Rescue of a Folding Defective Glycoprotein by Evolution
To examine the role of a single conserved structure element in folding, we generated a mutant virus lacking the conserved disulfide bond at the base of the V4 domain of HIV-1 gp120 (A). We found that removal of this disulfide bond by replacing the cysteines by alanines (C385A/C418A) led to Env misfolding (van Anken et al., 2008
). Whereas the defective phenotype is no surprise considering the conserved nature of the disulfide, the folding-defective mutant did mediate some cell entry when placed in the context of the complete virus, albeit insufficient to cause a spreading virus infection (van Anken et al., 2008
). A minority of the mutant Env molecules apparently folded correctly, exited the ER, and reached the virion surface to mediate attachment and membrane fusion.
Because of its residual function, we used this mutant for protein evolution studies, to identify and characterize escape routes that result in restoration of Env folding and function in the absence of the conserved disulfide bond. For an 8-mo evolution experiment of mutant virus, SupT1 T-cells were transfected with 10 μg of the molecular clone of the HIV-1LAI
isolate (pLAI) containing the C385A and C418A substitutions (mut
), and cells were passaged until they were wasted due to viral infection. Virus was then passaged cell free onto fresh cells, and the process was repeated for 73 d. At day 73, the env
gene was PCR amplified and sequenced. The sequences revealed two reversions: A418V and T415I (, B and C). The PCR fragment was recloned into pLAI and individual pLAI clones were sequenced, revealing two clones with the A418V and T415I reversions (R2
), whereas one clone only contained the T415I reversion (R1
), indicating that the latter reversion occurred first and that the R2
virus did not constitute the entire virus population yet at day 73. Fresh SupT1 cells then were transfected with 10 μg of the molecular clone containing R2
Env, and evolution was continued for an additional 180 d. The env
gene was PCR amplified and sequenced at the time points indicated by an arrow in B. At week 24, a third reversion occurred: A385V (R3
). Both A385V and A418V were first-site reversions, but the wt
cysteines were not restored, which is consistent with our design of the mutants. Conversion of alanine codons back into a cysteine codon required at least two nucleotide changes, providing a high mutational threshold and favoring alternative repair pathways. The change from alanine to valine represents a relatively simple evolutionary event (GC
C to GT
C). Several other simple evolution routes could have been chosen starting from the alanine codons, but we only found the reversions to valine. Moreover, we did not observe further evolution of the valine codons (B; data not shown). The A385V reversion emerged again in an independent evolution experiment (data not shown), confirming that the evolution to valine is preferred. Inspection of gp120 sequences in the Los Alamos sequence database showed that an isoleucine at position 415 is found in ~6% of the virus isolates, whereas threonine is found in ~80%; the cysteines at positions 385 and 418 are absolutely conserved (http://www.hiv.lanl.gov/
). In summary, we observed the sequential appearance of three amino acid substitutions: T415I, A418V, and A385V, respectively (R1
, and R3
; , B and C).
To establish that the identified substitutions accounted for the revertant phenotype, the relevant env
fragments were recloned into the molecular clone HIV-1LAI
to produce virus stocks. SupT1 T cells were infected with wt
, mutant, and revertant viruses, and subsequent virus spread was monitored (D). The mutant virus did not cause a spreading infection (van Anken et al., 2008
), but viral replication improved increasingly for revertants R1
, and R3
. These results indicate that a three-step evolution process took place upon removal of the V4-base disulfide bond, with all three reversions contributing to the final revertant phenotype.
To further study the contribution of each reversion to the improvement in virus replication we performed quantitative single cycle infection experiments by using reporter cells carrying the luciferase gene under control of the HIV-1 LTR (Wei et al., 2002
). This assay provides a quantitative measure of Env-mediated viral entry, because upon entry the viral Tat protein transactivates the LTR and promotes luciferase expression. We used the assay to compare the different evolved variants and included a number of variants that did not emerge during the evolution experiment to provide a better understanding of each reversion.
