Journal: Journal of Virology

Article Title: A recurrent adaptive mutation in the transmembrane 2B protein of an insect picorna-like virus in a nonnative host

doi: 10.1128/jvi.01239-25

Figure Lengend Snippet: Sting colocalizes with the CrPV 2B protein. ( A ) Confocal microscopy images of Drosophila S2 cells transfected with a combination of the indicated plasmids. Cells were fixed 48 h after transfection and stained with antibodies against the V5 tag and the cis Golgi protein GM130. ( B ) Violin plot of Pearson’s correlation coefficient (R) of Sting with eGFP ( n = 27), 2B-eGFP ( n = 22), or 2B(D29N)-eGFP ( n = 22) in individual cells, calculated in Fiji using the Costes method. The median is represented with a solid black line and the quartiles with dashed lines. Adjusted P -values from Tukey’s multiple comparison test are indicated with asterisks: ** P < 0.01, *** P < 0.001. ( C ) Co-immunoprecipitation of Sting with 2B and 2B(D29N). S2 cells were transfected with the indicated plasmids, and the cell lysates (input) and eGFP immunoprecipitates (eGFP ImmP) were analyzed by western blot using eGFP, V5, and GAPDH antibodies. The white arrow heads indicate Sting-V5 (expected size 43.1 kDa), black arrow heads indicate 2B-eGFP and 2B(D29N)-eGFP (43.3 kDa), and green arrow heads indicate eGFP. As previously reported for human STING, Drosophila Sting showed distinct migrating bands, which might be due to post-translational modifications . M, size marker.

Article Snippet: Membranes were stained with antibodies against GFP (rat, 1:1000, ChromoTek, 3h9-150), V5 tag (mouse, 1:2000, Invitrogen, 46-0705), or GAPDH (rabbit, 1:1000, Bioss, bs-8778R), followed by secondary antibodies IRDye 800CW goat anti-rat IgG (LICORBio, 926-32219), IRDye 800CW goat anti-mouse IgG (LICORBio, 926-32210), and IRDye 800CW goat anti-rabbit IgG (LICORBio, 926-32211), respectively.

Techniques: Confocal Microscopy, Transfection, Staining, Comparison, Immunoprecipitation, Western Blot, Marker

Journal: PLOS Pathogens

Article Title: piR-bmo-796514 facilitates the proliferation of exogenous DNA virus (baculovirus) by targeting the host E3 ubiquitin ligase RNF181

doi: 10.1371/journal.ppat.1013848

Figure Lengend Snippet: (A) Sequence complementarity between piR-bmo-796514 and its target gene RNF181. The CDS of RNF181 was found to be complementary to the seed region (nucleotides 2-7) of piR-bmo-796514, with additional base-pairing occurring further downstream. (B) Analysis of the conserved domains showed that RNF181 possessed a typical RING finger conserved domain. (C) Detection of the transcriptional level of the RNF181 gene in BmN cells after transfection with piR-bmo-796514-mimics. (D) Detection of the transcriptional level of the RNF181 gene in BmN cells after transfection with piR-bmo-796514-inhibitor. (E-F) Dual-luciferase reporter gene assays to test the regulatory effect of piR-bmo-796514 on RNF181 using piRNA-796514 mimics and inhibitor. (G) Quantitative detection of RNF181 transcriptional levels in silkworm fat bodies after BmNPV infection. (H) Quantitative detection of RNF181 transcriptional levels in silkworm fat bodies after injection with piR-bmo-796514-antagomir and subsequent BmNPV infection. (I) Quantitative detection of RNF181 transcriptional levels in BmN cells after BmNPV infection. (J) Quantitative detection of RNF181 transcriptional levels in BmN cells after transfection with piR-bmo-796514-inhibitor and subsequent BmNPV infection. (K) Detection of silencing of RNF181 after transfection with dsRNA-RNF181 in BmN cells. (L) Detection of the transcriptional level of the viral vp39 gene in BmN cells after transfection with dsRNA-RNF181 and subsequent BmNPV infection. (M) Detection of the DNA load of the viral gp41 gene in BmN cells after transfection with dsRNA-RNF181 and subsequent BmNPV infection. (N) Western blot detection of RNF181 expression after transfection of BmN cells with pIEX-RNF181-V5. (O) Detection of the transcriptional level of the viral vp39 gene in BmN cells after overexpression of RNF181 and subsequent BmNPV infection. (P) Detection of the DNA load of the viral gp41 gene in BmN cells after overexpression of RNF181 and subsequent BmNPV infection. p -value <0.05 was considered as statistically significant. “NC” (“negative control”) refers to the control group that has not been infected with the virus. “NC-mimics”, “NC-inhibitor”, and “NC-antagomir” denote the negative control groups for the corresponding piRNA mimics, inhibitors, and antagomirs.

Article Snippet: The expression of RNF181-V5 and Integrin α2b-like-His proteins was detected by Western blotting using mouse anti-V5 antibody (Thermofisher, USA) and mouse anti-His (Proteintech, China). α-Tubulin was also detected as an internal reference.

Techniques: Sequencing, Transfection, Luciferase, Infection, Injection, Western Blot, Expressing, Over Expression, Negative Control, Control, Virus