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, 16 (1), e1008277
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Structures of Immature EIAV Gag Lattices Reveal a Conserved Role for IP6 in Lentivirus Assembly

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Structures of Immature EIAV Gag Lattices Reveal a Conserved Role for IP6 in Lentivirus Assembly

Robert A Dick et al. PLoS Pathog.

Abstract

Retrovirus assembly is driven by the multidomain structural protein Gag. Interactions between the capsid domains (CA) of Gag result in Gag multimerization, leading to an immature virus particle that is formed by a protein lattice based on dimeric, trimeric, and hexameric protein contacts. Among retroviruses the inter- and intra-hexamer contacts differ, especially in the N-terminal sub-domain of CA (CANTD). For HIV-1 the cellular molecule inositol hexakisphosphate (IP6) interacts with and stabilizes the immature hexamer, and is required for production of infectious virus particles. We have used in vitro assembly, cryo-electron tomography and subtomogram averaging, atomistic molecular dynamics simulations and mutational analyses to study the HIV-related lentivirus equine infectious anemia virus (EIAV). In particular, we sought to understand the structural conservation of the immature lentivirus lattice and the role of IP6 in EIAV assembly. Similar to HIV-1, IP6 strongly promoted in vitro assembly of EIAV Gag proteins into virus-like particles (VLPs), which took three morphologically highly distinct forms: narrow tubes, wide tubes, and spheres. Structural characterization of these VLPs to sub-4Å resolution unexpectedly showed that all three morphologies are based on an immature lattice with preserved key structural components, highlighting the structural versatility of CA to form immature assemblies. A direct comparison between EIAV and HIV revealed that both lentiviruses maintain similar immature interfaces, which are established by both conserved and non-conserved residues. In both EIAV and HIV-1, IP6 regulates immature assembly via conserved lysine residues within the CACTD and SP. Lastly, we demonstrate that IP6 stimulates in vitro assembly of immature particles of several other retroviruses in the lentivirus genus, suggesting a conserved role for IP6 in lentiviral assembly.

