Since many years the CD4bs is considered a very interesting target to block HIV-1 infection. This vulnerable and conserved site on the otherwise genetically diverse envelope protein is essential for the viral entry process. Many studies are being carried out to precisely define the binding surface of CD4 and to pinpoint critical amino acids involved in the gp120-CD4 interaction. So far it is defined as a discontinuous epitope with several distinct regions of gp120 involved [11
]. More than half of the gp120-CD4 interaction surface is formed by the gp120 residues 365–371 and 425–430 as well as the amino acids lining the Phe43-cavity (Trp112, Val255, Thr257, Glu370, Phe382, Tyr384, Trp427, Met475 and the main chains of 256 and 375–377). Although these lining residues contribute little to the direct interaction with CD4, they can certainly have an effect on the gp120-CD4 interaction or on the binding of CD4bs antibodies [11
]. Most of the interactions between CD4 and the envelop protein are dedicated to the outer domain with the CD4 binding loop as central focus. This loop, formed by ten continuous amino acids (364–373), is essential for the CD4-gp120 binding. Several CD4bs binders, neutralizing and non-neutralizing, have been described to date [26
]. In general, shared regions involved in binding of CD4 and most CD4bs mAbs are the amino acids 257, 368–370, 421–427, and 457. Changes in amino acids Asp368 and Glu370 disrupt the binding of CD4 in CD4bs mAbs and are therefore critical for the CD4bs epitope [11
Here, we describe mutations found in viruses resistant to the miniCD4 proteins M48 and M48U1, two highly active CD4bs inhibitors. Resistance was induced to evaluate the evolution of the virus under miniCD4 pressure. Overall, most mutations found are situated in the outer domain, which makes up the major part of the CD4bs. Two mutated residues, V255 and S375, both highly conserved, contact the Phe43-cavity and are known to influence the interaction of CD4 or CD4bs antibodies with the envelope protein [11
]. A methionine at position 255, found in one of the SF162 M48U1 resistant viruses, together with a S375N and L494V substitution presumably destabilizes and/or occluded the Phe43-cavity (Figure A). A decrease in affinity of M48 towards the SF162 gp120 V255M mutant suggests that the CD4bs region changed to some extent. Previously, a V255E substitution was shown to be responsible for in vitro
selected sCD4 resistant viruses [54
]. The polar amino acid serine at position 375 is mutated in all M48U1 resistant viruses. The arginine side chain at this position is predicted to fill the gp120 Phe43-cavity, the main target of M48U1, implying a steric hindrance to the approach of the cyclohexylmethoxy moiety harbored by the modified Phe23 of M48U1 (Figure B). As the ancestor miniCD4 protein M48 is lacking this extra moiety, it does not penetrate as deep into the Phe43-cavity, and therefore attachment of this miniCD4 protein is still possible in the presence of Arg375 (Figure B). In concordance, our binding studies revealed a significant decrease in affinity of M48U1, compared to the wild type gp120 affinities, towards the SF162 gp120 S375R mutant, but the same was not observed for M48. In addition, we showed that an asparagine instead of a serine on 375, which does not obstruct the Phe43-cavity, had no dramatic effect on the interaction between both M48U1 and M48 and the mutant gp120. Previously, other groups have reported on the importance of position 375 in interactions of gp120 with CD4 and with CD4bs inhibitors. McKeating et al.
described a virus where a single S375N substitution conferred the virus resistant to a neutralizing human serum containing CD4bs antibodies and another group reported on this substitution in viruses resistant towards sCD4 and NBD-556, a small molecule that mimics CD4 [54
]. Next, a tryptophan substitution on 375 fills the Phe43-cavity and forces gp120 into a CD4-bound conformation, which seems to contradict with the observed cross-resistance against 17b in this study [57
]. This mutation was also involved in resistance towards some CD4bs compounds from the BMS family of entry inhibitors (BMS-806, #155, and BMS-488043) [54
Figure 5 Close-up views of both V255M and S375R mutants in interaction with M48U1. (A) Close-up of the Val255 residue in space fill (left) and of the mutant Met255 residue in space fill (right) showing a steric clash with the cyclohexylmethoxy moiety at the para (more ...)
The histidine at position 105, highly conserved and part of the inner domain, was only found mutated in the M48 BaL resistant virus; but nevertheless H105Y conferred resistance, not only to M48, but also to M48U1 and sCD4. However, since this mutation was only found in BaL, it may be strain-specific.
