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Autophagy facilitates antibody-enhanced dengue virus infection in human pre-basophil/mast cells.

Fang YT, Wan SW, Lu YT, Yao JH, Lin CF, Hsu LJ, Brown MG, Marshall JS, Anderson R, Lin YS - PLoS ONE (2014)

Bottom Line: Dengue virus (DENV) infection can cause severe hemorrhagic disease in humans.Our results demonstrate a significant induction of autophagy in antibody-enhanced DENV infection of pre-basophil-like KU812 and immature mast cell-like HMC-1 cells.Given the importance of ADE and FcR-bearing cells such as monocytes, macrophages and basophil/mast cells in dengue disease, the results provide insights into dengue pathogenesis and therapeutic means of control.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, National Cheng Kung University Medical College, Tainan, Taiwan.

ABSTRACT

Background: Dengue virus (DENV) infection can cause severe hemorrhagic disease in humans. Although the pathogenic mechanisms underlying severe DENV disease remain unclear, one of the possible contributing factors is antibody-dependent enhancement (ADE) which occurs when sub-neutralizing antibodies derived from a previous DENV infection enhance viral infection through interaction between virus-antibody complexes and FcR-bearing cells, such as macrophages and basophil/mast cells. Although recent reports showed that DENV induces autophagy, the relationship between antibody-enhanced DENV infection and autophagy is not clear.

Methodology/principal findings: We showed that sub-neutralizing antibodies derived from dengue patient sera enhanced DENV infection and autophagy in the KU812 pre-basophil-like cell line as well as the HMC-1 immature mast cell line. Antibody-enhanced DENV infection of KU812 cells increased the number of autophagosome vesicles, LC3 punctation, LC3-II accumulation, and p62 degradation over that seen in cells infected with DENV alone. The percentages of DENV envelope (E) protein-positive cells and LC3 puncta following antibody-enhanced DENV infection of KU812 cells were reduced by the autophagy inhibitor 3-MA. Antibody-enhanced DENV infection of HMC-1 cells showed co-localization of DENV E protein and dsRNA with autophagosomes, which was inhibited by 3-MA treatment. Furthermore, DENV infection and replication were reduced when KU812 cells were transfected with the autophagy-inhibiting Atg4BC74A mutant.

Conclusions/significance: Our results demonstrate a significant induction of autophagy in antibody-enhanced DENV infection of pre-basophil-like KU812 and immature mast cell-like HMC-1 cells. Also, autophagy plays an important role in DENV infection and replication in these cells. Given the importance of ADE and FcR-bearing cells such as monocytes, macrophages and basophil/mast cells in dengue disease, the results provide insights into dengue pathogenesis and therapeutic means of control.

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Antibody-enhanced DENV infection and autophagosome formation in KU812 cells.(A and B) Cells were inoculated with medium alone (Mock), with DENV alone (MOI = 1), or with DENV in the presence of a sub-neutralizing dilution (1∶10,000) of dengue patient sera at 4°C for 1.5 h. After washing, cells were resuspended in fresh medium and incubated at 37°C for 24 h. The DENV E protein (A) and NS4B protein (B) were detected by flow cytometry. Infection of UV-inactivated DENV (iDENV) was served as negative control. The means of three independent experiments ± SD are shown. (C and D) After 24 h post-infection, the culture supernatant (C) and the mixture of cell and culture supernatant (D) were collected to determine viral titers by plaque assay. The means ± SD of three independent experiments are shown. (E) After infection for 24 h, cells were fixed and observed under TEM. We analyzed five cells by TEM in each condition including mock, DENV alone, and ADE. One section per cell was obtained to quantify the autophagosome vesicles. The black square areas in the left panels were amplified (×40000) and shown in right panels. The arrowheads indicate the autophagosomes. Cy: cytoplasm; N: nucleus. (F) The quantification of autophagosome numbers in each section from (E) is shown. The means ± SD of three independent experiments are shown. **P<0.01.
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pone-0110655-g001: Antibody-enhanced DENV infection and autophagosome formation in KU812 cells.(A and B) Cells were inoculated with medium alone (Mock), with DENV alone (MOI = 1), or with DENV in the presence of a sub-neutralizing dilution (1∶10,000) of dengue patient sera at 4°C for 1.5 h. After washing, cells were resuspended in fresh medium and incubated at 37°C for 24 h. The DENV E protein (A) and NS4B protein (B) were detected by flow cytometry. Infection of UV-inactivated DENV (iDENV) was served as negative control. The means of three independent experiments ± SD are shown. (C and D) After 24 h post-infection, the culture supernatant (C) and the mixture of cell and culture supernatant (D) were collected to determine viral titers by plaque assay. The means ± SD of three independent experiments are shown. (E) After infection for 24 h, cells were fixed and observed under TEM. We analyzed five cells by TEM in each condition including mock, DENV alone, and ADE. One section per cell was obtained to quantify the autophagosome vesicles. The black square areas in the left panels were amplified (×40000) and shown in right panels. The arrowheads indicate the autophagosomes. Cy: cytoplasm; N: nucleus. (F) The quantification of autophagosome numbers in each section from (E) is shown. The means ± SD of three independent experiments are shown. **P<0.01.