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Effect of IP5 and IP6 on in vitro assembly of EIAV Gag, EIAV GagΔMA, and HIV GagΔMA.
(A,C,E) Representative low and high magnification images of respective proteins assembled in the absence (red) or presence of 10 μM IP5 (pink) or 10 μM IP6 (blue) at pH 6. Examples of wide tubes (T:W), narrow tubes (T:N), and spheres (S) are indicated by green and purple triangles. (B,D,F). The number of VLPs (spheres-purple, tubes-green) per 55μm2 for no fewer than five representative images for each condition. Center lines show the medians; box limits indicate the 25th and 75th percentiles as determined by R software; whiskers extend to minimum and maximum values; data points are plotted as circles. Please note the different Y-axis ranges for the bar chart plots in B, D and F. The low magnification images are representative of the distribution of spheres and tubes assembled under different conditions, while the high magnification images were selected to illustrate their morphology. The mean value of counted particles is given in italics in the bar charts.
Fig 2
Fig 2. Cryo-electron microscopy of EIAV GagΔMA assemblies.
(A) Sum of 10 computational slices through gaussian-filtered tomograms containing EIAV GagΔMA tubes and spheres assembled at pH6 and pH8. Protein density is black. Scale bar is 50nm; note the significantly smaller diameter of tubes assembled at pH6. (B) Isosurface representations of the subtomogram averages derived from the different EIAV GagΔMA assemblies at pH6 and pH8. In all cases, the CANTD and CACTD-SP are colored cyan and orange, respectively. The IP6 density is colored in pink. The symmetry-independent copies of CA-SP are denoted with number 1,2,3 and 1’,2’,3’ for the tubes at pH6 and pH8, respectively. For the spheres, only one monomer is colored as all monomers in the hexamer are symmetry-related. In all structures, the helical pitch and densities for larger and several smaller side chains are visible in the EM-density, in good correspondence with the observed resolutions.
Fig 3
Fig 3. Structural variability of immature EIAV GagΔMA assemblies.
(A) Superimposition of the seven conformations adopted by CA in spheres and tubes. All monomers are aligned on the CACTD. For the monomer in the spheres the CANTD and CACTD-SP are colored in cyan and orange, respectively. For the tubular assemblies the symmetry-independent copies (1,2,3 and 1’,2’,3’ for tubes assembled at pH6 and pH8, respectively) are colored pale cyan, aquamarine and teal for the CANTD and light orange, bright orange and orange for the CACTD. The color coding is identical for all panels. (B) The refined models of the different EIAV GagΔMA assemblies are shown as seen from the outside of the virus particle, centered above the CA-SP hexamer. The top panel shows only the CANTD assembly, the bottom panel only the CACTD-SP assembly. The numbering for the symmetry independent copies is annotated (as described in Fig 2B). The 6-fold, 3-fold and 2-fold symmetry axes are annotated by a hexamer, triangle, and oval respectively. The distortion of the CA hexamer in the tubes at the CANTD is clearly visible and leads to a separation of the two halves of the immature hexamer. There is significantly less distortion at the CACTD. Colored rectangles indicate regions enlarged in Panels C-G. (C-G) In order to show structural similarities and variations within the immature EIAV assembly in tubes and spheres the models from the tubes were triplicated. The symmetry independent monomers in each of the triplicated models were then aligned against the CA monomer determined in the spheres. This allows visualization of the differences that symmetry independent CA monomers can adopt in relation to their neighbors. (C-E) All symmetry independent copies have been aligned on the CANTD. Models are shown as seen from outside the virus. (F-G) All symmetry independent copies have been aligned on the CACTD. Models are shown in a 90-degree rotation compared to (C-E). (C) the trimeric interface stabilizing the interhexameric interactions. This trimeric interface involving helices 2 (colored in red) is rigid and almost no structural changes can be seen. (D) Interactions around the hexameric ring are shown. Helices 4 of one monomer of the symmetry independent copies are colored in red to show the large structural variation of the intra-hexameric interactions at the CANTD. (E) The dimeric interface between helices 1 (shown in red) is variable as seen by the increasing separation of the helices and the lack of alignment of adjacent monomers. (F) The inter-hexameric interactions at the CACTD are maintained via a dimeric interface involving helices 9 in two adjacent monomers. The distance between the dimeric interface stays fixed, despite small changes in angular relation between the monomers forming the dimeric interface. (G) The interactions around the 6HB and the hexameric ring involving residues in the MHR are maintained, but show a variable degree of flexibility to adapt to the varying diameter in the tubes and spheres.
Fig 4
Fig 4. Conserved structural interactions in EIAV and HIV-1.
Comparison of structural features in EIAV and HIV-1. The HIV-1 CA-SP1 model derived from HIV-1 GagΔ16–99Δp6 (pdb 5l93, referred to as ΔMACANCΔp6 in [15]) is shown on the left. The EIAV CA-SP model derived from EIAV GagΔMA is shown on the right. S3 Movie shows a guided tour of this comparison. (A) Side view on the CASP lattice of EIAV and HIV-1 CASP. One monomer is highlighted, surrounding monomers of the lattice are shown with reduced opacity. The residues are colored according to the conservation between the two viruses. The color legend is indicated in panel (A). (B) Interactions and structural features in the CANTD. The trimeric interface in EIAV and HIV-1 is similar (Left). The Helix 1 in EIAV is shorter than in HIV-1 (Right). The extent of helix 1 in EIAV approximately corresponds to the conserved residues in HIV-1. (C) Comparison of the dimeric CACTD interface. Both lentiviruses use hydrophobic residues in helix 9 to stabilize the dimeric interface. F308/L309 and W316/M317 are annotated in EIAV and HIV-1, respectively. (D) Conserved residues in the MHR and helix 11 contribute to interactions around the hexameric ring to stabilize the immature CA assembly (Left). In HIV-1 residues D329, P356 and H358 form an important three-way interaction linking the CACTD base and the CA-SP1 helix of two adjacent CA monomers to each other. The equivalent residues in EIAV are E321, T348 and Q350 (Right). (E) The EIAV CASP 6HB is shorter than its counterpart in HIV-1. In EIAV and HIV-1 the CA-SP cleavage site is located within the helix, while the SP-NC cleavage site is located below the helix. Proteolytic cleavage sites are annotated by dashed lines.
Fig 5
Fig 5. IP6 stabilizes the immature EIAV CASP lattice.
(A) EIAV CASP and the IP6 molecule are shown as seen from the outside of the VLP and additionally rotated by 90°. IP6 sits in the center of the hexamer and is coordinated by a ring of six lysines in the MHR (K282) and six lysines in the CASP 6HB (K351). An isosurface representation of the IP6 density is shown in pink. The densities for the individual equatorial and the axial phosphate groups are clearly visualized. The non-occupied phosphate group is caused by the 6-fold symmetry applied during processing and the fact that IP6 can sit in the binding site in 6 rotationally equivalent positions. (B) Relative infectious particle production in 293FT cells of VSV-G-pseudotyped provirus of wild type EIAV Gag (WT) and Gag with point mutations. Graphs show the average and standard deviation of three independent experiments; dots show individual data points. (C,E,G) Representative low and high magnification images of GagΔMA WT, K282A, and K351A proteins assembled in the absence (red) or presence (blue) of 10 μM IP6 at pH 6. (D,F,H) The number of VLPs (spheres-purple, tubes-green) per 55μm2 for no fewer than five representative images for each condition. Center lines show the medians; box limits indicate the 25th and 75th percentiles as determined by R software; whiskers extend to minimum and maximum values; data points are plotted as circles. The mean value of counted particles is given in italics in the bar charts.
Fig 6
Fig 6. The effect of IP6 on other lentiviruses.
(A) Comparison of the MHR sequence and the CASP junction of lentiviruses. Blue bars indicate the location of known (EIAV and HIV-1) and predicted IP6 interacting lysine residues. (B-E) In vitro assembly results of HIV-2, SIV, FIV, and BIV Gag constructs without (red) and with (blue) IP6 at 22°C. (B, C) HIV-2 and SIV assembly was done in 50 mM Tris pH 8, 100 mM NaCl, with GT25 oligo by dialysis. (D) FIV assembly was done in 50 mM Bis-Tris propane, 150 mM NaCl, GT50 oligo by dilution. (E) BIV assembly was done in 50 mM MES pH 6.5, 100 mM NaCl, GT50 oligo by dilution. The mean value of counted particles is given in italics in the bar charts.

