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Previous Article | Next Article 
Journal of Virology, June 2008, p. 5951-5961, Vol. 82, No. 12
0022-538X/08/$08.00+0 doi:10.1128/JVI.00214-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
and
Rebecca C. Craven1*
Department of Microbiology and Immunology,1 Department of Biochemistry and Molecular Biology, The Pennsylvania State University, College of Medicine, Hershey, Pennsylvania 17033,2 Cedar Crest College, Allentown, Pennsylvania 181043
Received 30 January 2008/ Accepted 28 March 2008
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In spite of limited sequence similarity, the three-dimensional structure of mature CA is highly conserved among retroviruses and consists of the two mostly 
-helical domains, NTD and CTD, connected by an interdomain linker (8, 18, 19, 24, 29, 36, 37). After maturation is completed, the final capsid shell consists of a lattice of CA hexamers, established by NTD-NTD interactions and linked by CTD-CTD dimerization (24, 25, 27, 43, 48, 51, 53, 79). The dimer interface is formed by the dimerization helix, the second helix of the CTD. A third interface, an NTD-CTD interdomain interaction that forms during maturation, was originally predicted by a genetic study of the Alpharetrovirus Rous sarcoma virus (RSV) and subsequently confirmed and mapped by biochemical and structural studies examining human immunodeficiency virus (HIV) (10, 27, 43, 44). Mutagenesis in HIV and the binding of retroviral inhibitors have documented that this interdomain interface is essential for capsid integrity and infectivity (23, 28, 35, 67, 70, 73, 75).
The start of the CTD contains the highly conserved major homology region (MHR) motif that has been implicated in both the immature and mature stages of virus assembly. Numerous substitutions to the three absolutely conserved polar residues (Gln, Glu, and Arg) and the conserved hydrophobic residues cause severe defects in immature virus assembly and a loss of infectivity, presumably by disrupting key steps in Gag assembly (16, 20, 46, 50, 56, 68). Certain conservative substitutions at many of the same positions, however, appear to compromise the formation of the mature capsid shell instead of Gag assembly (16, 20, 46, 50). In RSV, the loss of infectivity caused by such substitutions was traced to an improperly formed core structure, resulting in a failure of genome replication (10, 16).
The conserved residues of the MHR are not directly involved in any of the known intermolecular interfaces in the mature capsid shell, but instead, these residues contribute to the structure of the CTD through intramolecular interactions (27, 29, 37, 43, 48, 79). Residues downstream of the MHR are important for the CTD-CTD and NTD-CTD interfaces (24, 27, 43, 44, 48, 79). Many second-site suppressors in RSV that restore infectivity to noninfectious CA mutants are located downstream of the MHR motif and in other regions of CA that are implicated in maturation and the formation of the mature capsid (10, 49, 65). This observation suggests that the conserved MHR substitutions perturb capsid formation by affecting the folding or final structure of the CTD during maturation.
A possible explanation for the role of the MHR in Gag assembly and maturation was recently suggested by a crystal structure of a novel dimer interface in the HIV CTD (32, 33, 39). A mutation in the CTD caused a relaxation of the bend between the first and second helices, allowing dimerization by a "domain swap" arrangement wherein the structural elements containing the MHR and the following helix form a largely hydrophobic interface between two molecules. This model is supported by the homology between the Gag protein and certain mammalian transcription factors that dimerize via this domain swap mechanism (32, 39). Cryoelectron tomographic and cryoelectron microscopic analyses of immature particles, however, have been of insufficient resolution to confirm this model (12, 14, 77, 80, 81).
