Resistance to the CCR5 Inhibitor 5P12-RANTES Requires a Difficult Evolution from CCR5 to CXCR4 Coreceptor Use

Viral resistance to small molecule allosteric inhibitors of CCR5 is well documented, and involves either selection of preexisting CXCR4-using HIV-1 variants or envelope sequence evolution to use inhibitor-bound CCR5 for entry. Resistance to macromolecular CCR5 inhibitors has been more difficult to demonstrate, although selection of CXCR4-using variants might be expected. We have compared the in vitro selection of HIV-1 CC1/85 variants resistant to either the small molecule inhibitor maraviroc (MVC) or the macromolecular inhibitor 5P12-RANTES. High level resistance to MVC was conferred by the same envelope mutations as previously reported after 16–18 weeks of selection by increasing levels of MVC. The MVC-resistant mutants were fully sensitive to inhibition by 5P12-RANTES. By contrast, only transient and low level resistance to 5P12-RANTES was achieved in three sequential selection experiments, and each resulted in a subsequent collapse of virus replication. A fourth round of selection by 5P12-RANTES led, after 36 weeks, to a “resistant” variant that had switched from CCR5 to CXCR4 as a coreceptor. Envelope sequences diverged by 3.8% during selection of the 5P12-RANTES resistant, CXCR4-using variants, with unique and critical substitutions in the V3 region. A subset of viruses recovered from control cultures after 44 weeks of passage in the absence of inhibitors also evolved to use CXCR4, although with fewer and different envelope mutations. Control cultures contained both viruses that evolved to use CXCR4 by deleting four amino acids in V3, and others that maintained entry via CCR5. These results suggest that coreceptor switching may be the only route to resistance for compounds like 5P12-RANTES. This pathway requires more mutations and encounters more fitness obstacles than development of resistance to MVC, confirming the clinical observations that resistance to small molecule CCR5 inhibitors very rarely involves coreceptor switching

Fitness Epistasis and Constraints on Adaptation in a Human Immunodeficiency Virus Type 1 Protein Region

Fitness epistasis, the interaction among alleles at different loci in their effects on fitness, has potentially important consequences for adaptive evolution. We investigated fitness epistasis among amino acids of a functionally important region of the human immunodeficiency virus type 1 (HIV-1) exterior envelope glycoprotein (gp120). Seven mutations putatively involved in the adaptation of the second conserved to third variable protein region (C2–V3) to the use of an alternative host-cell chemokine coreceptor (CXCR4) for cell entry were engineered singly and in combinations on the wild-type genetic background and their effects on viral infectivity were measured. Epistasis was found to be common and complex, involving not only pairwise interactions, but also higher-order interactions. Interactions could also be surprisingly strong, changing fitness by more than 9 orders of magnitude, which is explained by some single mutations being practically lethal. A consequence of the observed epistasis is that many of the minimum-length mutational trajectories between the wild type and the mutant with highest fitness on cells expressing the alternative coreceptor are selectively inaccessible. These results may help explain the difficulty of evolving viruses that use the alternative coreceptor in culture and the delayed evolution of this phenotype in natural infection. Knowledge of common, complex, and strong fitness interactions among amino acids is necessary for a full understanding of protein evolution

"Resistance" to PSC-RANTES revisited: two mutations in human immunodeficiency virus type 1 HIV-1 SF162 or simian-human immunodeficiency virus SHIV SF162-p3 do not confer resistance

Resistance of human immunodeficiency virus type 1 (HIV-1) to small-molecule CCR5 inhibitors is well demonstrated, but resistance to macromolecular CCR5 inhibitors (e.g., PSC-RANTES) that act by both CCR5 internalization and receptor blockade had not been reported until recently (3). The report of a single simian-human immunodeficiency virus SHIV(SF162-p3) variant with one V3 and one gp41 sequence change in gp160 that conferred both altered replicative fitness and resistance to PSC-RANTES was therefore surprising. We introduced the same two mutations into both the parental HIV-1(SF162) and the macaque-adapted SHIV(SF162-p3) and found minor differences in entry fitness but no changes in sensitivity to inhibition by either PSC-RANTES or the small-molecule allosteric inhibitor TAK-779. We attribute the earlier finding to confounding fitness effects with inhibitor sensitivity

Conserved changes in envelope function during human immunodeficiency virus type 1 coreceptor switching

We studied the evolution of human immunodeficiency virus type 1 (HIV-1) envelope function during the process of coreceptor switching from CCR5 to CXCR4. Site-directed mutagenesis was used to introduce most of the possible intermediate mutations in the envelope for four distinct coreceptor switch mutants, each with a unique pattern of CCR5 and CXCR4 utilization that extended from highly efficient use of both coreceptors to sole use of CXCR4. Mutated envelopes with some preservation of entry function on either CCR5- or CXCR4-expressing target cells were further characterized for their sensitivity to CCR5 or CXCR4 inhibitors, soluble CD4, and the neutralizing antibodies b12-IgG and 4E10. A subset of mutated envelopes was also studied in direct CD4 or CCR5 binding assays and in envelope-mediated fusion reactions. Coreceptor switch intermediates displayed increased sensitivity to CCR5 inhibitors (except for a few envelopes with mutations in V2 or C2) that correlated with a loss in CCR5 binding. As use of CXCR4 improved, infection mediated by the mutated envelopes became more resistant to soluble CD4 inhibition and direct binding to CD4 increased. These changes were accompanied by increasing resistance to the CXCR4 inhibitor AMD3100. Sensitivity to neutralizing antibody was more variable, although infection of CXCR4-expressing targets was generally more sensitive to neutralization by both b12-IgG and 4E10 than infection of CCR5-expressing target cells. These changes in envelope function were uniform in all four series of envelope mutations and thus were independent of the final use of CCR5 and CXCR4. Decreased CCR5 and increased CD4 binding appear to be common features of coreceptor switch intermediates

