Journal: Journal of Virology

Article Title: Tetraspanin CD81 serves as a functional entry factor for porcine circovirus type 2 infection

doi: 10.1128/jvi.01408-24

Figure Lengend Snippet: CD81 promotes PCV2 replication. ( A ) pCAGGS-CD81-Flag recombinant plasmids were transfected into PK-15 cells, followed by inoculation with 0.1 MOI PCV2. After 24 h of infection (hpi), Western blot was conducted to detect the expression levels of corresponding proteins. The protein bands were quantified using NIH ImageJ software. ( B ) TCID 50 assay was performed to determine the influence of overexpressing CD81 on the progeny of PCV2. ( C ) Indirect IFA was conducted to assess the infection status of PCV2 on PK-15 cells after overexpression of CD81. Scale bar, 50 µm. Quantification of CoraLite488 and DAPI (4',6-diamidino-2-phenylindole) fluorescence-positive cells was performed using the ImageJ plugin, respectively. The proportion of PCV2-positive cells relative to the total number of cells within the field of view was statistically analyzed. The vector in A–C is the pCAGGS control plasmid. ( D ) Western blot was conducted to detect the interference effect of CD81 siRNA. siNC (siRNA negative control) is the negative-control interference fragments. ( E–G ) RNA interference. PK-15 cells transfected with CD81 siRNA were infected with 0.1 MOI PCV2 for 36 h. Western blot was conducted to detect the expression levels of PCV2 Cap and endogenous CD81 ( F ). The protein bands were quantified using ImageJ software. TCID 50 assay was conducted to determine the influence of silencing CD81 gene on the progeny PCV2 ( E ). IFA was performed to evaluate the infection of PCV2 on PK-15 cells after silencing CD81 gene. Also, quantification of CoraLite488 and DAPI fluorescence-positive cells was performed using the ImageJ plugin, respectively. The proportion of PCV2-positive cells was statistically analyzed. ( G ). Scale bar, 50 µm. Data represent the mean ± SD of three independent replicate experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: Using the shRNA online design tool ( https://rnaidesigner.thermofisher.com ), shRNAs targeting porcine CD81 and RhoA genes were designed and the sequences were submitted to Nanjing GenScript Company for synthesis.

Techniques: Recombinant, Transfection, Infection, Western Blot, Expressing, Software, Over Expression, Fluorescence, Plasmid Preparation, Control, Negative Control

Journal: Journal of Virology

Article Title: Tetraspanin CD81 serves as a functional entry factor for porcine circovirus type 2 infection

doi: 10.1128/jvi.01408-24

Figure Lengend Snippet: CD81 is involved in PCV2 internalization into PK-15 cells. ( A–C ) CD81 knockdown (shCD81) and negative-control shRNA (shNC) PK-15 cells were infected with 0.1 MOI PCV2. After 36 h, RNA and protein samples were collected. qPCR was performed to detect the expression levels of CD81 mRNA ( A ) and PCV2 Cap mRNA ( B ). Western blot was conducted to detect the expression levels of PCV2 Cap protein and endogenous CD81. The protein bands were quantified using ImageJ software ( C ). TCID 50 assay was used to determine the viral titer of PCV2 ( D ). ( E ) The virus adsorption assay. CD81 knockdown and shNC PK-15 cells were inoculated with 5 MOI PCV2 and incubated for 1 h at 4°C. DNA samples were extracted, and qPCR was performed to detect the relative content of PCV2, with mtDNA (Mitochondrial DNA) as an internal control. The y-axis represents the fold change in viral DNA content between the experimental and the control groups, calculated using the 2 -ΔΔCT formula based on the CT values of the target gene Cap and the reference gene mtDNA (Mitochondrial DNA). ( F ) The virus internalization assay. CD81 knockdown and shNC PK-15 cells were inoculated with 5 MOI PCV2 and incubated for 1 h at 4°C, and then transferred to 37°C for an additional 2 h. DNA samples were extracted, and qPCR was performed to detect the relative content of PCV2, with mtDNA as an internal control. The y-axis represents the fold change in viral DNA content between the experimental and control groups, calculated using the 2 -△△CT formula based on the CT values of the target gene Cap and the reference gene mtDNA. Data represent the mean ± SD of three independent replicate experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: Using the shRNA online design tool ( https://rnaidesigner.thermofisher.com ), shRNAs targeting porcine CD81 and RhoA genes were designed and the sequences were submitted to Nanjing GenScript Company for synthesis.

Techniques: Knockdown, Negative Control, shRNA, Infection, Expressing, Western Blot, Software, Virus, Adsorption, Incubation, Control

Journal: Journal of Virology

Article Title: Tetraspanin CD81 serves as a functional entry factor for porcine circovirus type 2 infection

doi: 10.1128/jvi.01408-24

Figure Lengend Snippet: Syndecan-1 collaborates with CD81 to facilitate PCV2 infection. ( A ) The interaction between CD81 and Syndecan-1 was detected with Co-IP. ( B ) The interaction between PCV2 Cap protein and Syndecan-1 was detected with Co-IP. ( C ) The endogenous CD81 and Syndecan-1 were detected in PK-15 cells with Co-IP. PK-15 cells were infected with PCV2 for 36 h, lysed, and immunoprecipitated with anti-Syndecan-1 antibody. The whole-cell lysates and immunoprecipitated products were analyzed by Western blot. ( D ) Immunofluorescence confocal microscopy was used to detect co-localization of CD81 and Syndecan-1 at the cell membrane. Scale bar, 5 µm. ( E ) Western blot was conducted to detect the interference effect of three siRNAs targeting Syndecan-1. ( F–J ) PK-15 cells transfected with the siRNA targeting CD81 and Syndecan-1 were infected with 0.1 MOI PCV2 or 5 MOI PCV2. Western blot was performed to detect the expression levels of PCV2 Cap protein, and the protein bands were quantified using ImageJ software ( F ). qPCR was performed to detect the expression levels of CD81 mRNA ( G ), Syndecan-1 mRNA ( H ), PCV2 Cap mRNA ( I ), and the relative content of adsorbed PCV2, with mtDNA (Mitochondrial DNA) as an internal control ( J ). The y-axis represents the fold change in viral DNA content between the experimental and control groups, calculated using the 2 -ΔΔCT formula based on the CT values of the target gene Cap and the reference gene mtDNA (Mitochondrial DNA). Data represent the mean ± SD of three independent experiments. *, P < 0.05, **, P < 0.01, ***, P < 0.001.

Article Snippet: Using the shRNA online design tool ( https://rnaidesigner.thermofisher.com ), shRNAs targeting porcine CD81 and RhoA genes were designed and the sequences were submitted to Nanjing GenScript Company for synthesis.

Techniques: Infection, Co-Immunoprecipitation Assay, Immunoprecipitation, Western Blot, Immunofluorescence, Confocal Microscopy, Membrane, Transfection, Expressing, Software, Control