The domain swap model proposes that certain MHR residues may directly mediate an intermolecular interaction in the immature virus. In the mature form, however, the same residues participate in only intramolecular interactions. Accordingly, the conserved hydrophobic MHR residues must move from an exposed position (in the domain swap arrangement) into the interior of the CTD during maturation (24, 27, 33, 79). Such a reorganization of the CTD was previously suggested based on results observed with an anti-MHR antibody that recognized uncleaved Gag protein but not mature CA protein (20). However, the molecular events of capsid assembly occurring during maturation (i.e., the precise nature of the structural transformations, the sequence of events or intermediates in the capsid assembly pathway, and the roles of specific residues in controlling these events) have remained largely uncharted. Given the importance of the MHR to the maturation process and the demonstrated potential of maturation inhibitors as effective antiviral therapies (47, 62, 64, 67, 70, 73), an understanding of the molecular details of this process remains a critical unmet need.
The MHR and suppressor mutations provide a powerful tool to allow the genetic and biochemical dissection of events occurring during retroviral maturation. To examine the ability of the MHR to control mature capsid assembly independently of any role in immature assembly, we selected MHR mutations that cause a loss of core integrity and infectivity without any obvious consequence on Gag function. Overall, the results reveal that certain residues of the MHR, in particular, the conserved hydrophobic amino acids, have a critical role during maturation in facilitating an early step in capsid assembly which likely involves interdomain interactions.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The CA protein was expressed in Escherichia coli (BL21 DE3) by using the autoinduction system developed previously by Studier (69). Isotopically labeled protein was produced by bacterial growth in MD-5052 minimal medium supplemented with [15N] NH4Cl. The purification protocol was adapted from a previously described protocol (40). The bacterial pellet was resuspended in buffer containing 20 mM Tris-HCl, pH 7.5, 0.5 M NaCl, 10% glycerol, 1 mM EDTA, 10 mM dithiothreitol, Complete protease inhibitor cocktail (Roche), and lysozyme (Sigma). The suspension was sonicated, treated with Benzonase (Novagen), and clarified by centrifugation at 21,000 x g for 30 min. Soluble material was precipitated with 35% ammonium sulfate. The pellet was resuspended in 20 mM Tris-HCl, pH 7.5, 100 mM NaCl, and 1 mM EDTA and dialyzed against the same buffer. Insoluble material was removed by centrifugation, and the soluble fraction was loaded onto a DEAE column equilibrated with buffer A (50 mM Tris-HCl, pH 7.5, 1 mM dithiothreitol, and 0.1 mM EDTA). CA does not bind DEAE under these conditions. The column was washed with buffer A, and the flowthrough was collected and concentrated by precipitation with 50% ammonium sulfate. The protein pellet was resuspended and dialyzed against 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 0.1 mM ETDA. CA was further purified by size exclusion chromatography (Superdex 75), and the fractions containing monomeric CA were pooled and concentrated to approximately 10 mg/ml by using iCON concentrators (Pierce). CA was monitored at each step of purification by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and staining was carried out with Coomassie blue. Expression, solubility, and purification of the mutant proteins were similar to those of the WT protein. The following extinction coefficient values were used: 24,980 M–1 cm–1 for the WT and the I190V, L171V, L171V/A38V, A38V, E162Q, E162Q/F193L, F193L, and D52A mutant proteins; 26,470 M–1cm–1 for the F167Y, F167Y/I190V, and D155Y mutant proteins; 31,970 M–1cm–1 for the D155Y/R185W mutant protein; and 30,480 M–1cm–1 for the R185W mutant protein.
CD spectroscopy, intrinsic tryptophan fluorescence, and unfolding equilibrium. For circular dichroism (CD) spectroscopy, a Jasco J-710 spectropolarimeter operating in the far UV region of the spectrum was used for the determination of secondary structure. The protein was diluted in 50 mM sodium phosphate (NaPO4), pH 7.5, 50 mM NaCl, and 0.1 mM EDTA to a final concentration of 0.1 mg/ml. Intrinsic tryptophan fluorescence was performed using a 290-nm excitation wavelength on a PTI QuantaMaster luminescence spectrometer. Protein stability was measured as a function of the guanidine hydrochloride (Gdn-HCl) concentration (0 to 5.5 M Gdn-HCl) in 20 mM Tris-HCl, pH 7.5, and 0.1 mM EDTA. Analysis was performed by fitting the data to a two-state unfolding model as described previously (60).
