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. 2021 Jun 11;10:e67321. doi: 10.7554/eLife.67321

Figure 1. Partial knockdown of IRSp53 decreases HIV-1 Gag particle release by arresting assembly at the cell plasma membrane.

(a) siRNA knockdown of IRSp53 expression in Jurkat T lymphocytes leads to a significant decrease in pNL4-3ΔEnv Gag particle release (see graph and immunoblots for IRSp53, p = 0.00265, Student’s t-test, and loading controls beneath the graph). (b) Similarly, knockdown of IRSp53 expression in Gag expressing HEK293T cells led to a significant decrease in HIV-1 Gag particle release (p = 0.00487, Student’s t-test), as compared to siRNA IRTKS (p = 0.0116, Student’s t-test). On the other hand, knockdown of IRTKS expression (a closely related I-BAR protein) did not have a significant effect on particle release (p = 0.0924, Student’s t-test, upper graph, immunoblots for IRSp53, IRTKS, and loading controls beneath the graph) (n = 3 independent experiments). Another multiple comparisons statistical test ANOVA was applied to compare the three siRNA conditions showing a significative difference with a p value = 0.0089. (c) Transmission electron microscopy images of HEK293T cells expressing HIV-1 Gag with siRNA control (upper panel) and siRNA IRSp53 (lower panel). Scale bar is 0.5 µm (upper image) and is 1 µm (lower image). (d) Transmission electron microscopy zoomed images of viral buds from HIV-1 Gag expressing cells treated with siRNA-mediated knocked down of IRSp53 (lower panel) showing arrested buds at the plasma membrane as compared to the siRNA control cells (upper panel) which display a normal range of buds in different stages of assembly and budding (scale bar = 100 nm). (e) Measurement of the bud dimensions (height and width median with interquartile) in the control siRNA and siRNA IRSp53 conditions (n = 145 buds from 14 different cells for each condition, n = 2 independent experiments). The knocked down cells exhibit a narrow range of heights corresponding to the arrested buds visible in the images, while the control cells display a wider range of heights corresponding to assembly progression (left graph, ‘Height of bud’). Distribution of the height values in the two conditions is significantly different (p = 1.05 × 10−28, Kolmogorov-Smirnov test). On the opposite, the widths of the buds in both conditions did not display significant differences in distributions (p = 0.0609, Kolmogorov-Smirnov test).

Figure 1—source data 1. Immunoblot Quantification using Fiji for Figure 1B graph.
Figure 1—source data 2. Heights and Widths of HIV-1 Gag bud measurements for Figure 1E graph.

Figure 1.

Figure 1—figure supplement 1. Effect of siRNA-based knockdown of IRSp53 and IRTKS gene expression on HIV-1 Gag particle release.

Figure 1—figure supplement 1.

(a) Expression of HIV-1 Gag in HEK293T cells (from molecular clones pNL43ΔPolΔEnv or pGag – optimized HIV-1 Gag) and a schematic representation of the viral Gag polyprotein. The HIV-1 Gag Pr55 polyprotein is known to have four main domains, the matrix MA interacting with cell membrane thanks to its lipid-binding site and myristate (myr), the capsid CA for Gag-Gag oligomerization, the nucleocapsid NC interacting with the viral genomic RNA, and p6 recruiting Tsg101 (ESCRT protein) for particle budding. (b) Validation of siRNA targeting IRSp53 gene expression. siRNA targeting IRSp53 diminishes the expression of IRSp53-GFP as seen by immunoblots (left panel, lane 1: siCTRL; lane 2: siIRSp53) and fluorescence imaging of transfected HEK293T cells expressing ectopic IRSp53-GFP with the corresponding siRNA (right panel). (c) Validation of siRNA targeting ectopic IRTKS. siRNA targeting IRTKS diminishes expression of IRTKS-GFP as seen by immunoblots (left panel, lane 1: siCTRL; lane 3: siIRTKS) and fluorescence imaging of transfected HEK293T cells expressing IRTKS-GFP with siRNA, as indicated (right panel). (d) Reduced viral release is seen in cells knockdown for endogenous IRSp53 expression (representative immunoblots, lane 1: siCTRL; lane 2: siIRSp53). (e) Endogenous IRTKS knockdown has no significant effect on HIV-1 Gag particle release (representative immunoblots, lane 1: siCTRL; lane 3: siIRTKS). Arrows (<) show the corresponding proteins of interest.
Figure 1—figure supplement 2. Protein sequence comparison of IRSp53 and IRTKS.

Figure 1—figure supplement 2.

(a) IRSp53 and IRTKS are membrane curving I-BAR proteins. (b) IRSp53 (Query, accession Q9QB8) shares a 40% sequence homology with IRTKS (Subject, accession Q9UHR4) sequence alignment performed with NCBI Protein BLAST. Most of this homology is centered in the I-BAR/IMD domain of the three proteins and the C-terminal SRC homology 3 (SH3) domain common to the two proteins.
Figure 1—figure supplement 3. Transmission electron microscopy of siRNA treated HEK293T cells expressing HIV-1 Gag.

Figure 1—figure supplement 3.

siRNA control (a) or siRNA IRSp53 (b) treated cells reveal that IRSp53 protein depletion in cells display HIV-1 Gag arrested assembly at the cell plasma membrane as shown by transmission electron microscopy. Scale bar is 0.5 µm or 1 µm, as indicated in the images.