Critical role of conserved hydrophobic residues within the major homology region in mature retroviral capsid assembly

Abstract

During retroviral maturation, the CA protein undergoes dramatic structural changes and establishes unique intermolecular interfaces in the mature capsid shell that are different from those that existed in the immature precursor. The most conserved region of CA, the major homology region (MHR), has been implicated in both immature and mature assembly, although the precise contribution of the MHR residues to each event has been largely undefined. To test the roles of specific MHR residues in mature capsid assembly, an in vitro system was developed that allowed for the first-time formation of Rous sarcoma virus CA into structures resembling authentic capsids. The ability of CA to assemble organized structures was destroyed by substitutions of two conserved hydrophobic MHR residues and restored by second-site suppressors, demonstrating that these MHR residues are required for the proper assembly of mature capsids in addition to any role that these amino acids may play in immature particle assembly. The defect caused by the MHR mutations was identified as an early step in the capsid assembly process. The results provide strong evidence for a model in which the hydrophobic residues of the MHR control a conformational reorganization of CA that is needed to initiate capsid assembly and suggest that the formation of an interdomain interaction occurs early during maturation.

FIG. 1.

Ribbon diagram of the monomeric structure of RSV CA. The locations of the MHR (D155, E162, F167, and L171) and suppressor (A38, R185, I190, and F193) residues mutated in this study are shown in a ball-and-stick fashion. The diagram was made using PBD ID 1EM9 (NTD) and 1EOQ (CTD); the broken line represents the interdomain linker.

FIG. 2.

Far UV analysis and protein stability of WT and mutant CA proteins. (A) The secondary structure was determined by CD from 195 to 255 nm. (B) Protein stability was determined by monitoring the α-helical content of the protein at 222 nm from 0 to 5.5 M Gdn-HCl.

FIG. 3.

NaPO₄-induced assembly of purified WT and D52A proteins. (A) The assembly reaction of WT and D52A protein was followed by turbidity, as described in Materials and Methods. (B to J) EM of WT protein assembled at 0.5 M NaPO₄, pH 7.5, at 78 μM. (K) D52A protein assembled under identical conditions. (L to N) WT protein assembled at 0.5 M NaPO₄, pH 8.0, at 156 μM. Scale bars, 100 nm.

FIG. 4.

Determination of in vitro assembly phenotypes of the MHR and suppressor mutations. Shown are the F167Y-I190V series (A), L171V-A38V series and F167Y/A38V double mutant (B), D155Y-R185W series (C), and E162Q-F193L series (D). The reactions were stopped when the maximum ΔOD₄₅₀ was reached.

FIG. 5.

Examination of assembled mutant proteins by EM. All proteins were assembled using standard conditions (0.5 M NaPO₄, pH 7.5, and 78 μM protein). Arrows in panel K (E162Q/F193L) point toward structures that contain or overlap smaller spheroidal structures. DY/RW, D155Y/R185W; EQ/FL, E162Q/F193L; FY/AV, F167Y/A38V. Scale bars, 100 nm.

FIG. 6.

Assembly of L171V-A38V series of mutant proteins examined by differential centrifugation. At 5, 100, 200, and 300 min after initiation of assembly, 5-μl samples were removed from the 100-μl reaction mixtures and centrifuged at 18,000 × g for 1 min. The supernatant was removed, and the pellet was resuspended in a final volume equivalent to the supernatant fraction. Both fractions were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. S, supernatant fraction; P, pelleted fraction.

FIG. 7.

Rescue of F167Y nonassembly phenotype by WT protein. (A) WT protein was mixed with either F167Y mutant protein (○) or BSA (⋄) at equimolar concentrations of 39 μM each. Also shown are the turbidimetric profiles of unmixed WT (▵), F167Y (▿), and BSA (—) proteins at 39 μM and WT protein at 78 μM (□). For clarity, only every 20th data point is shown. (B and C) MS analysis of unlabeled WT protein (B) and ¹⁵N-labeled F167Y protein (C) that were not mixed together and not subjected to NaPO₄ assembly conditions. (D) ¹⁵N-labeled F167Y mutant protein mixed with WT protein at 39 μM each was induced to assemble and prepared for MS analysis as described in the text.

FIG. 8.

Sensitivity of WT and suppressor mutant proteins to NaPO₄. The assembly rate was determined by fitting the growth phase of the turbidity curve to a linear equation and calculating the ΔOD/Δtime, where ΔOD was the final OD₄₅₀ minus the initial OD₄₅₀ of the growth phase, and Δtime was the final time minus the initial time of the growth phase. The log of the rate was graphed as a function of NaPO₄ concentration.