The goal of this study was to determine whether HIV-1 can adapt to changes in residues that are important for maintaining CA structure without losing all biological activity. Our approach was to make mutant constructs that might retain the ability to replicate and thereby present an opportunity to isolate second-site suppressors. Out of 13 mutations in Trp23, two in Phe 40 (F40W, F40Y), and a double mutation (W23F/F40Y), we found only one substitution, W23F, that could satisfy this requirement. This observation emphasizes the critical contribution of Trp23 and Phe40 to the local structure of helices I and II, respectively. Note the proximity of these residues in the view shown in and the central location of Phe in the hydrophobic core ().
The data clearly indicate a strict requirement for an aromatic residue at position 23 and it is interesting that even Tyr cannot substitute for Trp23 (or Phe40), presumably because the OH group of Tyr makes it too hydrophilic. The finding that Trp cannot replace Phe40 may reflect a steric requirement for a less bulky residue at position 40. These results are consistent with the crucial role of residues in helices I and II in the assembly of the HIV-1 capsid (Ganser et al., 2003
; Ganser-Pornillos et al., 2004
; Lanman et al., 2003
; Li et al., 2000
). In this regard, it is also of interest to note that deuterium exchange studies have identified a CA peptide consisting of residues 23 to 40, which appears to be involved in N-terminal homotypic domain interactions in the in vitro
assembled CA protein as well as in immature and mature HIV-1 virus-like particles (Lanman et al., 2004
; Lanman and Prevelige, 2005
Isolation of second-site suppressors is a powerful genetic technique to obtain additional insights into the structural and functional effects of particular mutations. However, there are only a few reports of second-site suppressors of CA mutations (see below), presumably because most mutations in CA are not well tolerated (Auerbach et al., 2003
; von Schwedler et al., 2003
) and the possibilities for suppressing these mutations may be limited. Until now, second-site suppressors have been described for CA mutations in (i) the HIV-1 CypA binding loop (Aberham, Weber, and Phares, 1996
); (ii) an 11-residue segment (Guo et al., 2005
) at the C-terminus of HIV-1 CA that is required for virus replication (Liang et al., 2002
; Liang et al., 2003
; Melamed et al., 2004
); and (iii) the RSV MHR (Bowzard, Wills, and Craven, 2001
In the present study, despite the low probability of success, we were able to isolate two second-site suppressor mutants, W23F/V26I and W23F/V26I/R154K, after prolonged passage in MT-4 cells. However, it appears that only the V26I substitution contributes significantly to suppression of the W23F mutation (), although the additional R154K mutation might have a subtle effect ( and ). The suppression event was specific for MT-4 cells, since W23F could not replicate even with delayed kinetics in other cell lines (see above). Transmission electron microscopy showed that many of the suppressor mutant particles isolated from MT-4 cells have the appearance of WT virus and contain conical cores (; ). Although the infectivity of these mutants (produced in either HeLa or MT-4 cells) was higher than that of W23F, it was still much lower than the WT value (). The reduced infectivity in CEM174 cells, which are used in the LuSIV assay (Roos et al., 2000
), might be related to the fact that similar cells (CEM (12D7)) do not support long-term replication of any of our mutants (data not shown).
The original W23A and F40A mutants (Tang et al., 2003b
) and the HIV-1 His mutant, H84A (Scholz et al., 2005
), were found to have abnormally high amounts of CA in their cores. In the case of W23F, the CA content is somewhat higher than normal, but the amount of CA in the cores of the two second-site suppressor mutants is essentially like that of WT, i.e., ~30% core-associated CA protein () (Tang et al., 2003b
). (A WT value of ~17% has also been reported (Forshey and Aiken, 2003
).) These data are in accord with recent physical determinations indicating that of the ~5000 copies of Gag in the immature virion, only 1000-1500 copies of CA are associated with the core of the mature particle (Briggs et al., 2003
; Briggs et al., 2004
; Lanman et al., 2004
; Vogt and Simon, 1999
Another striking feature of W23A and F40A cores is a reduction in RT content from the WT value of ~30% of total virion RT () (Khan et al., 2001
; Tang et al., 2003b
) to ~5% (Tang et al., 2003b
). Similar findings have been reported for the H84A mutant (Scholz et al., 2005
) and an RSV MHR mutant L171I (Cairns and Craven, 2001
). Surprisingly, the WT level of core-associated RT was not restored in either W23F or in the suppressor mutants () and this might account for the low level of infectivity observed in the LuSIV assay (). More detailed studies of the structure and organization of the ribonucleoprotein complex in HIV-1 cores will be needed to fully understand the relationship between RT levels in cores, RT function, and the ability to generate infectious particles.
Finally, we ask: “How can we explain why V26I partially rescues the W23F mutation?” This question is especially relevant since Val26 is partly exposed on the surface of helix 1, whereas Trp23 is buried within the hydrophobic core () (Momany et al., 1996
; Tang, Ndassa, and Summers, 2002
). Interestingly, the NMR structure of the N-terminal domain of CA (Tang, Ndassa, and Summers, 2002
) indicates that Val26 and Trp23 are actually close to each other. Specifically, among the family of models in the structure file (1GWP.pdb), the smallest separation between the side chain heavy atoms of Trp23 and Val26 ranges from 3.8 to 4.7 Å, with a mean of 4.2 Å. Based on this information, a model of the W23F/V26I structure was constructed such that the additional non-polar methylene group in Ile (CH-CH2
), i.e., compared with Val (CH-CH3
), contacts the Phe side chain and at least partially fills the cavity created by the W23F mutation () (S. R. Durell, personal communication).
Fig. 7 Structural models of WT CA and the W23F/V26I mutant illustrating contacts between the side chains of Trp23 and Val26 or the substituted Phe23 and Ile26 residues. (A) WT. Model number 10 in the 1GWP.pdb structure file of the N-terminal CA domain (Tang, (more ...)
In conclusion, we have shown that it is possible to make a substitution in a residue critical for maintaining HIV-1 CA structure and function, i.e., W23F, which results in virions with a low level of infectivity in a single-round replication assay and moderate loss of the WT phenotype. Since the N-terminal CA structure is defined by stringent parameters and only rarely accommodates even small perturbations at certain residues, it is especially surprising that a second-site suppressor of the W23F mutation could be isolated. These findings are novel and demonstrate that despite the limits imposed on assembly of proper CA structure, HIV-1 is able to partially adapt to even severe structural disruptions.