Reconstitution of selective HIV-1 RNA packaging in vitro by membrane-bound Gag assemblies
- University of California, Berkeley, United States
- Howard Hughes Medical Institute, United States
- University of Maryland Baltimore County, United States
- Howard Hughes Medical Institute, University of California, Berkeley, United States
- Lawrence Berkeley National Laboratory, United States
Figures
Figure 1 with 1 supplement
In vitro reconstitution of selective RNA packaging by HIV-1 Gag.
(a) Confocal fluorescence micrograph of GUVs containing 5% PI(4,5)P2. 100 nM HIV-1 Gag-ATTO594 and 0.5 nM HIV-1 5’UTR-Alexa488 were premixed and added to the exterior of the GUVs which were imaged 10 min later. Upper panel, membrane in red and Gag in white/cyan. Lower panel, RNA. (b–c) As (a) but with fluorescent HIV-1 RRE and a 378 nt control ssRNA (RNA378), respectively. (d–f) As (a–c) but with 5 nM non-fluorescent RNA378 added together with the fluorescent RNAs. (a–f) White arrows mark Gag clusters on GUVs, and their position on the corresponding RNA images. Scale bar for all images, 10 µm. (g) Quantitation of fluorescent RNA binding to Gag clusters. Gag clusters on membranes were identified in 10 z-stacks each containing ~10 GUVs, using an unsupervised script which calculated the average RNA fluorescence within the clusters. Gag clusters with an average RNA fluorescence >2.0 times that of the surrounding membrane were counted as positive for colocalization. All measurements were conducted on the same batch of GUVs and error bars indicate standard deviation between three repeats on separate GUV preparations. n.s./*, not significant, and significant, respectively, at p<0.05 level by Student's t-test.
Figure 1—figure supplement 1
RNA recruitment by clusters of Gag with altered NC domain.
(a) Confocal micrograph of GUV, 10 min after adding 100 nM ΔNC Gag-ATTO594 and 0.5 nM 5’UTR -Alexa488. Upper panel, membrane in red and Gag in white/cyan. Lower panel, RNA. (b) As (a), but with full-length Gag treated for 15 min at 37°C with 100 µM of the Zn-finger disrupting compound AT-2. (c) As (b), but Gag treated with DMSO only as negative control for (b). (a–c) White arrows mark Gag clusters on GUVs, and their position on the corresponding RNA images. Scale bar for all images, 10 µm.
Figure 2
Effect of 5’UTR alterations on packaging by HIV-1 Gag.
(a) Schematic of the HIV-1 5’UTR indicating introduced mutations. Blue indicates the position of the GCGCGC to GAGA mutation in the dimerization motif creating the monomerized construct '5’UTR mono'. Red indicates the position of the splice donor site G290. Replacement of nucleotides after G290 with the sequence from a spliced RNA created the construct ‘5’UTR env-1’. Cyan indicates the position of packaging-critical unpaired and weakly paired guanines mutated to adenines in the construct '5’UTR 3way1'. PBS, primer binding site. DIS, dimerization signal. SD, splice donor site. ψ, psi stem loop. The schematic shows one monomer of the dimerizing 5’UTR. (b–f) Confocal micrographs of GUVs, 10 min after adding 100 nM Gag-ATTO594, 0.5 nM 5’UTR -Alexa488, and 5 nM non-fluorescent competitor RNA as indicated. Upper panels, membrane in red and Gag in white/cyan. Lower panel, RNA. White arrows mark Gag clusters on GUVs, and their position on the corresponding RNA images. Scale bar for all images, 10 µm. (g) Quantitation of fluorescent RNA binding to Gag clusters, performed as for Figure 1g but using a lower threshold of 1.25 for counting Gag clusters as positive for fluorescent RNA. n.s./*, not significant, and significant, respectively, at p<0.05 level by Student's t-test.
Figure 3
CA domain mutants affect Gag clustering on membranes.
(a) Schematic of a hexagonal lattice showing the Gag CA domain at the two-, three- and six fold symmetry contacts, as arranged in the immature Gag lattice in HIV-1 assembly sites and immature virions (Schur et al., 2015) (PDB entry 4USN used in panel a–d). (b) Model of the CA domain at the two fold, with mutated residues in “Gag two-fold” marked in red. (c–d) As (b), for Gag mutated at the three- and six-fold symmetry contacts, respectively. (e) Confocal micrograph of GUVs, 10 min after adding 100 nM Gag-ATTO594. Membrane in red and Gag in white/cyan. (f) As (e), using Gag-ATTO594 two-fold. GUVs with diffuse Gag binding are denoted with white asterisks. (e–f) White arrows mark Gag clusters on GUVs. Scale bar, 10 µm. (g) Type of Gag binding to GUVs. For each protein, GUVs were counted in 10 confocal z-stacks, and classified according to having no Gag fluorescence, only clustered Gag fluorescence (black), or a diffuse Gag fluorescence covering the entire membrane (with or without additional brighter clusters, gray). All measurements were conducted on the same preparation of GUVs, with error bars indicating the standard deviation between three repeats conducted on separate GUV preparations. *significant at p<0.05 level by Student's t-test.