In vitro capsid assembly. Capsid assembly was monitored by optical density at 450 nm (OD450) by using a SpectraMax Plus384 spectrophotometer (Molecular Devices). Unless otherwise noted, assembly conditions were 78 µM (2 mg/ml) CA protein in 10 mM Tris-HCl, pH 7.5, 75 mM NaCl, and 0.05 mM ETDA and assembly was initiated by the addition of 0.5 M NaPO4, pH 7.5 (final concentrations). Each reaction was performed in a 100-µl volume in a 96-well UV transparent microplate (BD Falcon). Typically, 1 min elapsed between the initiation of assembly and the first reading. The plate was mixed for 5 s prior to the first reading and 3 s prior to each subsequent reading.
The change in OD450 (
OD450) was calculated by subtracting the OD450 value of the in vitro assembly reaction mixture lacking NaPO4 from the OD450 value of a parallel reaction mixture containing 0.5 M NaPO4. For most proteins, the OD450 graph was a sigmoidal curve resembling a multiphasic reaction with lag, growth, and stationary phases. The modified Gompertz equation was used to determine the lag time of the assembly reactions (82). For Clarity, only 1 of 10 data points is shown on most of the graphs.
EM. The products of the in vitro assembly reactions were examined by electron microscopy (EM). Upon reaching the maximum OD450, the samples were removed from the spectrophotometer and stored at 4° for 12 to 16 h. During storage, the assembled protein settled at the bottom of the tube, allowing for concentration without centrifugation. The settled samples were transferred to a Formvar-coated grid, washed with water, stained with 2% uranyl acetate, and visualized using a 120-kV transmission electron microscope operating at 60 kV. The capsid-like structures retained heavy amounts of stain; thus, the contrast of the resulting EM images was adjusted by using Adobe Photoshop.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-helical, as observed by the large negative peaks at 222 nm and 208 nm (18, 37, 66) (Fig. 2A). All of the mutant proteins had CD spectra that were indistinguishable from that of the WT, indicating that they were properly folded, without any large-scale defects in secondary structure (Fig. 2A and data not shown). Next, the effects of the MHR and suppressor substitutions on protein stability were evaluated. The WT CA protein was unfolded by Gdn-HCl with a midpoint concentration of 
2.2 M, as determined by either the relative ellipticity at 222 nm or the intrinsic tryptophan fluorescence at 325 nm (Fig. 2B and Table 1). No increased sensitivity to Gdn-HCl was observed for any of the mutant proteins, indicating the lack of gross destabilization (Table 1).
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NaPO4-induced in vitro RSV CA multimerization.
To dissect the importance of particular MHR residues for capsid assembly, a turbidimetric assay was utilized (22, 45, 57, 59, 63). Coupled with EM analysis, this allowed for a quantitative and qualitative evaluation of the assembly capabilities of the MHR and suppressor mutant proteins. In this system, monomeric CA protein at 2 mg/ml (78 µM) was stimulated to assemble by the addition of 0.5 M NaPO4. After a lag period, assembly was evidenced by an increase in OD450 due to light scattering by the large structures (Fig. 3A). Under these conditions, the lag time for the WT protein was 190 min and a maximum optical density was reached after an additional 150 min. The duration of the lag time and the OD450 reached were dependent upon both NaPO4 and the protein concentration (data not shown).
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85 nm (Fig. 3B). Other cylindrical structures appeared to be completely formed tubes, with diameters of 50 nm (Fig. 3L), rather than curled planar arrays. Both the planar arrays and tubes had various lengths (200 to 1,500 nm). Particularly notable was the presence of angular structures (Fig. 3D to J and L to N) similar to the irregular polyhedra observed in native RSV capsids (15, 38). Many of the assembled structures were multilamellar (Fig. 3D to I). Apparently, such angular structures were not formed under the nonphysiological, low pH, in vitro conditions used previously (37). A similar range of structures was described for HIV in both virions and in vitro-assembled CA protein (6, 11, 13, 30, 45, 48, 74, 76). The shapes and varieties of the structures formed under conditions of 156 µM CA protein and pH 8.0 (Fig. 3L to N) appeared identical to ones formed under conditions of 78 µM and pH 7.5 (Fig. 3B to J). The formation of angular capsid-like structures indicates that RSV CA has the ability to form authentic CA-CA interfaces at a near neutral pH under the NaPO4-induced assembly conditions and supports the validity of this assay to evaluate the effect of CA mutations on capsid assembly.
