Journal: Journal of Virology

Article Title: Syntaxin-6 restricts SARS-CoV-2 infection by facilitating virus trafficking to autophagosomes

doi: 10.1128/jvi.00002-25

Figure Lengend Snippet: The proximal proteome of ACE2 protein during SARS-CoV-2 infection. ( A ) The schematic diagram of the study. ( B ) Venn diagrams of the ACE2 proximal proteins identified in mock and SARS-CoV-2-infected group. ( C ) The KEGG pathway enrichment analysis was performed with the ACE2 proximal proteins identified in virus-infected cells using the KOBAS 3 online tool. ( D ) The protein–protein interaction network revealed the clustering of SNARE proteins. ( E and F ) H1299-ACE2 cells were infected with SARS-CoV-2 (MOI = 50) for 1 h and fixed by paraformaldehyde. Endogenous STX6, viral Spike ( E ), and N ( F ) were stained by antibodies. The white frames indicated the colocalization of STX6 with Spike or N. Scale bars, 20 µm.

Article Snippet: The following antibodies were used in this study: Rabbit anti-syntaxin 6 polyclonal antibody (10841–1-AP), rabbit anti-ACE2 polyclonal antibody (21115–1-AP), rabbit anti-AXL polyclonal antibody (13196–1-AP), mouse anti-TFRC monoclonal antibody (66180–1-Ig), mouse anti-Beta Actin monoclonal antibody (66009–1-Ig), mouse anti-GAPDH monoclonal antibody (60004–1-Ig), mouse anti-EEA1 monoclonal antibody (68065–1-Ig), rabbit anti-Rab7A polyclonal antibody (55469–1-AP), and mouse anti-LAMP1 monoclonal antibody (67300–1-Ig) were obtained from Proteintech.

Techniques: Infection, Virus, Staining

Journal: Journal of Virology

Article Title: Syntaxin-6 restricts SARS-CoV-2 infection by facilitating virus trafficking to autophagosomes

doi: 10.1128/jvi.00002-25

Figure Lengend Snippet: Syntaxin-6 inhibits SARS-CoV-2 infection. ( A and B ) H1299-ACE2 cells were transfected with siRNA negative control (siNC) (−), siSTX6 at 10 nM (+) or 50 nM (++) for 36 h ( A ), or transfected with vector (−), low concentration (+), or high concentration (++) Flag-STX6 plasmid for 24 h ( B ) and then infected with SARS-CoV-2 at an MOI of 0.1. The virus N mRNA level, the N protein level in cells, and the virus titer in the supernatant were quantified at 24 hpi. ( C ) H1299-ACE2 cells were first transfected with siRNAs, then complemented with vector or Flag-STX6 plasmid. Virus infection was performed at 24 h post plasmid transfection. The virus N mRNA level, N protein level in cells, and the virus titer in the supernatant were quantified at 24 hpi. ( D and E ) A549-ACE2 ( D ) and Huh7 ( E ) cells were transfected with siRNAs and then infected with SARS-CoV-2 at an MOI of 0.1. The virus N mRNA level and N protein level in cells were analyzed. The differences among groups were determined by a one-way analysis of variance followed by Tukey’s post hoc test for multiple comparisons. Data are shown as the mean ± SD. * P < 0.05, ** P < 0.01, and *** P < 0.001; ns, not significant; n = 3.

Article Snippet: The following antibodies were used in this study: Rabbit anti-syntaxin 6 polyclonal antibody (10841–1-AP), rabbit anti-ACE2 polyclonal antibody (21115–1-AP), rabbit anti-AXL polyclonal antibody (13196–1-AP), mouse anti-TFRC monoclonal antibody (66180–1-Ig), mouse anti-Beta Actin monoclonal antibody (66009–1-Ig), mouse anti-GAPDH monoclonal antibody (60004–1-Ig), mouse anti-EEA1 monoclonal antibody (68065–1-Ig), rabbit anti-Rab7A polyclonal antibody (55469–1-AP), and mouse anti-LAMP1 monoclonal antibody (67300–1-Ig) were obtained from Proteintech.

