Journal: Nature Microbiology

Article Title: Influenza virus uses mGluR2 as an endocytic receptor to enter cells

doi: 10.1038/s41564-024-01713-x

Figure Lengend Snippet: a , Potassium channels involved in influenza virus replication were identified by screening siRNA libraries of human ion channels. b , Viral titres in different potassium channel-knockdown A549 cells. The data were summarized from the screen by combining the results from different siRNAs presented in a . c , The mRNA level of KCa1.1 in KCa1.1-knockdown A549 cells was significantly lower than that in control cells. d , KCa1.1 knockdown significantly reduced H1N1 virus replication in A549 cells. e , Paxilline treatment did not affect the viability of A549 cells. f , Paxilline treatment significantly reduced H1N1 virus replication in A549 cells. g , Paxilline affected the early stage of influenza infection. h , KCa1.1-knockdown and paxilline treatment did not affect the attachment of H1N1 virus to A549 cells. i , KCa1.1-knockdown or paxilline treatment affected the internalization of H1N1 virus into A549 cells. j , KCa1.1-knockdown affected the internalization of H1N1 virus confirmed by a three-dimensional microscopy-based assay under permeabilized and unpermeabilized conditions with z-stacks and a membrane marker. Cell nucleus, blue; viral HA, turquoise; DiD, red. k , KCa1.1 knockdown affected the internalization of H1N1 virus into A549 cells confirmed by a microscopy-based assay under permeabilized and unpermeabilized conditions. Cell nucleus, blue; viral HA, green. The solid line represents median and dashed lines represent quartiles of the data in the violin graphs of k . l , KCa1.1 overexpression did not increase H1N1 virus internalization. The images in j and k are representative of three independent experiments. Error bar in panels b – i and l indicates the standard deviation. The data shown in a – i , k and l are means ± s.d. ( n = 3 biologically independent experiments). Statistical analysis was performed using unpaired, two-tailed Student’s t -test. NS, not significant. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. Exact P values are available in Source Data.

Article Snippet: Rabbit anti-KCa1.1 pAb (CN APC-151, 1:100) was from Alomone Labs.

Techniques: Virus, Knockdown, Control, Infection, Microscopy, Membrane, Marker, Over Expression, Standard Deviation, Two Tailed Test

Journal: Nature Microbiology

Article Title: Influenza virus uses mGluR2 as an endocytic receptor to enter cells

doi: 10.1038/s41564-024-01713-x

Figure Lengend Snippet: a , Schematic representation of the domain structures for wild-type HA of H1N1 and a 6×His-tagged form of HA (HA-His). b , Structural prediction of HA-His by Alphafold2 and visualized by Pymol. c , KCa1.1 does not interact with HA. Direct interaction of KCa1.1 and HA was tested by use of a pull-down assay with the anti-Myc antibody coupled agarose beads. The images in panel c are representative of three independent experiments.

Article Snippet: Rabbit anti-KCa1.1 pAb (CN APC-151, 1:100) was from Alomone Labs.

Techniques: Pull Down Assay

Journal: Nature Microbiology

Article Title: Influenza virus uses mGluR2 as an endocytic receptor to enter cells

doi: 10.1038/s41564-024-01713-x

Figure Lengend Snippet: a , Effect of seven different membrane proteins that interact with KCa1.1 on influenza virus replication evaluated using an RNAi assay. b , mGluR2 knockdown reduced the internalization of H1N1 virus into A549 cells. c , mGluR2 knockdown reduced the internalization of H1N1 virus into A549 cells, as confirmed using a microscopy-based assay. Cell nucleus, blue; viral HA, green. d , H1N1 virus replication in A549 cells with different treatments. e , Interaction of mGluR2 and KCa1.1 confirmed by co-immunoprecipitation with agarose beads coupled with anti-Flag antibodies. f , Co-overexpression of KCa1.1 and mGluR2 increased H1N1 virus internalization. g , KCa1.1 did not internalize with H1N1 virus into A549 cells. Cell nucleus, blue; KCa1.1, green. h , mGluR2 internalized with H1N1 virus into A549 cells. Cell nucleus, blue; mGluR2, green. i , KCa1.1 knockdown prevented internalization of mGluR2 with influenza virus into A549 cells. Cell nucleus, blue; mGluR2, green. The microscopy-based assays shown in c , g , h and i were performed under unpermeabilized conditions. The solid line represents median and dashed lines represent quartiles of the data in the violin graphs of c and g – i . The images in c , e , g , h and i are representative of three independent experiments. Error bar in panels b , d and f indicates the standard deviation. The data shown in a – d and f – i are means ± s.d. ( n = 3 biologically independent experiments). Statistical analysis was performed using the unpaired, two-tailed Student’s t -test; NS, not significant. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. Exact P values are available in the Source Data.