A mutation expected to disrupt the ability of CA to properly assemble was tested to further validate the in vitro assay. Upon the cleavage of CA from Gag, the amino terminus of the protein refolds, forming a salt bridge between the terminal Pro and a conserved Asp residue (9, 29, 74). The disruption of this salt bridge by an Asp-to-Ala substitution results in a loss of in vitro CA assembly and infectivity (61, 72, 74). In RSV, the behavior of the D52A mutant CA protein deviated from the WT protein by rapidly forming light-scattering material within 15 min (Fig. 3A). However, the final OD450 value was lower than that for WT protein. No capsid-like structures were formed by the D52A protein, instead, EM revealed only dark-staining regions of amorphous protein precipitates (Fig. 3K), with only a rare inchoately formed cylindrical structure interspersed within the precipitate. These observations are likely due to uncontrolled aggregation of the D52A protein triggered by NaPO4, which is consistent with the behavior of the analogous HIV mutant (74).
F167Y and L171V mutant proteins lack the ability to properly assemble, but assembly is restored by intradomain and interdomain suppression.
In the mature form of CA, F167 and L171 are located proximally to one another within the hydrophobic core of the CTD monomer. Both mutations dramatically inhibited the assembly of CA protein in vitro. The F167Y mutant protein formed a small amount of light-scattering material after a greatly lengthened lag time (550 min), and only amorphous precipitate was observed by EM (Fig. 4A and 5A). Similarly, the L171V protein showed little, if any, assembly under NaPO4-induced conditions (Fig. 4B and 5D). These assembly defects imply that the F167 and L171 residues of the MHR are essential for proper capsid assembly.
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OD450. Likewise, the suppressor of the L171V mutation (the A38V mutation) restored the assembly capability to protein containing the L171V substitution (Fig. 4B) by shortening the long lag phase of L171V protein to a WT-like level.
The ability of the suppressor mutations to restore assembly to the MHR mutant proteins was also demonstrated by the presence of organized structures in the turbid samples. However, in neither case (F167Y/I190V or L171V/A38V) was the full range of morphologies seen in WT protein reproduced with the double mutant. The F167Y/I190V protein predominantly assembled into spheroids of 30 nm, often lacking distinct edges and well-defined centers (Fig. 5B). In contrast, only tube-like structures with diameters of 50 nm were seen in the assembled L171V/A38V protein (Fig. 5E). These structures resembled the tubes and planar arrays seen in the WT protein, but many were considerable longer, measuring up to 2 µm in length (data not shown).
Interestingly, the two suppressor mutations dramatically increased CA multimerization when the MHR substitutions were not present. The I190V protein assembled very quickly, with a lag of only 3.5 min (Fig. 4A). Like the F167Y/I190V mutant protein, the I190V protein predominately assembled into 30-nm spheroids (Fig. 5C). Relative to those formed by the F167Y/I190V protein, many of the I190V spheroids were more distinct, with easily discerned edges and centers. The effects of the A38V suppressor mutation were even more striking. The rise in turbidity was so rapid that the lag time could not be calculated since the observed curve was not sigmoidal under these conditions (Fig. 4B). Spheroids, short tubes, and capsid-like structures similar to those of the WT were formed by the A38V protein (Fig. 5F). The results indicate that the I190V and A38V suppressor mutations strongly increased the propensity of CA to multimerize.
