Article Figures & Data
Figures
- Fig. 1.
Effects of mutations on virus particle assembly and release. (A) Shown is a pair of CA proteins (Protein Data Bank [PDB] accession no. 3GV2) in HIV-1 CA hexamers viewed roughly from the center of the hexamer outward. NTDs (blue) and CTDs (green) are marked, as are the β-hairpin (β) and cyclophilin A binding (CypA) loops and helices 1 to 11 on one of the capsid proteins. H62 residues are indicated in red. (B) HEK 293T cells were transfected with the indicated HIVLuc constructs. At 72 h posttransfection, virus samples were collected, and Gag proteins were detected after gel electrophoresis by immunoblotting using a primary anti-CA antibody. Full-length PrGag and CA bands are as indicated. (C) For quantification of viral particle release at 72 h posttransfection, virus and cell samples were collected and subjected to electrophoresis and immunoblotting using an anti-CA antibody. Cell and virus Gag levels were quantitated densitometrically; raw virus release levels (Virus Gag/Cell PrGag) were calculated and are shown, normalized to WT HIV release levels. Results derive from two (H62K and H62W), three (H62C and H62F), four (H62Y), five (H62A), or eight (WT) separate experiments. Standard deviations are as shown.
- Fig. 2.
Analysis of virus infectivity. Luciferase gene-transducing HIV viruses were produced by cotransfection of HEK 293T cells with a VSV-G expression vector plus WT and H62 mutant HIVLuc constructs (A) or the indicated HIV variants that derived from selection of H62A or H62F second-site revertants (B). At 3 days posttransfection, virus-containing medium supernatants were collected, filtered through 0.45-μm sterile filters, and used to infect HiJ cells. At 3 days postinfection, HiJ cells were collected and processed for luciferase assays. Results are depicted as normalized luciferase activity signals in infected cells, relative to signals from cells infected with WT HIVLuc virus in parallel: they are not corrected for variations observed in virus particle release. Results are derived from 46 (WT), 16 (H62A and H62F), 2 (H62C and H62F/G208A), 3 (H62K), 4 (H62W and H62Y), 5 (G208A), 8 (G208R and H62F/SP1P10L), and nine (H62A/G208R) separate experiments. Standard deviations are as shown.
- Fig. 3.
Effects of H62 CA mutations on virus-like-particle morphologies. The indicated HIVLuc viruses from transfected cells were sedimented through 20% sucrose cushions, resuspended, adhered to carbon-coated EM grids, and visualized by EM. Conical and tubular cores in panels A, G, and H are indicated by three white arrowheads each. The size bar for all panels is provided at the bottom of panel G, and observed core morphologies are tabulated in Table 1.
- Fig. 4.
In vitro assembly of HIV capsid proteins. WT or variant CA proteins were expressed in bacteria, purified, and employed in in vitro assembly reactions. Following assembly incubations, samples were processed and imaged by EM at low magnification (A, B, D, E, G, H, J, and K; size bar in panel J) or high magnification (C, F, I, and L; size bar in panel L). For each capsid variant, assembly efficiency was assessed by the appearance of capsid tubes, and tube assembly was quantified by tube coverage in EM images as described in Materials and Methods. Average tube coverage percentages, normalized to the WT level, were calculated from 12 pictures each and are as follows: WT, 100%; H62A, 0%; H62A/G208R, 11%; G208R, 68%.
- Fig. 5.
Replication kinetics of HIV mutants. Stocks of WT and CA variant viruses on an NL4-3 HIV background were generated by transfection of constructs into HEK 293T cells and were normalized with respect to Gag levels. CEM-SS (A) or MT4 (B to D) cells were infected in parallel with normalized virus stocks of WT or variant viruses and passaged at 3- to 4-day intervals. Virus spread was monitored by measuring Gag levels in aliquots of infected cells by immunoblotting and densitometry. Replication is plotted as relative Gag level in infected cells versus number of days postinfection.
- Fig. 6.
Proximity of mutant and revertant residues on models of HIV CA. The locations of CTD helices 10 and 11 and residues H62 (red) and G208 (yellow) on pairs of CA proteins in HIV CA hexamers (PDB accession no. 3GV2) are depicted. Panel A is viewed roughly parallel to the plane of capsid coats, with the predicted outer capsid surface up and the inner surface down. Panel B is viewed roughly perpendicular to the capsid surface from the outside. Light and dark blue N-terminal domains and light and dark green C-terminal domains are as labeled.
Tables
- Table 1.
Morphologies of HIV particlesa
Capsid variant % conical or cylindrical No. viewed WT 55 64 H62A 19 52 H62C 19 73 H62K 17 41 H62F 17 24 H62W 21 34 H62Y 47 15 H62A/G208R 47 30 ↵a Morphologies of virus-like particles. Virus-like particles from cells transfected with the indicated HIVLuc viruses were sedimented through 20% sucrose cushions, resuspended, and processed for EM. The percentages of particles with discernibly roughly conical or cylindrical cores were determined from the indicated numbers of separate virus particle images.