Techniques: Infection, Transfection, Negative Control, Plasmid Preparation, Concentration Assay, Virus

Journal: Journal of Virology

Article Title: Syntaxin-6 restricts SARS-CoV-2 infection by facilitating virus trafficking to autophagosomes

doi: 10.1128/jvi.00002-25

Figure Lengend Snippet: Syntaxin-6 inhibits viral invasion. ( A–C ) Luciferase activity in cell lysates was determined at 24 h post-SARS-CoV-2 spike pseudovirus transduction. ( A ) H1299-ACE2 cells were transfected with indicated siRNAs for 36 h and then transduced with SARS-CoV-2 spike pseudovirus. The knockdown efficiency was monitored by western blotting (right). ( B ) H1299-ACE2 cells were first transfected with siRNAs, then complemented with vector or Flag-STX6 plasmid, and transduced with SARS-CoV-2 spike pseudovirus. ( C ) H1299-ACE2 cells with or without ectopic expression of TMPRSS2 were transfected with Flag-STX6 plasmid, treated or untreated with E-64d (20 µM), and then transduced with SARS-CoV-2 spike pseudovirus. ( D ) H1299-ACE2 cells were transfected with indicated siRNAs, precooled, and incubated with pre-chilled SARS-CoV-2 at an MOI of 10 for 30 min at 4°C. For binding analysis, the attached virus was determined by reverse transcription real-time quantitative PCR (qRT-PCR) after removing the unbound virus. For internalization analysis, cells were transferred to 37°C and maintained for 30 min to allow internalization. Uninternalized virus particles were removed by treating the cells with 0.05% trypsin. qRT-PCR was used to detect the relative amount of internalized virus. ( E ) H1299-ACE2 cells transfected with indicated siRNA were infected by SARS-CoV-2 at an MOI of 10. SARS-CoV-2 positive- and negative-sense RNA were detected by strand-specific primers 30 min post-infection. ( F ) H1299-ACE2 cells were infected with SARS-CoV-2 at an MOI of 50, and double-stranded RNA (dsRNA) was detected with a monoclonal antibody at 30 min post-infection (left). The percentage of cells with dsRNA foci was calculated by high-content analysis (right). n = 12 image fields. Scale bars, 20 µm. The differences between the two groups were determined by two-tailed t tests. Data are shown as the mean ± SD. * P < 0.05, ** P < 0.01, and *** P < 0.001; ns, not significant; n = 3.

Article Snippet: The following antibodies were used in this study: Rabbit anti-syntaxin 6 polyclonal antibody (10841–1-AP), rabbit anti-ACE2 polyclonal antibody (21115–1-AP), rabbit anti-AXL polyclonal antibody (13196–1-AP), mouse anti-TFRC monoclonal antibody (66180–1-Ig), mouse anti-Beta Actin monoclonal antibody (66009–1-Ig), mouse anti-GAPDH monoclonal antibody (60004–1-Ig), mouse anti-EEA1 monoclonal antibody (68065–1-Ig), rabbit anti-Rab7A polyclonal antibody (55469–1-AP), and mouse anti-LAMP1 monoclonal antibody (67300–1-Ig) were obtained from Proteintech.

Techniques: Luciferase, Activity Assay, Transduction, Transfection, Knockdown, Western Blot, Plasmid Preparation, Expressing, Incubation, Binding Assay, Virus, Reverse Transcription, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Infection, High Content Screening, Two Tailed Test

Journal: Journal of Virology

Article Title: Syntaxin-6 restricts SARS-CoV-2 infection by facilitating virus trafficking to autophagosomes

doi: 10.1128/jvi.00002-25

Figure Lengend Snippet: Syntaxin-6 colocalized with the virus, and both colocalized with all endosomes tested. ( A ) H1299-ACE2 cells were transduced with S-EGFP pseudovirus and fixed by paraformaldehyde at 1 h post-transduction. Endogenous STX6 was stained by antibody. The white frames indicated the colocalization of STX6 with S-EGFP. ( B ) H1299-ACE2 cells were transfected with mCherry-STX6 plasmid and then transduced with S-EGFP pseudovirus. One hour post-infection, cells were fixed, and the endogenous EEA1, Rab5, and Rab7 were stained by antibodies. The LAMP1-Flag plasmid was first transfected together with mCherry-STX6 plasmid for LAMP1-Flag staining. White frames indicate colocalization of S-EGFP, mCherry-STX6, and different markers. ( C ) Mock and S-EGFP pseudovirus transduced H1299-ACE2 cells fixed at 1 h post-infection and stained with STX6 and EEA1, Rab5, Rab7, or Flag antibodies. ( D–G ) The subcellular distribution dynamics of endogenous STX6 in Mock and S-EGFP pseudovirus transduced H1299-ACE2 cells. ( D ) The Manders’ colocalization coefficients (MCCs) represent the proportion of STX6 colocalized with different endosome markers in each cell before and after pseudovirus transduction. ( E ) The number of STX6 + marker + puncta in ( D ) was counted. ( F ) The MCCs represent the proportion of marker colocalized with STX6 in each cell before and after pseudovirus transduction. ( G ) The MCCs represent the proportion of STX6 in marker + PP + puncta or in marker + PP − puncta in each cell post-S-EGFP pseudovirus transduction. ( H ) Live cell fluorescence imaging shows the colocalization process of S-EGFP pseudovirus and mCherry-STX6. ( I ) Representative images showing the localization of endogenous STX6 in H1299-ACE2 cells at 24 h post-SARS-CoV-2 infection (MOI = 0.01). The differences between the two groups were determined by two-tailed t tests. Data are shown as the mean ± SD. * P < 0.05, ** P < 0.01, and *** P < 0.001; ns, not significant; n = 20. Scale bars, 20 µm.