The L171V/A38V double mutant appears to assemble more slowly than the WT protein, as indicated by a decreased slope during the growth phase and a lower maximum OD450 (Fig. 4B). However, the abundance of long tubes and lack of other structures (Fig. 5E) suggest that some of these effects may be due to a lower inherent ability of long tubes to scatter light (7). As an alternative method to follow assembly, a differential centrifugation protocol that separated large assembled products from soluble CA was utilized (Fig. 6). As predicted from the turbidimetric assay, the WT protein was found mostly in the supernatant fraction at 5 and 100 min, in both fractions at 200 min, and mostly in the pellet at 300 min (Fig. 6). The assembly defective L171V protein was present only in the supernatant fraction at all time points. In contrast, a detectable amount of L171V/A38V protein was pelletable at early time points, consistent with a WT-like lag time. The amount of pelletable CA increased slowly with time, continuing to increase at 300 min (Fig. 6) and beyond (data not shown). Consistent with the turbidity measurements, A38V rapidly assembled into pelletable material (Fig. 6). This assay confirms that during the growth phase, the L171V/A38V protein assembled more slowly than did the WT, even though their lag times were similar. This difference in assembly rates may be due to the drastically different range of particle morphologies formed by these two proteins.
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D155Y MHR mutant protein retains partial multimerization ability. The D155 residue is located at the beginning of the MHR, near the junction of the interdomain linker and the CTD (Fig. 1). When tested for effects on in vitro assembly capacity, the D155Y substitution behaved like the F167Y and L171V mutations and crippled protein assembly as monitored by turbidity (Fig. 4C). Assembly was restored to a WT-like pattern by the secondary R185W mutation. As was true of the I190V and A38V suppressor mutants, the R185W substitution in the absence of an MHR mutation dramatically accelerated the formation of light-scattering material upon the addition of NaPO4 (Fig. 4C). Thus, the three MHR mutations and their suppressors exhibit very consistent effects on in vitro assembly.
Unlike F167Y and L171V, the D155Y protein retained some ability to form organized structures (Fig. 5G). Small spheroids (range, 16 to 50 nm; mean, 30 nm) were readily seen; some of these were observed in chain-like arrays aligned in a roughly cylindrical shape. The larger multilamellar or polyhedral structures commonly seen with the WT protein were rare (data not shown). The D155Y/R185W protein readily formed larger angular structures (Fig. 5H) in addition to small spheroids and chain-like arrays of small spheroids similar to the ones observed with the D155Y sample. The R185W mutation by itself favored the formation of large multilamellar, angular structures similar to ones seen frequently with the WT protein (Fig. 5I). Unlike the D155Y and D55Y/R185W proteins, the R185W protein formed tubular structures. Thus, the D155Y mutant possesses a limited ability to multimerize and can be restored to a more WT-like behavior by the R185W secondary substitution.
Lethal E162Q mutation is assembly competent in vitro.
The absolutely conserved Glu residue in the MHR (E162 in RSV and E159 in HIV) participates in an extensive intramolecular hydrogen bonding network in both the mature CA and in the domain swap model for immature Gag assembly (24, 33, 36, 37). In RSV, a Gln substitution at this position resulted in a loss of infectivity (16) that could be suppressed by the F193L second-site mutation (49). The E162Q substitution caused a slight reduction (40%) in virus release from infected cells (16). The F193L suppressor restored E162Q budding to WT-like levels (49), suggesting that these mutations cause significant impact on Gag function(s).
The E162Q protein readily assembled in vitro (Fig. 4D), forming structures, including the angular multilamellar structures, similar to those formed by WT protein (Fig. 5J). In the E162Q/F193L double-mutant protein, the F193L suppressor had little effect on in vitro assembly (Fig. 4D and 5K to M), only marginally increasing the turbidity over that achieved by the E162Q protein without improving the lag time. The E162Q/F193L protein formed structures that contained or overlapped smaller spheroidal structures (Fig. 5K) and resembled the nested capsids observed in some HIV and RSV particles (6, 11, 15). F193L was the only suppressor tested that failed to stimulate assembly in the context of a double-mutant protein (Fig. 4D). The F193L mutation alone had no detrimental or stimulatory effect on in vitro assembly (Fig. 4D). Therefore, the phenotypic effects of E162Q and F193L on virus replication are not related to alterations in mature capsid assembly, but these effects may be influencing Gag assembly by altering the domain swapping of the MHR residues.
