Journal: Cell host & microbe

Article Title: Modular HIV-1 Capsid Assemblies Reveal Diverse Host-Capsid Recognition Mechanisms

doi: 10.1016/j.chom.2019.07.007

Figure Lengend Snippet: (A) Schematics of several reported capsid oligomers. (B) Schematics of host capsid-binding proteins with their potential capsid-binding regions in red. (C-E). Capsid-host factor co-pelleting assays using A14C/E45C disulfide crosslinked CA tubes (top) and SDS-PAGE quantification of the reduction of protein in the soluble fraction (bottom). Error bars represent standard error of the mean of three independent experiments. (F-H) Size-exclusion chromatography co-elution assays between host factors and disulfide crosslinked hexamers (top) or CA monomers/dimers (bottom). A shift of the elution volume (red relative to others) indicates co-elution. (C) A soluble construct of TRIMCyp, BCCCyp, co-pellets with CA tubes, but not those containing P90A mutation. (F) BCCCyp co-elutes stably with CA hexamers, but marginally with individual CA. (D) An MxB truncation containing residues 1–83 co-pellets with CA tubes, but the MxB 11RRR13 to 11AAA13 mutation eliminates co-pelleting. (G) MxB1–83 does not co-elute with CA nor hexamers. (E) BCCSPRY or PCNA-SPRY constructs containing 2 or 3 SPRY domains of TRIM5α, respectively, co-pellet with CA tubes. (H) BCCSPRY does not co-elute with hexamers nor CA. See also Figure S1.

Article Snippet: All purification steps were monitored by SDS-PAGE gel electrophoresis (gels purchased from ConnSTEM and Invitrogen).

Techniques: Binding Assay, SDS Page, Size-exclusion Chromatography, Co-Elution Assay, Construct, Mutagenesis, Stable Transfection

Journal: Cell host & microbe

Article Title: Modular HIV-1 Capsid Assemblies Reveal Diverse Host-Capsid Recognition Mechanisms

doi: 10.1016/j.chom.2019.07.007

Figure Lengend Snippet: (A) Top: schematics of 1/3-hexamer and 1/2-hexamer design. Two types of disulfide-bonds (S-S) are colored (A14C/E45C in blue, A42C/T54E in red). Bottom: SDS-PAGE demonstrating formation of 1/3- and 1/2-hexamers. On the left, CA samples run in reducing conditions show ~25 kDa monomers. On the right, CA samples run in non-reducing conditions reveal dimer, trimer, and hexamer species as designed. (B) Size-exclusion chromatography (SEC) demonstrates well-behaved partial-hexamer assemblies (marked with schematics). (C) Orthogonal views of the crystal structures (ribbon) of 1/3-hexamerEE (top) and 1/2-hexamerEE-ΔCTD (bottom) in the hexamer envelope (grey surface, PDB ID: 3H47). The structures are highly homologous to the corresponding portions in known hexamer crystal structures. See also Figure S2, Table S1, and Table S2.

Article Snippet: All purification steps were monitored by SDS-PAGE gel electrophoresis (gels purchased from ConnSTEM and Invitrogen).

Techniques: SDS Page, Size-exclusion Chromatography

Journal: Cell host & microbe

Article Title: Modular HIV-1 Capsid Assemblies Reveal Diverse Host-Capsid Recognition Mechanisms

doi: 10.1016/j.chom.2019.07.007

Figure Lengend Snippet: (A)-(D) BCCCyp efficiently binds partial hexamers containing at least two wild-type (WT) binding sites with residue P90 (green circles). SEC co-elution is indicated by a shift of the elution volume (red relative to the others), with a corresponding shift of the elution fraction bands in SDS-PAGE (bottom). BCCCyp co-elutes with 1/3-hexamerEE (A) and 1/2-hexamerEE containing two WT binding sites (B). (C) Co-elution is abrogated for 1/2-hexamerEE with three or two P90A mutations (red crosses). (D) Restoration of a hexamer from two 1/2-hexamers (cyan and pink cartoons) each containing a single P90 site restores BCCCyp binding. (E) Schematic models of the two CypA domains on TRIMCyp (cartoon) bind to any two CA subunits within a hexamer (surface). See also Figure S3.

Article Snippet: All purification steps were monitored by SDS-PAGE gel electrophoresis (gels purchased from ConnSTEM and Invitrogen).