Somewhat controversial data were collected for the D474N substitution. The SF162 viruses resistant towards M48 and one of the SF162 viruses resistant against the combination of M48 and M48U1 (rCombiSF162_a) all selected for this D474N substitution as a single mutation. This mutation was shown to decrease the entry efficiency into TZM-bl cells by 39%, but no significant differences were found in binding affinities for M48 and M48U1 towards the D474N mutant SF162 gp120 protein. Furthermore, the pBRNL4.3 replication competent clone and the mutant pseudoviruses carrying D474N failed to reproduce the resistance observed with the SF162 resistant strains. Moreover, this mutation appears quite common in naturally occurring viruses (Table ). A previous study reported a D474A mutant with nearly wild type affinity for CD4-Ig, but with a marked decrease in neutralization sensitivity [59
]. This mutant was also shown to impair viral fusion and fitness, especially for the BaL strain [42
]. However, as observed in gp120 three-dimensional structures, Asp474 makes a strong hydrogen bond with Arg476, which is impossible with an alanine residue at this position. Notably, the D474A mutation was also not detected in naturally occurring viruses (Los Alamos HIV-sequences database). Taken together, we do not have a valuable mechanistic explanation for this D474N resistant mutant to date.
It is important to take into consideration the drawbacks of the different techniques used. First, in silico
modeling and fluorescence polarization binding studies using monomeric gp120 proteins are not fully representative for the native gp120 and gp41 structure; nor do they model the gp120-gp41 interaction, the interaction of the variable loops, and the interaction between the three units that make up a functional spike completely correct. Secondly, there is considerable evidence suggesting that the genetic environment is of importance for optimal envelope interactions and functioning [60
]. Expressing Env in a non-isogenic backbone could affect the quaternary structure of the envelope protein and hence its function.
By analyzing 3045 sequences of the Los Alamos Database, we found that most mutated amino acid residues are strongly conserved across HIV-1 clades. This conserved nature of the mutated positions strongly suggests that they are critical for the survival of the virus. So, the next question we wanted to address was if the different mutations had an impact on entry efficiency. Therefore, we infected TZM-bl cells with different pBRNL4.3 viruses containing the envelope from wild type or mutant SF162. We showed that all gp120 mutants tested entered target cells less efficiently compared to WT virus. Two mutations resulted in a severe reduction in entry efficiency, with almost no infection observed for H105Y and V255M. The S375R substitution was responsible for a 67% reduction in entry efficiency. Finally, the effect on entry was less pronounced for the viruses bearing the S375N, G471R, and D474N substitutions. Surprisingly, a D474A substitution was previously shown to have a severe effect on viral infectivity in a BaL pseudovirus, but the same was not true for a YU-2 pseudovirus [42
]. Again, these results show the importance of the envelope environment for the phenotype.
Skewing viruses towards a CD4 independent phenotype can be a concern when using CD4 mimics. Therefore, we evaluated the viral growth on CD4 negative HOS cells and on CD4low MDM. There was no viral growth observed on CD4 negative HOS cells, whereas all viruses were able to grow on HOS CD4+ CCR5+ cells. Evaluation of the growth kinetics in MDM revealed that WT viruses were more efficiently replicating in MDMs than the resistant ones. Taken together, there is no evidence that the mutations we have identified as key to the development of resistance against the miniCD4 proteins M48 and M48U1 are rendering the virus less dependent on CD4 for entry.
Finally, we wanted to know if the observed mutations had an impact on the inhibitory potential of other CD4bs inhibitors. Therefore, we tested the resistant viruses towards some other CD4bs inhibitors, the mAbs 4E10, 2F5, 17b, 447-52D and the NNRTI TMC120. Taken together, we observed cross-resistance towards all other CD4bs inhibitors to some extent. All viruses except one (rM48BaL) became cross-resistant towards the nanobody A12. The exact recognition site is not yet revealed, but A12 is considered to target a region within or close to the CD4bs since it competes with CD4 and b12 for gp120 binding [22
]. Our results indeed support this prediction. Furthermore, only the BaL viruses resistant towards M48U1 showed wild type levels of sensitivity towards sCD4, consistent with data published about the S375W mutation, while the other viruses showed some cross-resistance. Residues lining the Phe43-cavity, such as V255 and the main chain of S375, are known to possibly impact the binding of CD4 or CD4bs antibodies [11
]. Because glycine at position 471 and aspartic acid at 474 have been described to interact with CD4, b12, and VRC01 [11
], the observed cross-resistance of some viruses was not surprising. Some low level cross-resistance against the mAb b12 was observed for the M48 SF162 resistant viruses and for the viruses resistant towards the combination. All these viruses had the exact same position (Asp474) mutated which could account for the observed resistance.
All SF162 resistant viruses were cross-resistant towards the mAb 17b, which targets a CD4-induced region, overlapping the conserved co-receptor binding site. Also some cross-resistance towards the V3 mAb 447-52D was observed for all SF162 resistant viruses carrying the D474N mutation. These results, together with the decreased affinity for 17b, suggest a more occluded co-receptor region in the resistant viruses.
CD4 mimetics are interesting antiretrovirals, mainly because they target a highly conserved site of the HIV envelope protein and confer broad and extremely potent neutralizing capacity. A point of concern, as with many antivirals targeting the envelope, is the relative ease of resistance acquisition against these molecules. However, changes in the highly conserved CD4bs often come at a cost for the virus. Combining miniCD4s with other entry inhibitors or physically linking CD4 miniproteins with molecules targeting e.g. the CD4i site, may increase the barrier for resistance. These and other strategies are currently under investigation.