Defective multimerization ability of L171V and F167Y is rescued by the addition of WT protein. The ability of WT protein to rescue the assembly defects caused by the F167Y and L171V mutations was tested by mixing the WT protein with each of the mutant proteins at equimolar ratios (39 µM each). WT protein by itself at 39 µM exhibited a long lag and limited light scattering. Doubling the protein to 78 µM shortened the lag period and resulted in a maximum turbidity that was roughly twice that of the 39-µM sample (Fig. 7A). Adding the F167Y mutant protein to the WT protein (each at 39 µM) almost doubled the final turbidity without any significant change in the lag time (388 min for the WT versus 374 min for the mixed sample). Reducing the WT protein to 20 µM and increasing the mutant protein to 58 µM caused an approximately proportional increase in the lag time (data not shown). The above data suggest that the mutant protein was incorporated into growing structures initiated by WT protein. The mutant protein may stimulate WT assembly by acting as a crowding agent (21). However, bovine serum albumin (BSA) could not replicate the stimulatory effect of F167Y and actually caused a slight inhibition of WT assembly (Fig. 7A), arguing against this explanation.
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A38V, R185W, and I190V suppressor mutations increased the sensitivity of CA to NaPO4. Proteins containing the A38V, R185W, and I190V suppressor mutations without a lethal substitution exhibited dramatically increased assembly rates (Fig. 4A to C). The suppressor mutant proteins were compared to the WT protein over a range of NaPO4 concentrations (from 0.1 M to 1.0 M). All three proteins were more easily stimulated to assemble at lower concentrations of NaPO4 than was the WT or F193L protein (Fig. 8). The assembly rates of A38V, R185W, and I190V were greater than that of WT at every NaPO4 concentration tested, whereas the F193L protein showed a WT-like assembly rate across the entire range. Both the greatly reduced lag time (Fig. 4A to C) and the increased sensitivity to NaPO4 (Fig. 8) caused by the A38V, R185W, and I190V suppressor mutations are consistent with a stimulation of an early step of capsid assembly that occurs during the lag phase.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In vitro assembly of RSV CA. The CA protein formed into structures of diverse morphologies, spheroidal structures of various diameters, tubular structures with various lengths, and occasional large structures that have the angular features of authentic RSV cores (15, 38). An examination of authentic RSV particles by cryoelectron tomography documented a similar range of polymorphic structures, including one class of angular capsids whose members appear to contain genome and abundant glycoprotein spikes and are considered the best candidates for infectious virions (15). The broad range of structures, including angular capsid-like structures, observed in this study may be due to either the near neutral pH or the use of NaPO4. In contrast, a previous study showed CA assembling a more restricted range of structures (only small spheroidal and tubular forms) under distinctly nonphysiological conditions (pH 5.5 or lower, with NaCl at a level of 0.6 M or greater) (37).
Several recent studies have suggested that the normal regulation of capsid assembly in maturing virions may involve additional virion components, such as the lipid membrane, Env glycoprotein tails, genomic RNA, or partially cleaved Gag (4-6, 11, 15). The formation of structures that are distinctly different from authentic capsids (i.e., long tubes) (Fig. 5E and M) may reflect the lack of regulation of capsid assembly by these components in the in vitro reaction. Regardless, the results illustrate the intrinsic ability of RSV CA to form capsid-like structures without the requirement of any other viral component, as has been shown for HIV CA under high-ionic-strength conditions (30, 45, 48).