In line with the replication data, viral infectivity and hence Env function gradually improved in R1-R3 compared with mut, with R3 displaying infectivity as high as 68% of wt (). To examine the individual and pairwise contribution of each reversion to the revertant phenotype of R3, we constructed viruses lacking the first reversion, T415I, but containing the second or third reversion or both (AV, VA, and VV). Infectivity of both AV and VV was lower than that of R2 and R3, indicating that T415I did contribute to the revertant phenotype of R2 and R3 (). In contrast, the VV variant was not more infectious than the VA variant, suggesting that the second reversion (A418V), although obviously beneficial in R2, did not contribute to the improved phenotype of R3 ().
Figure 2. Reversions restore virus infectivity. TZM-bl reporter cells (~17 × 103; confluence of 70–80%) were infected with 5.0 ng of mutant or wt virus in the presence of SQV in a 96-well plate, and luciferase activity was measured after (more ...)
Restoration of Env Folding and Incorporation into Virions
Because folding-defective Env mutants such as C385A/C418A are retained in the ER, they produce virions virtually devoid of Env molecules (van Anken et al., 2008
). We therefore determined the content of revertant Env molecules on virions, expressed as gp120/p24 ratio, relative to wt
(D, inset). Only ~10% of C385A/C418A Env was found on virus particles relative to wt
, consistent with the severe folding defect measured for this mutant (van Anken et al., 2008
). Revertants R1
, and R3
displayed gp120 virion contents of 17, 62, and 109%, suggesting that protein folding had improved with each successive substitution.
To confirm that improved folding of the revertants accounted for the gain in Env incorporation into virions, we monitored gp120 maturation in HeLa cells by pulse-chase analysis. We compared maturation kinetics of wt
, mutant, and revertants by using three readouts we have developed previously (Land et al., 2003
): 1) formation of disulfide bonds detectable through mobility changes of cellular gp120 in nonreducing SDS-PAGE, 2) signal peptide cleavage visible via reducing SDS-PAGE, and 3) secretion of gp120.
Immediately after the pulse, all gp120 variants displayed the same mobility in reducing and nonreducing gels (), indicating that only few disulfide bonds had formed. After 2 h of chase, wt gp120 migrated faster in the nonreducing gel, as the fully oxidized native state. In contrast, most gp120 molecules of the mutant and revertants remained in the original, relatively unfolded, state, whereas a minority was present as faster migrating “smear,” especially in R3, representing partially oxidized folding intermediates. The mutant did not display detectable levels of the native state even after 4 h, but the revertants did reach a native-like state. Whereas for R1 and R2 the native band was faint, a considerable fraction of R3 reached native after 4 h of chase. Approximately 50% of R3 Env was still immature after 4 h, perhaps because only ~50% folds correctly. If so, this 50% would be incorporated into the virion very efficiently (D, inset). Alternatively, the R3 protein folds slower than wt and will continue to fold successfully after 4 h. We have not studied later time points because the pulse-chase data in this expression system become less reliable after 4 h. Regardless of the scenario, the reversions did not completely compensate for the lack of a disulfide bond.
Figure 3. Reversions restore gp120 folding. HeLa cells were infected with VVT7 and transfected with plasmids encoding wt gp120, mut, revertant (R1, R2, or R3) or two additional mutants FG1 and FG2. Cells were pulse labeled for 5 min and chased for the indicated (more ...)
An unusual property of Env is that it must undergo some oxidative folding before its signal peptide can be removed (Land et al., 2003
). We therefore used signal peptide cleavage as an additional measure for successful folding. In reducing gels, the single band present at the end of the pulse, corresponding with the preprotein form of gp120 (Ru; reduced uncleaved), changed into two bands, the lower one corresponding to cleaved gp120 (Rc; reduced cleaved). After 4 h of chase, uncleaved species were no longer detectable for wt
. Signal peptide cleavage was significantly reduced for the mutant and R1
, but R2
and in particular R3
displayed restored cleavage.
The third readout confirmed restoration of productive R3
folding: unlike the mutant, R1
, and R2
, a substantial fraction of R3
gp120 molecules had been secreted after 8 h (, bottom). Secreted wt
occurred as a compact band, but secreted R3
smeared out, most likely because of changed glycan modifications (Trombetta and Parodi, 2003
). These results are consistent with the Env incorporation and virus replication experiments, and they confirm that virus-driven evolution resulted in a gradual repair of gp120 folding.