CCR5 Mutations Distinguish N-Terminal Modifications of RANTES (CCL5) with Agonist versus Antagonist Activity

CCR5 is the major HIV-1 entry coreceptor. RANTES/CCL5 analogs are more potent inhibitors of infection than native chemokines; one class activates and internalizes CCR5, one neither activates nor internalizes, and a third partially internalizes without activation. Here we show that mutations in CCR5 transmembrane domains differentially impact the activity of these three inhibitor classes, suggesting that the transmembrane region of CCR5, a key interaction site for inhibitors, is a sensitive molecular switch, modulating receptor activity

Evolution of CCR5 Use before and during Coreceptor Switching▿ †

The envelope gene (env) of human immunodeficiency virus type 1 (HIV-1) undergoes rapid divergence from the transmitted sequence and increasing diversification during the prolonged course of chronic infection in humans. In about half of infected individuals or more, env evolution leads to expansion of the use of entry coreceptor from CCR5 alone to CCR5 and CXCR4. The stochastic nature of this coreceptor switch is not well explained by host selective forces that should be relatively constant between infected individuals. Moreover, differences in the incidence of coreceptor switching among different HIV-1 subtypes suggest that properties of the evolving virus population drive the switch. We evaluated the functional properties of sequential env clones from a patient with evidence of coreceptor switching at 5.67 years of infection. We found an abrupt decline in the ability of viruses to use CCR5 for entry at this time, manifested by a 1- to 2-log increase in susceptibility to CCR5 inhibitors and a reduced ability to infect cell lines with low CCR5 expression. There was an abnormally rapid 5.4% divergence in env sequences from 4.10 to 5.76 years of infection, with the V3 and V4/V5 regions showing the greatest divergence and evidence of positive selection. These observations suggest that a decline in the fitness of R5 virus populations may be one driving force that permits the emergence of R5X4 variants

Development of resistance to 5P12-RANTES (5P12).

<p><b>A.</b> Weekly increases in p24 capsid antigen during four successive rounds of selection: 5P12 1 (blue, weeks 1–15); 5P12 2 (green, weeks 13–25); 5P12 3 (purple, weeks 24–33); and 5P12 4 (brown, weeks 30–44). Control cultures with no inhibitor are shown in black filled circles. <b>B.</b> Increasing concentrations of 5P12 expressed as multiples of the 90% inhibitory concentration (IC₉₀ = 0.12 nM) for each of the four rounds of selection, with colors matching panel A. <b>C.</b> Replication of viruses from indicated weeks of selection on activated CD4⁺ T cells from a CCR5Δ32 homozygous donor. Values are mean p24 capsid antigen levels (± SE of triplicate cultures) after 7 days of culture. <b>D.</b> Viruses from the indicated weeks of 5P12-RANTES round 4 of selection (5P12 4) or control cultures with no inhibitors were used to infect activated CD4⁺ T cells from normal donors in the presence of the CXCR4 blocking agent AMD3100 (AMD). The percent inhibition by AMD3100 of p24 capsid antigen levels after 7 days of culture is plotted versus the week of virus isolation.</p

V3 sequences of control, maraviroc-resistant, and 5P12-RANTES-selected CC1/85 HIV-1 viruses.

a<p>Log₁₀ relative light units (RLU) in single cycle infection of NP-2.CD4.CCR5 cells mediated by envelope (<i>env</i>) clones with the indicated V3 sequence. Mean values for multiple <i>env</i> clones with the same V3 sequence, representative single values for individual clones. Note that <i>env</i> clones with the same V3 sequence may differ in sequence in other regions of envelope.</p

Env (gp160) sequence evolution to CXCR4 use.

<p><b>A.</b> A phylogenetic tree representing the <i>env</i> clones that evolved from CCR5 to CXCR4 use. The tree is rooted with one of two variants found in the starting CC1/85 virus population with a TNNTxN motif sequence at position 459–465 (HXB2 numbering) in C5 instead of NDTSGT. All <i>env</i> clones that developed CXCR4 use were derived from this founder sequence. The weeks at which the <i>env</i> clones were isolated is indicated by the symbol legend, and the V3 sequence is indicated by the color given in the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022020#pone-0022020-g003" target="_blank">Fig. 3</a>. All <i>env</i> clones from week 36 and later of 5P12-RANTES selection were capable of using CXCR4 (see below), whereas only a subset of control <i>env</i> clones from week 44 or later were capable of entry via CXCR4. <b>B.</b> Entry data for <i>env</i> clones from weeks 36, 42, and 44 either from control cultures (open symbols) or 5P12-RANTES containing cultures (closed symbols, weeks depicted as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022020#pone-0022020-g004" target="_blank">Fig. 4A</a>). The symbols are color coded as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022020#pone-0022020-g003" target="_blank">Figs. 3</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022020#pone-0022020-g004" target="_blank">4A</a>.</p