The RSV CA assembly reaction had a pattern of an extended lag phase with a subsequent growth phase (Fig. 3A and 4A to D). Similar results have been reported for HIV CA multimerization by using NaCl-induced assembly conditions (4, 45). This pattern is consistent with a nucleation step, followed by the elongation and closure steps that have previously been proposed for HIV and RSV capsid assembly in vivo (6, 11, 15). Similar nucleation-elongation polymerization mechanisms have been well documented for other protein assembly processes, including those of tobacco mosaic virus capsid, T4 and P22 procapsids, and microtubules (22, 42, 57, 59, 63). The precise events occurring during the lag period with RSV CA could not be observed directly by using optical light scattering, but they are being pursued using methods with greater sensitivity.
A role for the MHR in controlling capsid assembly. The lethal F167Y and L171V substitutions caused a complete loss of in vitro CA assembly (Fig. 4A and B and 5A and D), and this deficiency was reversed by secondary mutations (Fig. 4A and B and 5B and E) that were originally isolated due to their ability to restore infectivity (10). The conservative F167Y and L171V alterations cause no observable diminishment of immature virus release in vivo (10, 16, 20), nor do they interfere with in vitro Gag assembly (J. Phillips and V. Vogt, unpublished data). Additionally, the D155Y substitution also limited mature capsid assembly. This mutation caused a less severe multimerization defect (Fig. 4C and 5G), signifying that this mutation is in some way qualitatively different from that of F167Y and L171V. In total, these findings argue that certain MHR residues, particularly the conserved hydrophobic residues, have a critical role in the assembly of the mature capsid shell that is independent of any additional Gag function(s) they may serve.
MHR mutations and suppressors act upon an early event of CA assembly. The increased lag period of the F167Y and L171V proteins (Fig. 4A and B) and the ability of these mutants to be incorporated into assembling structures in the presence of WT protein (Fig. 7A to D) lead us to hypothesize that the F167 and L171 residues are involved in an early step of capsid assembly. Three second-site substitutions (A38V, R185W, and I190V) by themselves greatly accelerate assembly (Fig. 4A to C) and increase the sensitivity of the protein to NaPO4-induced multimerization (Fig. 8), consistent with an improved ability to initiate capsid assembly. Thus, the stimulation of a nucleation event by these suppressors appears to provide a plausible explanation for their ability to suppress the F167Y and L171V mutations and suggests that a block in nucleation is the primary reason for the loss of infectivity observed for these mutant viruses (20).
The ability of the A38V mutation in the NTD to efficiently suppress the assembly defect caused by both the L171V and F167Y mutations in the CTD suggests that an NTD-CTD interaction is essential for the initiation event. The isolation of NTD suppressors (A38V and an additional P65Q mutation) of lethal MHR substitutions in RSV provided the first evidence of an interdomain interaction (10), an observation that has since been amply confirmed by studies with the HIV CA protein (27, 43). Further support for the ability of the NTD suppressor to correct a defect in the CTD is derived from the observation that the F167Y/A38V mutant virus is infectious (49). The lethal F167Y phenotype can also be suppressed by a substitution located in the C-terminal cleavage site of CA that alters the rate of release of mature CA from Gag (10), further suggesting a role of the MHR participating in virus maturation.
CA structural flexibility, assembly, and capsid morphology.
The plasticity of CA is demonstrated by multiple crystal structures, each containing differing arrangements of intersubunit contacts (8, 24, 34, 73, 73, 79). Indications of the dynamic behavior of the CTD comes from the high degree of flexibility in the second helix of this domain in an HIV protein that contained an engineered mutation that prevented dimerization (2, 78). During maturation, a refolding of the first 20 residues of the NTD further illustrates the dynamic nature of CA (9, 29, 54, 71, 74). The inherent plasticity of the NTD and CTD is likely due to the various structural roles of CA during multiple stages of the viral replication cycle.
The flexibility of CA has been well documented, but little is known about the role of the MHR in determining the dynamic behavior of CA. The MHR residues are involved in the organization of the CTD, rather than being directly involved in the intermolecular CA-CA interfaces (18, 27, 36, 37, 48). Thus, the F167Y, L171V, and D155Y MHR mutations are probably altering the ability of the CTD to adopt one or more conformations essential for the initiation of capsid formation. By extension, the suppressors (A38V, I190V, and R185W) may correct the defect in assembly by causing an increased propensity of CA to assemble by stabilizing CA in a proassembly conformation.