To analyze the outcome of the R3
folding process, we performed neutralization experiments with the wt
viruses by using reagents that are dependent on gp120 conformation. The viruses were preincubated with the respective reagents and subsequently added to target cells containing a luciferase reporter gene under control of the HIV-1 LTR (Wei et al., 2003
). First, we tested the ability of CD4 to inhibit these viruses. CD4 binding is highly dependent on conformation making contacts with both the inner and outer domain of gp120 (Kwong et al., 1998
were equally sensitive to inhibition by CD4-IgG2, indicating that the structure of the CD4 binding site on these viruses was similar (A). b12 is a potent broadly neutralizing antibody with a conformational epitope that overlaps with the CD4 binding site, although most contacts are made with the outer domain of gp120 only (Pantophlet et al., 2003
; Zhou et al., 2007
). The b12 epitope is close to the 385–418 disulfide bond and residues in this area, e.g., N386, are known contact sites for b12 (Pantophlet et al., 2003
; Zhou et al., 2007
). Overall, the inhibition curves of wt
were similar, although R3 was slightly less sensitive to inhibition by b12 (B). We next performed neutralization experiments with 2G12, which binds to a conformation-dependent mannose epitope on N-linked carbohydrates (Trkola et al., 1996
; Scanlan et al., 2002
; Sanders et al., 2002b
). The carbohydrate at N386, located next to the 385–418 disulfide bridge, is involved in 2G12 binding (Trkola et al., 1996
; Sanders et al., 2002b
; Scanlan et al., 2002
) and local structural perturbations can affect the binding of 2G12 (Sanders et al., 2008
). We did not observe major differences in inhibition by 2G12 (C). Combined these results indicated that the R3
gp120 folded into a structure that is similar to the wt
Figure 4. wt and R3 viruses show similar sensitivities for conformational antibodies. Single cycle infection experiments in TZM-bl cells containing a luciferase reporter construct under control of the HIV-1 LTR were carried out in the presence of escalating concentrations (more ...)
Molecular Dynamics Simulations Reveal Restoration of Local β-Sheet Structure
To understand improved folding and function of the revertants at the atomic level, we performed MD simulations of gp120 variants. Complete sampling of complete protein (un)folding by MD simulation would require enormous amounts of computational capacity, which cannot be mustered for a protein the size of gp120. We therefore carried out a comparative study of individual variants in their folded state at a residue-specific level. Changes in dynamics and stability in the gp120 native state have implications for its folding as the quality control recognition system is closely associated with structural integrity. Events of local unfolding, which are reflected in the loss of intramolecular hydrogen bonding and increased structural fluctuation, are indicative of the tendency toward global unfolding.
We performed simulations with wt gp120 and all variants (mut, R1, R2, and R3, and two additional mutants FG1 and FG2; see below). Starting from identical conformations, simulations of gp120 variants were conducted in explicit solvent (water) for an extended period (10 ns at 300 K). Despite the small differences in primary sequence (2 or 3 of 317 amino acids), the time to reach equilibrium required for each variant varied significantly as judged from the backbone (N, Cα, and C′) positional root-mean-square deviation (RMSD) evolution profiles (Supplemental Figure 1). Apparently, point mutations can cause subtle structural perturbations that require a longer time scale to reach equilibrium. We therefore focused subsequent analyses on the 5- to 10-ns segments, for which all variants were stable with little variation in secondary structure and nonbonded energies (Supplemental Table 1). Still, overall protein structure seemed very similar throughout the simulations for wt, mut, and revertants. We also carried out several simulations for wt, mut, R1, R2, and R3 at elevated temperatures (400K). Overall dynamics were similar as judged from the overall secondary structure content (with slightly higher RMSD and relative root-mean-square fluctuation [RMSF]). We concluded that neither the lack of disulfide bond 385–418 nor the reversions caused major structural changes in the native structure of gp120. We therefore focused on local perturbations of the structure in the vicinity of the mutated residues.