Techniques: Binding Assay, Co-Elution Assay, SDS Page

Journal: Cell host & microbe

Article Title: Modular HIV-1 Capsid Assemblies Reveal Diverse Host-Capsid Recognition Mechanisms

doi: 10.1016/j.chom.2019.07.007

Figure Lengend Snippet: (A) Left: Schematic of the six CA monomers (purple) centered at the interface between three CA hexamers. Middle: Design of hexamer-2 (purple cartoon) as a trimer of 14C/45C (yellow spheres) disulfide-crosslinked 1/3-hexamers. The 42E/54E solubilizing mutations are shown as cyan spheres. Right: Side view of hexamer-2foldon design highlighting the fused trimeric foldon domain at the C-terminus of CA. (B) SDS-PAGE (left) and SEC (right) analysis of purified 1/3-hexamer and hexamer-2foldon assemblies. In non-reducing SDS-PAGE analysis 1/3-hexamers and hexamer-2foldon assemblies run as ~50 kDa dimers. In reducing conditions, they run as ~25 kDa monomers. (C) Negative-stain EM micrograph (top), 2D class averages (middle), and 3D reconstruction (bottom) of hexamer-2foldon. (D) Crystal structure of hexamer-2foldon with the 2Fo-Fc electron density as grey surface (1□ level). (E) Superposition of the hexamer-2foldon crystal structure with the corresponding regions in native (PDB ID: 4XFX) and cross-linked (PDB ID: 3H47) CA crystal structures. Most differences between the structures are due to flexibility at the NTD-CTD hinge that connects the two CA domains. See also Figure S4 and Table S3.

Article Snippet: All purification steps were monitored by SDS-PAGE gel electrophoresis (gels purchased from ConnSTEM and Invitrogen).

Techniques: SDS Page, Purification, Staining

Journal: Cell host & microbe

Article Title: Modular HIV-1 Capsid Assemblies Reveal Diverse Host-Capsid Recognition Mechanisms

doi: 10.1016/j.chom.2019.07.007

Figure Lengend Snippet: (A) A schematic of di-hexamer assembly (left) and SDS PAGE analysis of the CASpyCat-CASpyTag reaction (right). The SpyTag/SpyCatcher reaction proceeded essentially to completion. (B) Hexamer-SpyCatcher/Tag assembly and purification. CA and CA-Spy fusions are mixed at an appropriate ratio to assemble hexamers (top), purified by anion-exchange chromatography (middle), and analyzed by non-reducing SDS-PAGE (bottom). Hexameric species containing specific amounts of CA-SpyTag/Catcher fusion molecules were efficiently separated. (C) Schematics of producing Tri-hexamer (top) and Hepta-hexamer (bottom right) assemblies. (D) Schematics of pentamer-hexamer assemblies. (E-H) SEC (SEC-MALS in (E)), non-reducing SDS-PAGE, and negative-stain EM analysis of assembled Di-hexamers (E), Tri-hexamers (F), Hepta-hexamers (G), and Pentamer-hexamers (H). Sample micrographs are shown, as indicated, with associated 2-D class averages. In (E) and (F), the 2-D class averages of constructs containing 184A/185A mutant interfaces show non-contacting hexamer subunits. See also Figure S5.

Article Snippet: All purification steps were monitored by SDS-PAGE gel electrophoresis (gels purchased from ConnSTEM and Invitrogen).

Techniques: SDS Page, Purification, Chromatography, Staining, Construct, Mutagenesis

Journal: Cell host & microbe

Article Title: Modular HIV-1 Capsid Assemblies Reveal Diverse Host-Capsid Recognition Mechanisms

doi: 10.1016/j.chom.2019.07.007

Figure Lengend Snippet: (A) MxB1–83 (left), but not the 11AAA13 mutant (right), co-elutes with hexamer-2foldon (red) in SEC (top) and SDS-PAGE (bottom) analyses. (B) MxB1–83 does not co-elute with di-hexamers in SEC. (C) MxB1–83 binds to hexamer-2foldon with a 9.6 ± 1.1 μM Kd by ITC. (D) Model of full-length MxB (cartoon) wedging its unstructured N-termini into two disparate three-fold inter-hexamer interfaces (surface). (E) A PCNA trimer fused to the SPRY domain of rhesus TRIM5α co-elutes with hepta-hexamers (red) in SEC. (F) BCCCyp shows marginal binding to a di-hexamer containing only one wild-type P90 site (left), but significant binding to a di-hexamer with two wild-type P90 sites on adjacent hexamers (right). P90 is indicated with green circles and P90A as red crosses. (G) A model of flexible TRIMCyp (cartoon) binding between hexamers (surface), and within hexamers (Figure 2). See also Figure S6.

Article Snippet: All purification steps were monitored by SDS-PAGE gel electrophoresis (gels purchased from ConnSTEM and Invitrogen).

Techniques: Mutagenesis, SDS Page, Binding Assay