The particular locations of the suppressors in CA are consistent with this scenario. The A38 residue lies on the surface of the second helix of the NTD in a location where it may modulate NTD conformation and indirectly influence hexamer formation and/or the NTD-CTD interface. This interpretation is further supported by the observation that the A38V suppressor corrects the defects caused by multiple MHR mutations in a temperature-sensitive fashion (49). I190 lies on a slight bulge, a conserved feature in the middle of the dimerization helix (18, 36, 37), that is predicted to form upon the processing of CA from Gag (33). Although I190 is not directly involved in the dimer interface (R. Kingston, personal communication), the I190V substitution may conceivably alter the conformation of the helix and thus affect CTD dimerization and/or NTD-CTD interaction. The identification of multiple mutations with suppressor capabilities near I190 provides further evidence that the control of the conformation of this helix is vital to proper capsid assembly (10, 49, 65). The R185 residue is located near the "top" of the dimerization helix in a position analogous to a Lys residue that is involved in the NTD-CTD interface in HIV (43). Thus, the R185W suppressor substitution is likely influencing the NTD-CTD interface, although an effect on the domain structure is also likely. The increased resistance of the R185W protein to denaturation (Fig. 2B and Table 1) is consistent with the hypothesis that this substitution is acting by stabilizing the CTD domain in an assembly competent form.
The R185 residue may also contribute to the maturation process. In the domain swap model, R185 is located near a bend between the first two helices of the CTD which has been proposed to undergo a transition during maturation (33, 39). The domain swap model, which also predicts that the conserved hydrophobic residues (in RSV, these are F167, L171, and the neighboring F164) undergo a refolding event during maturation, fits the data showing that the hydrophobic MHR mutations disrupt an early event of capsid assembly.
Flexibility in either the conformation of the CA monomer or the intermolecular interactions upon multimerization is needed to build retroviral capsid shells, using fullerene principles (6, 26, 28, 48, 55). Therefore, if the MHR mutations and suppressors alter the range of conformations available to the CA monomer, effects on morphology are predicted. Indeed, many of the double-mutant and suppressor mutant proteins had a limited range of morphologies, e.g., the tubular structures formed by the L171V/A38V double mutant or the spheroids formed by the I190V protein. The effects of MHR and suppressor mutations on an early step of assembly and on the morphologies of the final products formed in vitro support the idea that the shape of retroviral capsids is determined in part by the nucleation process, as recently suggested from the cryoelectron tomographic study of RSV conducted by Butan et al. (15).
Implications for the development of antiviral therapies and drug resistance. The results from the MHR and suppressor mutations emphasize how genetic alteration of CA can drastically influence the multimerization ability of CA protein and alter virus infectivity. These mutations provide valuable tools for probing the structural transformations involved in maturation. Equally important, an understanding of the mechanism whereby suppression works to restore assembly to a maturation-defective virus is likely to have important implications for drug resistance to capsid assembly inhibitors. Two inhibitors, CA-I and CAP-1, block HIV-1 replication by binding to CA (35, 67, 70, 73). Although mutations in CA that alter the binding ability of the inhibitors may confer resistance, the results from the second-site suppressors suggest that drug resistance may also be achieved by mutations in other regions of CA that increase the assembly propensity of the protein.
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ACKNOWLEDGMENTS |
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This work was funded by grant CA100322 from the National Institutes of Health (R.C.C.) and by the Pennsylvania Tobacco Settlement Fund through the PA Department of Health (R.C.C., J.M.F., and I.J.R.).
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FOOTNOTES |
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Published ahead of print on 9 April 2008.
Present address: Department of Pathology, Johns Hopkins School of Medicine, Baltimore, MD 21205.
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) or BSA (
) at equimolar concentrations of 39 µM each. Also shown are the turbidimetric profiles of unmixed WT (
), F167Y (
). 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.