Our previous studies identified side chains in the NTD and CTD that are critical for HIV-1 capsid stability
[11, 47]. At the time of those studies, the positions of the mutations in the CA hexamer were not known, as the high-resolution structure of the hexamer had not yet been reported. Subsequent studies showed that several of the NTD substitutions that perturbed capsid stability (P38A, E45A, T54A) were located at the NTD-NTD interface
, while some of the CTD substitutions (K203A, Q219A) were present at the 3-fold CTD-CTD interhexameric interface
. In the present study, we focused on the role of the NTD-CTD intersubunit interface, which was not well characterized at the time of the previous studies, in HIV-1 capsid structure and function.
High-resolution structural studies provided the foundation for this investigation. The CA hexamer structure suggested that the NTD-CTD interface is likely to be important for either capsid assembly or stability. Previous structural and biochemical studies of HIV-1 and other retroviral capsids demonstrated that this interface is present in the capsid lattice formed by assembly of CA in vitro[6, 8, 24–28, 34]. However, the role of the NTD-CTD interface in the virion and its contribution to capsid function had not been demonstrated.
Our engineered disulfide crosslinking data confirms that the NTD-CTD interface is a component of the native HIV-1 capsid lattice. Crosslinking between Cys residues at positions 68 and 212 resulted in the formation of monomer, dimer, trimer, tetramer, pentamer and hexamer bands. Lack of crosslinked CA species in the single mutants (M68C and E212C) showed that the banding results from disulfide bonding between the engineered cysteines (Figure
1D). Further evidence for the NTD-CTD hexameric interface was also provided by crosslinking observed in the Q63C/Y169C, A64C/L211C, M144C/M215C and M68C/L211C mutants, though at reduced efficiency (Figure
1B), which was not enhanced upon chemical oxidation with copper O-phenanthroline (our unpublished observations). Engineered crosslinking provides an experimental probe for the formation of an NTD-CTD interface in mutant virions. We have recently employed this approach to study the formation of the individual capsid intersubunit interfaces during maturation
, and it may also prove useful for studying the effects of small molecules on capsid assembly and structure.
We also observed that amino acid substitutions at the NTD-CTD interface affect capsid assembly. Three of the mutants (A64D, M68A, D166A) exhibited normal Gag processing, and produced particles at wild type levels. However, the virions had significant reductions in the number of conical capsids (Figure
1). D166 is an interdomain helix-capping amino acid, whose interactions with helix 4 of an adjacent subunit provides an amino terminal capping for the NTD-CTD interaction
. Other substitutions at the interface, including H62A, K140A and M215A, also perturbed capsid structure. However, these three substitutions led to altered processing of Gag, suggesting that the changes may affect capsid structure through maturation defects. A previous study of H62A had also reported that this mutant is impaired for HIV-1 capsid assembly
. H62 occupies the hydrophobic core of the NTD-CTD interface forming stacking interactions with F32 and Y145
[6, 8]. The phenotype of the H62A mutant is also reminiscent of the effects of CAP-1, a small molecule capsid assembly inhibitor that binds the NTD pocket and disrupts the packing of the Phe32 and Tyr145 aromatic side chains
[8, 49]. K140 and M215 lie in close proximity to the helix-capping amino acids R143 and Q219, respectively
. Their proximity to helix-capping amino acids suggest that substitutions at these positions may alter contacts at the NTD-CTD interface and perturb capsid assembly. Previous work from another group had shown that the NTD-CTD interface substitutions Y169A and L211A also induced severe Gag processing defects
, which may result from the generation of novel cleavage sites or from structural alterations induced by the mutations.
A major finding from this work is that substitutions at the NTD-CTD interface alter capsid stability (Table
1). Most of the substitutions at the NTD-CTD interface produced virions with unstable capsids, all of which were poorly infectious. The mutations that destabilized the capsid in vitro also resulted in accelerated uncoating in target cells, as indicated by the reduced ability of the mutant particles to abrogate restriction by TRIMCyp. The in vitro uncoating data agreed well with the abrogation of restriction results. However, Q63A, R167A, and E180A were exhibited rapid uncoating in vitro, but these mutants abrogated restriction with wild type efficiency. Our understanding of the mechanism of restriction abrogation is limited; however, we have shown that addition of a second-site suppressor mutation restores the abrogation activity of the P38A mutant without reversing its intrinsic capsid instability
, suggesting that mutations can perturb uncoating in target cells without altering the biochemical stability of the capsid. The Q179A substitution also had no apparent effect on capsid stability or viral infectivity. Structurally, Q179 is located at the end of helix 8 and is near N57, Q63 and M66 in the NTD. Currently, it is not clear why the Q179A substitution was not deleterious to capsid function; we surmise that the location of this side chain near the edge of the interface may be a factor.
The R162A mutant exhibited conical capsids that were unstable (Figure
3D and Figure
5C). The mutant was impaired for reverse transcription in target cells (Figure
6), was poorly infectious and was impaired for abrogation. We attribute the phenotype of this mutant to a capsid stability defect. Structurally, R162 is near the stack formed by Y145, H62 and F32; the R162 guanidinium group is thought to stabilize the stacking of these aromatic side chains
. It is plausible that the substitution of alanine at this position changes the juxtaposition of the Y145 and F32 side chains at the NTD-CTD interface, thus altering the structure at the interface and rendering the capsid unstable.
In an earlier study we reported that E45A exhibited an impairment in reverse transcription in target cells
. More recently, we observed that this mutant is, in fact, competent for reverse transcription in target cells
. In the present work, we observed that the hyperstable V165A mutant is impaired for reverse transcription, like the unstable mutants (Figure
6). Preliminary analysis of the cores purified from V165A by immunoblotting revealed that the mutant cores are enriched in unprocessed Gag and Gag-Pol (our unpublished observations). We speculate that the presence of these intermediates prevented reverse transcription by interfering with the activity of the processed RT or by perturbing capsid structure.
Our results highlight the NTD-CTD intersubunit interface as a potentially attractive target for therapeutic intervention. Recently, small molecule inhibitors targeting HIV-1 CA have been identified
[50–53]. One of these, PF-3450074 (PF74), binds to a pocket in the NTD located near the NTD-CTD interface and destabilizes the capsid
[41, 52]. Substitutions in the interface can lead to resistance by blocking the binding of PF74
, but these mutations may also result in reductions in viral fitness. Another small molecule, CAP-1, has been shown to alter subunit interactions at the NTD-CTD interface
[8, 49, 53]. Our finding that many NTD-CTD interface mutants exhibit impaired replication suggests that inhibitors targeting this interface may exhibit a high barrier to emergence of resistance. Further efforts to design capsid-targeting HIV-1 inhibitors will benefit from a thorough understanding of the structure and function of the viral capsid.