Article Snippet: The following antibodies were used in this study: Rabbit anti-syntaxin 6 polyclonal antibody (10841–1-AP), rabbit anti-ACE2 polyclonal antibody (21115–1-AP), rabbit anti-AXL polyclonal antibody (13196–1-AP), mouse anti-TFRC monoclonal antibody (66180–1-Ig), mouse anti-Beta Actin monoclonal antibody (66009–1-Ig), mouse anti-GAPDH monoclonal antibody (60004–1-Ig), mouse anti-EEA1 monoclonal antibody (68065–1-Ig), rabbit anti-Rab7A polyclonal antibody (55469–1-AP), and mouse anti-LAMP1 monoclonal antibody (67300–1-Ig) were obtained from Proteintech.

Techniques: Virus, Transduction, Staining, Transfection, Plasmid Preparation, Infection, Marker, Fluorescence, Imaging, Two Tailed Test

Journal: Journal of Virology

Article Title: Syntaxin-6 restricts SARS-CoV-2 infection by facilitating virus trafficking to autophagosomes

doi: 10.1128/jvi.00002-25

Figure Lengend Snippet: Syntaxin-6 is involved in the autophagy pathway to inhibit virus entry. ( A–D ) H1299-ACE2 cells transfected with siNC or siSTX6 were transduced with S-EGFP pseudovirus and fixed at 30 min and 90 min post-infection. Colocalization of S-EGFP pseudovirus with early endosome (left in A and B), late endosome (left in C), and lysosome (left in D) in each cell was visualized by confocal microscopy. Mander’s colocalization coefficients (MCCs) were used to calculate the proportion of S-EGFP pseudovirus colocalized with different markers (right in A–D). ( E ) H1299-ACE2 cells were transduced with S-EGFP pseudovirus after transfection of mCherry-STX6 for 24 h and were fixed at 1 hpi. The cells were subjected to immunofluorescence with antibodies against endogenous LC3B and visualized by confocal microscopy. The white frames indicated the colocalization of mCherry-STX6 with LC3B. ( F and G ) H1299-ACE2 cells transfected with siNC or siSTX6 were transduced with S-EGFP pseudovirus and fixed at 30 min and 90 min post-infection. Colocalization of S-EGFP and autophagosome (LC3B) in each cell was visualized by confocal microscopy (left in G) and calculated by MCC (right in G). The number of red puncta in ( G ), which represents LC3B puncta, was counted in F. ( H and I ) H1299-ACE2 cells were treated with 0.1 µM (+) and 1 µM (++) rapamycin ( H ) or 1 µM MHY-1485 ( I ) for 24 h, and cells were transduced with SARS-CoV-2 spike pseudovirus for 24 h. Luciferase activity in cell lysates was determined. ( J ) H1299-ACE2 cells overexpressing vector or Flag-STX6 plasmid were treated with dimethyl sulfoxide (DMSO) and MHY-1485 (1 µM) for 24 h. Cells were transduced with SARS-CoV-2 pseudovirus for 24 h. Luciferase activity in cell lysates was determined. The differences between the two groups were determined by a two-tailed t test. Data are shown as the mean ± SD. * P < 0.05, ** P < 0.01, and *** P < 0.001; ns, not significant; n = 20 ( A–D, F, and G ), n = 3 ( H–J ). Scale bars, 20 µm.

Article Snippet: The following antibodies were used in this study: Rabbit anti-syntaxin 6 polyclonal antibody (10841–1-AP), rabbit anti-ACE2 polyclonal antibody (21115–1-AP), rabbit anti-AXL polyclonal antibody (13196–1-AP), mouse anti-TFRC monoclonal antibody (66180–1-Ig), mouse anti-Beta Actin monoclonal antibody (66009–1-Ig), mouse anti-GAPDH monoclonal antibody (60004–1-Ig), mouse anti-EEA1 monoclonal antibody (68065–1-Ig), rabbit anti-Rab7A polyclonal antibody (55469–1-AP), and mouse anti-LAMP1 monoclonal antibody (67300–1-Ig) were obtained from Proteintech.