Figure 4 with 1 supplement
RNA selectivity of CA domain mutants.
(a) Confocal micrograph of GUV, 10 min after adding 100 nM Gag-ATTO594 and 0.5 nM 5’UTR -Alexa488. Upper panel, membrane in red and Gag in white/cyan. Lower panel, RNA. (b–e) As (a) using Gag two-fold, Gag three-fold, Gag six-fold and Gag two-, three-, six-fold, respectively. (f–j) As (a–j) with the addition of 5 nM non-fluorescent RNA378. (a–j) White arrows mark Gag clusters on GUVs, and their position on the corresponding RNA images. Scale bar for all images, 10 µm. (k) Quantitation of RNA binding to Gag clusters, performed as for Figure 1g. n.s./*, not significant, and significant, respectively, at p<0.05 level by Student’s t-test.
Figure 4—figure supplement 1
Type of Gag binding to GUVs in the presence of RNA.
Statistics of Gag membrane binding was performed as for Figure 3g, using the data presented in Figure 4f–j (ten z-stacks per condition, in three independent repeats).
Figure 5 with 2 supplements
Probing Gag-induced structural changes on the HIV 5’UTR using SHAPE.
(a) Secondary structure of the HIV 5’UTR RNA with SHAPE reactivities labeled. The secondary structure is based on the reported NMR structure (Keane et al., 2015) (PDB 2N1Q). Nucleotides exhibiting high, medium, low, or no SHAPE reactivities are labeled in red, orange, cyan, and black, respectively. SHAPE handles are labeled by gray shadows. The 3D structure colored based on the SHAPE profile using the same color scheme is shown as an insert. U5, U5 region of long terminal repeat. PBS, primer binding site. DIS, dimerization signal. SD, splice donor site. ψ, psi stem loop. AUG, start codon of gag gene. The nucleotides of SD that would be in a loop in the alternative conformation (289–292) are underlined with a gray box. The schematic shows one monomer of the dimerizing 5’UTR. (b) SHAPE profiles of the 5’UTR either alone (in gray) or in complex with the following Gag variants: the NC domain of Gag (in cyan), ΔMA-Gag (in blue), and Gag assembled on GUVs (in red). The regions not showing significant SHAPE changes are masked. (c) SHAPE changes between the 5’UTR alone and the 5’UTR bound by the Gag on GUV. Nucleotides exhibiting reduced or increased SHAPE value upon complex formation are labeled in cyan and dark red, respectively. Corresponding nucleotide are also labeled on the NMR 3D structure model using the same color scheme.
Figure 5—figure supplement 1
SHAPE changes for protein addition in solution and a longer 5’UTR RNA.
(a) Changes in SHAPE reactivity are plotted as in Figure 4c, for addition of ΔMA-Gag and NC, respectively, and between a longer 356 nt 5’UTR and the 345 nt 5’UTR used throughout the study. (b) Secondary structure of the 356-nt HIV 5’UTR RNA with SHAPE reactivities labeled as in Figure 4a.
Figure 5—figure supplement 2
Gag binding to GUVs under conditions of the SHAPE analysis.
Gag and GUVs were prepared as for SHAPE analysis. In the final step DMSO without SHAPE reagent was added and GUVs were imaged by confocal microscopy. A representative field of view is shown.
Figure 6
Effect of tRNA and viral RNA on Gag membrane association.
(a) Confocal micrograph of GUVs, 10 min after adding 100 nM Gag-ATTO594. (b) Confocal micrograph of GUVs, 10 min after adding 100 nM Gag-ATTO594, premixed with 1 µM tRNA. (a–b) Membrane in red and Gag in white/cyan. White arrows mark Gag clusters on GUVs. Scale bar, 10 µm. (c) Amount of Gag fluorescence in the vesicle fraction after a vesicle flotation assay with 100 nM Gag-ATTO488, tRNA at 0–20 µm, and 0.4 mg/mg 1.0 µm LUVs. (d) Amount of Gag fluorescence in the vesicle fraction after a vesicle flotation assay with 100 nM Gag-ATTO488 and RNAs at 5 nM. (e) Amount of Gag fluorescence in the vesicle fraction after a vesicle flotation assay with 100 nM Gag-ATTO488, 20 µm tRNA, and other RNAs at 5 nM. (c–e) All measurements shown in the same panel were conducted on the same LUV preparation, with error bars indicating the standard deviation between three repeats conducted on separate LUV preparations. n.s./*, not significant, and significant, respectively, at p<0.05 level by Student's t-test.
Figure 7
A model for selective HIV-1 genome packaging.
Cytosolic Gag (light gray triangles) is inhibited from membrane association by its association with tRNA (yellow). Gag (triangles and hexagons, colored dark gray where forming specific contacts with the packaging signal) binding viral genomic RNA (red; thick portions correspond to the 5’UTR, thin portions to the remainder, not shown to scale) has a higher probability to overcome this inhibition and associate with the plasma membrane. The selectivity of membrane-bound Gag for the viral genome necessitates CA-CA immature lattice contacts.
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Reconstitution of selective HIV-1 RNA packaging in vitro by membrane-bound Gag assemblies
eLife 5:e14663.
https://doi.org/10.7554/eLife.14663