Mentions: Recent reports demonstrated that autophagy is observed in several kinds of DENV-infected cells [42]. However, the relationship between antibody-enhanced DENV infection and autophagy is still unclear. Human pre-basophil-like KU812 cells were inoculated with DENV (MOI = 1) in the presence or absence of dengue patient sera (1∶10,000 dilution). Detection of DENV infection in KU812 cells was performed by flow cytometry (Figure 1A and 1B) and plaque assay (Figure 1C and 1D). Our previous study indicated very few virus-positive cells when KU812 cells were inoculated with DENV in combination with normal (i.e. dengue non-immune) human sera [43]. In addition, although the possibility of non-specific effect of antibody-containing serum to other RNA viruses cannot be completely ruled out, it was previously reported that infection of KU812 cells with another virus (RSV) was not enhanced by human RSV-positive, dengue-negative sera [43]. We herein showed the intracellular expression of E protein and NS4B protein at 24 h post-infection was significantly enhanced by antibody-enhanced DENV infection (Figure 1A and 1B). In addition, virus titers from the supernatant of antibody-enhanced DENV-infected KU812 cells (Figure 1C) and the combined supernatant and infected cells (Figure 1D) were increased than those from KU812 cells infected with DENV alone. UV-inactivated DENV (iDENV) served as negative control (Figure 1A-1D). Furthermore, we found that antibody-enhanced DENV infection of KU812 cells increased autophagosome formation as observed by electron microscopy (Figure 1E, arrowhead). The number of autophagosomes from one section in each cell was quantified from Figure 1E and shown in Figure 1F.


Autophagy facilitates antibody-enhanced dengue virus infection in human pre-basophil/mast cells.

Fang YT, Wan SW, Lu YT, Yao JH, Lin CF, Hsu LJ, Brown MG, Marshall JS, Anderson R, Lin YS - PLoS ONE (2014)