Atomic structures are available of the core of gp120 in different states: unliganded SIV gp120, CD4-bound HIV-1 gp120, and HIV-1 in complex with the neutralizing antibody b12 (Kwong et al., 1998
; Kwong et al., 2000
; Chen et al., 2005
; Zhou et al., 2007
). These structures reveal remarkable structural rearrangements of gp120 upon binding to CD4: the inner and outer domains of gp120 undergo large reorientation upon CD4 binding, whereby a four-stranded bridging sheet is formed. Despite the large differences in the free, CD4-bound and b12-bound states, the six-stranded β-barrel structure in the outer domain of gp120, which includes (the reverted) residues 385, 415, and 418, is particularly unaffected by these conformational rearrangements (A). The backbone Cα atoms of the β-barrel exhibited a marginal deviation between the states with a positional RMSD of 0.17 nm (data not shown). The six β-strands of the barrel (β16, β17, β19, β13, β12, and β22) are bridged by three disulfide bonds, one of which is formed by the C385/C418 pair (A). The first and last strands of the β-barrel (β16 and β22) are not linked through classical β-sheet interactions (i.e., interstrand backbone–backbone hydrogen bonds are absent), but only through the disulfide bond between C378 and C445. We turned to detailed analysis of the β-barrel for parameters that afford sufficient statistics, namely, RMSF and hydrogen bond occurrence. The local motions were sampled reliably over the equilibrated 5-ns trajectories. The RMSF and hydrogen bond occurrence analyses can provide evidence of local unfolding events, i.e., loss of native structures, which have implications for the folding process.
Figure 5. Reversions change dynamics within a six-stranded β-barrel in the outer domain of HIV gp120. (A) Structure of HIV gp120 core and the β-barrel structure in the outer domain. The four-stranded bridging sheet, which joins the inner and outer (more ...)
As a first measure for structural stability (or integrity) of the β-barrel, we evaluated the interstrand backbone–backbone hydrogen bond occurrence (percentage of time) between the individual strands of the barrel [τ (variant
), defined in the legend of ; Kabsch and Sander, 1983
]. The different side chains introduced by the mutations, and reversions resulted in large fluctuations of interstrand hydrogen bond occurrence within the 6-stranded β-barrel (B). The folding deficient mutant showed a substantial loss of total interstrand hydrogen bonding with a τTotal
) of 81.1% of wt
, whereas in the revertants the τTotal
was largely restored: (R1
) 90.7%, (R2
) 88.4%, and (R3
) 90.9% (B). This indicates that the backbone structure of the β-barrel is sensitive to elimination of the disulfide bond. Yet, only the first reversion contributed to a substantial gain in interstrand backbone–backbone hydrogen bonding.
Next, we explored whether the differences between wt, mut, and revertants correlated to alterations in side chain packing. We assessed the degree of flexibility of individual residues within the barrel by calculating their relative RMSF (i.e., the sum of atomic positional RMSF per residue normalized to the corresponding wt values). A lower relative RMSF value is indicative for improved ordering of a particular side chain. Taking wt as 100%, mut had the broadest distribution of per residue RMSF values with a maximum as large as 275% (C). The distributions of per residue RMSF as well as the overall averages of R1, R2, and R3 reduced progressively, reflecting improved side chain packing. To visualize this, we compared selected side chain structure ensembles of wt, mut, and R3 throughout the simulations (D). The conformations of several hydrophilic side chains were more heterogeneous in mut than in R3 and wt (e.g., Y384 and S375), implying more flexibility in the mutant and increased order in the revertant. In addition, the relative side chain orientations of R3 were more similar to those in wt than in mut (e.g., S375, N386, and R419), thereby restoring side chain–side chain hydrogen bond formation. For example, the hydrogen bond between the hydroxyl group of S375 (OHγ) and the tyrosyl group of Y384 (Oη) (highlighted by an orange arrow in D) was highly stable in wt with an occurrence of 95%, and it was destabilized in mut with an occurrence of only 16% (14% of which was bridged by a water molecule). In R3, this hydrogen bond was recovered (84% occurrence, without any additional water bridge), implying a higher degree of compactness. We found similar effects for hydrophobic side chain contacts in the interior of the barrel (data not shown).