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Grant support

This work was supported by National Institutes of Health (NIH, https://www.nih.gov/) grant R01-GM107013 and National Science Foundation (NSF, https://www.nsf.gov/) grant 1659534 to V.M.V., National Institute of Allergy and Infectious Diseases (NIAID, https://www.niaid.nih.gov/) grant R01-AI147890 to R.A.D., National Institute of General Medical Sciences (NIGMS, https://www.nigms.nih.gov/) grant P30-GM110758 and National Institute of Allergy and Infectious Diseases (NIAID, https://www.niaid.nih.gov/) grant P50AI150481 to J.R.P., NIAID grant AI142263 to M.C.J., European Research Council (ERC, https://erc.europa.eu/) under the European Union’s Horizon 2020 research and innovation programme (ERC-2014-CoG 648432 – MEMBRANEFUSION), Medical Research Council (https://mrc.ukri.org/) MC_UP_1201/16, Deutsche Forschungsgemeinschaft (https://www.dfg.de/) grant BR 3635/2-1 to JAGB, Austrian Science Fund (FWF, https://www.fwf.ac.at/en/) grant P31445 to FKMS. Molecular dynamics simulations were performed on the NCSA Blue Waters supercomputer, supported by the National Science Foundation grant number ACI-1548562. This work used the Extreme Science and Engineering Discovery Environment (XSEDE), which is supported by National Science Foundation grant number ACI-1548562. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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