Article Snippet: Rabbit anti-KCa1.1 pAb (CN APC-151, 1:100) was from Alomone Labs.

Techniques: Membrane, Virus, RNAi Assay, Knockdown, Microscopy, Immunoprecipitation, Over Expression, Standard Deviation, Two Tailed Test

Journal: Nature Microbiology

Article Title: Influenza virus uses mGluR2 as an endocytic receptor to enter cells

doi: 10.1038/s41564-024-01713-x

Figure Lengend Snippet: a , Adenovirus type 5 replication was significantly reduced in CLTC-knockdown A549 cells but not in mGluR2-knockdown A549 cells. Error bar in panels a indicates the standard deviation. The data shown in panels a is means ± s.d. (n = 3 biologically independent experiments). Statistical analysis was performed by using the unpaired, two-tailed Student’s t-test, ns, not significant, *** P < 0.001. Exact P values are available in Source Data. b , Formation of adenovirus type 5 CCPs was reduced in KCa1.1-knockdown or chlorpromazine-treated A549 cells but not in mGluR2-knockdown A549 cells. Green arrow, adenovirus type 5 CCPs; red arrow, attached adenovirus type 5. Scale bar, 200 nm. The images in b are representative of three independent experiments.

Article Snippet: Rabbit anti-KCa1.1 pAb (CN APC-151, 1:100) was from Alomone Labs.

Techniques: Knockdown, Standard Deviation, Two Tailed Test

Journal: Nature Microbiology

Article Title: Influenza virus uses mGluR2 as an endocytic receptor to enter cells

doi: 10.1038/s41564-024-01713-x

Figure Lengend Snippet: a , KCa1.1-knockdown, cyto D or paxilline treatment significantly reduced F-actin polymerization in H1N1-virus-internalized A549 cells. Cell nucleus, blue; phalloidin, green. b , Cyto D treatment or KCa1.1 knockdown prevented the influenza-virus-associated internalization of mGluR2 into A549 cells. Cell nucleus, blue; mGluR2, red. c , Cyto D or KCa1.1-knockdown treatment significantly reduced the internalization of H1N1 virus into A549 cells. d , KCa1.1 or mGluR2 knockdown and chlorpromazine treatment reduced the formation of CCPs of influenza virus in A549 cells. Green arrow, influenza virus CCP; red arrows, influenza virus attached. Scale bar, 200 nm. e , Model of the roles of mGluR2 and KCa1.1 in the CME of influenza virus. The microscopy-based assays shown in a and b were performed under unpermeabilized conditions. The solid line represents median and dashed lines represent quartiles of the data in the violin graphs of a and b . The images in a , b and d are representative of three independent experiments. Error bar in panels c indicates the standard deviation. The data shown in a – c are means ± s.d. ( n = 3 biologically independent experiments). Statistical analysis was performed using the unpaired, two-tailed Student’s t -test. NS, not significant. * P < 0.05; **** P < 0.0001. Exact P values are available in Source Data.

Article Snippet: Rabbit anti-KCa1.1 pAb (CN APC-151, 1:100) was from Alomone Labs.

Techniques: Knockdown, Virus, Microscopy, Standard Deviation, Two Tailed Test

Journal: Nature Microbiology

Article Title: Influenza virus uses mGluR2 as an endocytic receptor to enter cells

doi: 10.1038/s41564-024-01713-x

Figure Lengend Snippet: a and b , Replication of H5N6 ( a ) and H7N9 ( b ) viruses in KCa1.1-silenced or paxilline-treated A549 cells. c and d , Internalization of H5N6 ( c ) and H7N9 ( d ) viruses was significantly reduced in paxilline-treated or KCa1.1-knockdown A549 cells. Error bar in panels a – d indicates the standard deviation. The data shown in panels a – d are means ± s.d. (n = 3 biologically independent experiments). Statistical analysis was performed by using the unpaired, two-tailed Student’s t-test, * P < 0.05, ** P < 0.01, *** P < 0.001. Exact P values are available in Source Data.

Article Snippet: Rabbit anti-KCa1.1 pAb (CN APC-151, 1:100) was from Alomone Labs.

Techniques: Knockdown, Standard Deviation, Two Tailed Test