Techniques: Virus, Transfection, Transduction, Infection, Confocal Microscopy, Immunofluorescence, Luciferase, Activity Assay, Plasmid Preparation, Two Tailed Test

Journal: Journal of Virology

Article Title: Syntaxin-6 restricts SARS-CoV-2 infection by facilitating virus trafficking to autophagosomes

doi: 10.1128/jvi.00002-25

Figure Lengend Snippet: Proper localization of Syntaxin-6 is required in the viral restriction function. ( A ) Schematic diagram depicting the deletion mutants of STX6: full length (1–255), Δ N (delete 5–103), Δ SNARE (delete 158–225), and Δ TM (1–234). ( B ) Representative images showing the localization of full length and deletion mutants of STX6. ( C ) H1299-ACE2 cells were transfected with full length or deletion mutants of STX6 for 24 h, and cells were transduced with SARS-CoV-2 spike pseudovirus for 24 h. Luciferase activity in cell lysates was determined. ( D ) Representative images showing the localization of full length or deletion mutants of STX6 as well as S-EGFP in H1299-ACE2 cells at 1 h post-S-EGFP pseudovirus transduction. The differences among groups were determined by a one-way analysis of variance followed by Tukey’s post hoc test. Data are shown as the mean ± SD. * P < 0.05, ** P < 0.01, and *** P < 0.001; ns, not significant. n = 3. Scale bars, 20 µm.

Article Snippet: The following antibodies were used in this study: Rabbit anti-syntaxin 6 polyclonal antibody (10841–1-AP), rabbit anti-ACE2 polyclonal antibody (21115–1-AP), rabbit anti-AXL polyclonal antibody (13196–1-AP), mouse anti-TFRC monoclonal antibody (66180–1-Ig), mouse anti-Beta Actin monoclonal antibody (66009–1-Ig), mouse anti-GAPDH monoclonal antibody (60004–1-Ig), mouse anti-EEA1 monoclonal antibody (68065–1-Ig), rabbit anti-Rab7A polyclonal antibody (55469–1-AP), and mouse anti-LAMP1 monoclonal antibody (67300–1-Ig) were obtained from Proteintech.

Techniques: Transfection, Transduction, Luciferase, Activity Assay

Journal: Journal of Virology

Article Title: Syntaxin-6 restricts SARS-CoV-2 infection by facilitating virus trafficking to autophagosomes

doi: 10.1128/jvi.00002-25

Figure Lengend Snippet: Syntaxin-6 has antiviral activity against variants of concern (VOCs) and other endocytic intrusion viruses. ( A–D ) H1299-ACE2 cells were transfected with control siNC, siSTX6 for 36 h (left in A–D), or transfected with vector, flag-STX6 plasmid for 24 h (right in A–D) and then infected with BA.2 strain ( A ), BA.5 strain ( B ), XBB1.9 strain ( C ), and delta strain ( D ) at an MOI of 0.1. The virus N mRNA level was quantified at 24 hpi. ( E and F ) SARS-CoV-2 pseudovirus transduction ( E ) and SARS-CoV-2 infection ( F ) in H1299-ACE2 cells transfected with vector or human ( Homo sapiens ), mouse ( Mus musculus ), or bat ( Rhinolophus sinicus ) orthologs of STX6. ( G–I ) Hela ( G ), H1299-ACE2 ( H ), or Huh7.5.1 ( I ) cells were transfected with control siNC, siSTX6 for 36 h (left), or transfected with vector, flag-STX6 plasmid for 24 h (right) and then infected with EV71 ( G ), VSV-GFP ( H ), or HCV J399EM ( I ), respectively, at an MOI of 0.1. The virus RNA level was determined by qRT-PCR. The differences between the two groups were determined by two-tailed t test, except e and f (one-way analysis of variance followed by Tukey’s post hoc test). Data are shown as the mean ± SD. * P < 0.05, ** P < 0.01, and *** P < 0.001; ns, not significant; n = 3.

Article Snippet: The following antibodies were used in this study: Rabbit anti-syntaxin 6 polyclonal antibody (10841–1-AP), rabbit anti-ACE2 polyclonal antibody (21115–1-AP), rabbit anti-AXL polyclonal antibody (13196–1-AP), mouse anti-TFRC monoclonal antibody (66180–1-Ig), mouse anti-Beta Actin monoclonal antibody (66009–1-Ig), mouse anti-GAPDH monoclonal antibody (60004–1-Ig), mouse anti-EEA1 monoclonal antibody (68065–1-Ig), rabbit anti-Rab7A polyclonal antibody (55469–1-AP), and mouse anti-LAMP1 monoclonal antibody (67300–1-Ig) were obtained from Proteintech.

Techniques: Activity Assay, Transfection, Control, Plasmid Preparation, Infection, Virus, Transduction, Quantitative RT-PCR, Two Tailed Test