When examining each residue within the β-barrel, we did not find clear evidence of changes in relative RMSF between wt, mut, and R1–R3 in the strands in which the mutations were introduced (β17 and β19) (). Other interesting correlations did emerge, however, for residues that have a high RMSF in mut but show progressive RMSF reduction in R1–R3 (): 1) many residues in β16 (all residues except H374) and β22 (all residues except I443); 2) strand-end residues (C378, T413, T297, H330, and T450); 3) cysteines (or substituted residues; C378, C/A/V418, C296, and C445); and 4) residues flanking cysteines (N377, Y384, T297, N295, H330, P417, R440, R444, and S446). Because the barrel is buckled up by a number of disulfide bonds, the loss of one of these may lead to pronounced long-range perturbations, propagated through the barrel. This may explain the RMSF changes in cysteines and flanking residues distant from the mutated sites. The RMSF evolution of β16 and β22 seems somewhat surprising, but these strands are only connected by the 378–445 disulfide bond and not by sheet interactions. Hence, structural perturbations in the barrel may have a more pronounced impact on these two strands.
Absolute positional RMSF for individual residues within the β-barrel. Residues 385, 418, and 415 are labeled in bold and underlined.
The MD analysis hence revealed that the functional and biochemical data can be explained at the atomic level by improved backbone–backbone hydrogen bonding and local side chain packing within the β-barrel. Our results suggest that the disulfide bond, despite its conservation, can be functionally replaced by alternative local structural features within the β-barrel.
Strengthening β-Sheets or Filling the Gap?
During our evolution experiments, we consistently encountered introduction of β-branched amino acids (T415I, A418V, and A385V) to restore folding efficiency of gp120 (). The improvements we found in backbone hydrogen bonding and side chain packing within the β-barrel could be attributed to two factors. Knowledge-based predictions (Chou and Fasman, 1974
; Levitt, 1978
) and theoretical calculations (Rossmeisl et al., 2003
) have shown that β-branched amino acids such as valine and isoleucine exhibit high β-sheet propensities: they promote β-sheet formation. In contrast, introduction of a methyl group (when valine replaces alanine) may fill the gap introduced by replacement of cysteine by alanine. Both β-sheet propensity and “gap filling” could therefore be responsible for the improved folding of revertant gp120. To discriminate between these two scenarios, we constructed two additional hydrophobic mutants where one of the alanines was replaced by a non–β-branched leucine rather than valine, in the context of the R1
variant (i.e., including the T415I reversion; A): fill-gap mutant 1 (FG1
: C385A/C418L/T415I; ALI) and FG2
(C385L/C418A/T415I; LAI). Valine and leucine have similar biochemical properties (both are hydrophobic and the size difference is one methyl group), but valine is β-branched and is accommodated much better in β-sheets than leucine is. FG1
displayed minimal infectivity in single cycle infection experiments (), mirrored by minimal replication (B). The folding assays revealed that FG1
did not fold properly: negligible FG1
gp120 reached the native state and signal peptide cleavage was also minimal (). The folding, infectivity and replication of FG2
was better than of FG1
, but less than wt
, and R3
(, , and B), indicating that replacing A385 with leucine did improve protein folding to some extent. Both FG
variants showed decreased in silico stability compared with R1
, and R2
, as indicated by decreased hydrogen bonding and increased per residue RMSF ( and ).
Figure 7. Virus replication and folding of the non–β-branched fill-gap mutants FG1 (C385A/C418L/T415I) and FG2 (C385L/C418A/T415I). (A) Sequences of the V4 loop and flanking regions of wt, mutant, and revertant viruses. The original mutations are (more ...)
Thus, at position 418 a valine can improve folding and function, whereas a leucine cannot. In fact, leucine was inferior to alanine. At position 385, leucine conferred improved folding and function compared with alanine, but it was inferior to valine. The results imply that β-branching improves stability of the β-barrel and hence gp120 folding. Gap filling does contribute somewhat to improved gp120 folding, but only at position 385 and not 418.