Antibody-enhanced DENV infection and autophagosome formation in KU812 cells.(A and B) Cells were inoculated with medium alone (Mock), with DENV alone (MOI = 1), or with DENV in the presence of a sub-neutralizing dilution (1∶10,000) of dengue patient sera at 4°C for 1.5 h. After washing, cells were resuspended in fresh medium and incubated at 37°C for 24 h. The DENV E protein (A) and NS4B protein (B) were detected by flow cytometry. Infection of UV-inactivated DENV (iDENV) was served as negative control. The means of three independent experiments ± SD are shown. (C and D) After 24 h post-infection, the culture supernatant (C) and the mixture of cell and culture supernatant (D) were collected to determine viral titers by plaque assay. The means ± SD of three independent experiments are shown. (E) After infection for 24 h, cells were fixed and observed under TEM. We analyzed five cells by TEM in each condition including mock, DENV alone, and ADE. One section per cell was obtained to quantify the autophagosome vesicles. The black square areas in the left panels were amplified (×40000) and shown in right panels. The arrowheads indicate the autophagosomes. Cy: cytoplasm; N: nucleus. (F) The quantification of autophagosome numbers in each section from (E) is shown. The means ± SD of three independent experiments are shown. **P<0.01.
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pone-0110655-g001: Antibody-enhanced DENV infection and autophagosome formation in KU812 cells.(A and B) Cells were inoculated with medium alone (Mock), with DENV alone (MOI = 1), or with DENV in the presence of a sub-neutralizing dilution (1∶10,000) of dengue patient sera at 4°C for 1.5 h. After washing, cells were resuspended in fresh medium and incubated at 37°C for 24 h. The DENV E protein (A) and NS4B protein (B) were detected by flow cytometry. Infection of UV-inactivated DENV (iDENV) was served as negative control. The means of three independent experiments ± SD are shown. (C and D) After 24 h post-infection, the culture supernatant (C) and the mixture of cell and culture supernatant (D) were collected to determine viral titers by plaque assay. The means ± SD of three independent experiments are shown. (E) After infection for 24 h, cells were fixed and observed under TEM. We analyzed five cells by TEM in each condition including mock, DENV alone, and ADE. One section per cell was obtained to quantify the autophagosome vesicles. The black square areas in the left panels were amplified (×40000) and shown in right panels. The arrowheads indicate the autophagosomes. Cy: cytoplasm; N: nucleus. (F) The quantification of autophagosome numbers in each section from (E) is shown. The means ± SD of three independent experiments are shown. **P<0.01.
Mentions: Recent reports demonstrated that autophagy is observed in several kinds of DENV-infected cells [42]. However, the relationship between antibody-enhanced DENV infection and autophagy is still unclear. Human pre-basophil-like KU812 cells were inoculated with DENV (MOI = 1) in the presence or absence of dengue patient sera (1∶10,000 dilution). Detection of DENV infection in KU812 cells was performed by flow cytometry (Figure 1A and 1B) and plaque assay (Figure 1C and 1D). Our previous study indicated very few virus-positive cells when KU812 cells were inoculated with DENV in combination with normal (i.e. dengue non-immune) human sera [43]. In addition, although the possibility of non-specific effect of antibody-containing serum to other RNA viruses cannot be completely ruled out, it was previously reported that infection of KU812 cells with another virus (RSV) was not enhanced by human RSV-positive, dengue-negative sera [43]. We herein showed the intracellular expression of E protein and NS4B protein at 24 h post-infection was significantly enhanced by antibody-enhanced DENV infection (Figure 1A and 1B). In addition, virus titers from the supernatant of antibody-enhanced DENV-infected KU812 cells (Figure 1C) and the combined supernatant and infected cells (Figure 1D) were increased than those from KU812 cells infected with DENV alone. UV-inactivated DENV (iDENV) served as negative control (Figure 1A-1D). Furthermore, we found that antibody-enhanced DENV infection of KU812 cells increased autophagosome formation as observed by electron microscopy (Figure 1E, arrowhead). The number of autophagosomes from one section in each cell was quantified from Figure 1E and shown in Figure 1F.

Bottom Line: Dengue virus (DENV) infection can cause severe hemorrhagic disease in humans.Our results demonstrate a significant induction of autophagy in antibody-enhanced DENV infection of pre-basophil-like KU812 and immature mast cell-like HMC-1 cells.Given the importance of ADE and FcR-bearing cells such as monocytes, macrophages and basophil/mast cells in dengue disease, the results provide insights into dengue pathogenesis and therapeutic means of control.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, National Cheng Kung University Medical College, Tainan, Taiwan.

ABSTRACT

Background: Dengue virus (DENV) infection can cause severe hemorrhagic disease in humans. Although the pathogenic mechanisms underlying severe DENV disease remain unclear, one of the possible contributing factors is antibody-dependent enhancement (ADE) which occurs when sub-neutralizing antibodies derived from a previous DENV infection enhance viral infection through interaction between virus-antibody complexes and FcR-bearing cells, such as macrophages and basophil/mast cells. Although recent reports showed that DENV induces autophagy, the relationship between antibody-enhanced DENV infection and autophagy is not clear.

Methodology/principal findings: We showed that sub-neutralizing antibodies derived from dengue patient sera enhanced DENV infection and autophagy in the KU812 pre-basophil-like cell line as well as the HMC-1 immature mast cell line. Antibody-enhanced DENV infection of KU812 cells increased the number of autophagosome vesicles, LC3 punctation, LC3-II accumulation, and p62 degradation over that seen in cells infected with DENV alone. The percentages of DENV envelope (E) protein-positive cells and LC3 puncta following antibody-enhanced DENV infection of KU812 cells were reduced by the autophagy inhibitor 3-MA. Antibody-enhanced DENV infection of HMC-1 cells showed co-localization of DENV E protein and dsRNA with autophagosomes, which was inhibited by 3-MA treatment. Furthermore, DENV infection and replication were reduced when KU812 cells were transfected with the autophagy-inhibiting Atg4BC74A mutant.

Conclusions/significance: Our results demonstrate a significant induction of autophagy in antibody-enhanced DENV infection of pre-basophil-like KU812 and immature mast cell-like HMC-1 cells. Also, autophagy plays an important role in DENV infection and replication in these cells. Given the importance of ADE and FcR-bearing cells such as monocytes, macrophages and basophil/mast cells in dengue disease, the results provide insights into dengue pathogenesis and therapeutic means of control.

Show MeSH
Related in: MedlinePlus