human monocytes, suggest that the virus exploits the autophagic machinery to its own benefit in these cells. Taken together, these findings indicate that the early autophagic response induced by HSV-1 exerts a proviral role, improving viral production in a semi-permissive model such as THP-1 cells and human monocytes.

Figure 1. HSV-1 transiently induces autophagy during the early phases of the infection.

(a) Representative confocal images of THP-1 cells mock-infected or infected with HSV-1, collected at different times and stained for endogenous LC3. Quantification of LC3 puncta was performed using Imaris software; 50 cells were considered for each sample. (b) Representative images obtained using an epifluorescence microscope of THP-1 cells transiently transfected with GFP-LC3 plasmid for 6 h, then mock-infected or infected with HSV-1 and fixed at different times. GFP-LC3 puncta were quantified by counting them and 50 cells were considered for each sample. (c) Immunoblotting of LC3 and SQSTM1 in THP-1 cells infected or mock-infected at the indicated times. The densitometric analysis of the LC3II/actin and SQSTM1/actin ratios is reported. (d) Immunoblotting of LC3 and SQSTM1 in human monocytes infected or mock-infected at the indicated times. The results shown are representative of three independent donors. (e) Immunofluorescence images of THP-1 cells infected with EGFP-HSV-1 and stained to identify SQSTM1. Quantification of SQSTM1 puncta in cells expressing EGFP (EGFP-HSV-1 +) or not (EGFP-HSV-1 −). *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 2. The stimulation of autophagy by HSV-1 is not dependent on viral DNA.

THP-1 cells were transiently transfected with viral DNA for 1 h. As a control, transfection of THP-1 cells with GFP-LC3 plasmid and then mock-infected or infected with HSV-1 was used. (a) Amount of viral DNA inside THP-1 cells was determined by real-time PCR. (b) Representative images of SQSTM1 staining in THP-1 cells transfected with viral DNA. SQSTM1puncta were quantified as previously described. Twenty cells were analysed per assay. (c) Representative images of LC3 staining in THP-1 cells mock-infected or infected with VP26GFP HSV-1 for 1 h.

Figure 3. The MyD88-dependent pathway is involved in the early activation of HSV-1-mediated autophagy.

(a) TLR2 and MyD88 expression of THP-1 cells infected with HSV-1 and harvested at different times after infection. β-actin was used as a loading control. (b) MyD88 transcript levels were determined by real-time PCR. (c) THP-1 cells were transfected with GFP-LC3 and MyD88 plasmid or pcDNA3.1 as a control and infected with HSV-1 for 1 h. Quantification of GFP-LC3 puncta was performed; 20 cells were analysed for each condition (**p < 0.01). (d) Representative confocal images of infected or mock-infected MyD88−/− THP-1 cells stained to identify endogenous LC3 protein. Quantification of LC3 puncta was performed using Imaris software; 50 cells were analysed for each condition. (e) Immunoblot analysis of phosphorylated p38α in mock-infected and infected THP-1 cells. β-actin and H3 histone proteins were used as loading controls for the cytoplasmic fraction and the nuclear fraction, respectively.

Figure 4. Induction of autophagy mediated by HSV-1 has a proviral role.

Viral production was quantified in wt (**p < 0.01) (a) and MyD88−/− THP-1 cells (b) pre-treated with spautin-1 or 3-MA and infected with HSV-1 in the presence of the inhibitors for 24 h. (c) Amount of viral DNA in wt and MyD88−/− THP-1 cells was determined by real time PCR (**p < 0.01). (d) Immunofluorescence analysis showing gD-positive wt and MyD88−/− THP-1 cells 24 h pi. Hoechst 33342 was used to stain the nuclei. (e) Immunoblot analysis of ICP0, ICP8 and Us11 in wt and MyD88−/− THP-1 cells infected with HSV-1. GAPDH was used as a loading control. (f) THP-1 cells were nucleofected with Beclin 1 (BECN1) siRNA for 24 h and then infected with HSV-1. ICP0 and Us11 expression and viral production (g) were quantified by immunoblot analysis and plaque assay, respectively. The amount of viral DNA (h) was analyzed in THP-1 cells pre-treated with 3-MA and infected with HSV-1 in the presence of the inhibitor for 3 h (***p < 0.001). The same experiment was carried out in wt THP-1 and MyD88−/− cells and ICP0 expression was evaluated by immunoblot (i). ICP0 and Us11 expression in human monocytes nucleofected with BECN1 siRNA for 6 h and then infected with HSV-1 for additional 24 h (l), or pre-treated with 3-MA and infected with HSV-1 in the presence of the inhibitor for 3 h and 24 h (m). The results shown are representative of three independent donors.

Figure 5. Effect of endocytosis inhibitors on HSV-1 entry.

THP-1 cells pre-incubated with filipin or sucrose for 1 h were infected with HSV-1. After 24 h, cells were fixed and stained for gD. Evans Blue dye was used as a counter stain.