The pol gene encodes functional proteins that are targeted by RT inhibitors, and less genetic variation is observed in this gene compared to env. Nevertheless, some in vitro and in vivo observations suggest that genetically distinct viral variants may respond differently to certain antiretroviral drugs that target the pol gene. HIV-1 group O and HIV-2 strains are naturally resistant to non-nucleoside RT inhibitors (NNRTIs) . The rate of occurrence of nevirapine resistance-associated mutations after a single dose is significantly higher in women with HIV-1 subtype C than in women with subtype A or D . Many studies have also shown the existence of polymorphisms among non-B strains [18-20]. These accessory (or minor) mutations may not result in a significant decrease in susceptibility [21, 22], but may be associated with an increase in viral fitness (replication capacity) and/or increase in resistance level associated with major mutations, and thus long-term failure of therapy.
In the same way, since entry inhibitors target the highly variable env gene, it is likely that antiviral responses of entry inhibitors are even more influenced by the genetic diversity that exists among HIV-1 subtypes and CRFs. In addition to divergence among subtypes, a high intra-patient diversity is also seen; the overall rate of intra-patient divergence of the env gene is close to 1% per year. Slower rates of evolution are found in the other parts of the viral genome. These differences are likely driven by varying selective pressures rather than differences in the underlying mutation rate. Intra-host diversity reflects the successive fixation of advantageous mutations and the extinction of unfavorable lineages . HIV successively fixes mutations that allow it to escape immune responses in the host, or antiviral drugs.
Targeting the envelope glycoproteins (gp41 and gp120) represents a very great challenge because of their high levels of sequence diversity. The entry of HIV into a target cell represents the key initial step in the replication cycle of the virus and involves three different steps: (1) viral attachment of gp120 to CD4 receptor, (2) binding of gp120 to the co-receptors, and (3) fusion of viral and cellular membranes. The entry process thus involves a coordinated series of molecular interactions between the components of the virus glycoprotein complex (gp120/gp41) and the components of the receptor complex (CD4 and a chemokine co-receptor, CXCR4 or CCR5). Upon receptor and co-receptor binding to the surface subunit of gp120, subsequent rearrangements within gp41 allow fusion of viral and cellular membranes. It is important to note that the gp120 compromises five variable (V1-V5) domains interspersed with conserved (C1-C5) regions (see chapter by Tilton/Doms, Fig. 1).
Because of the diversity in the viral glycoproteins and host receptor molecules, their mechanism of action, and consequencely development of resistance, will also differ. Several CD4-gp120 inhibitors are under development. Apparently the degree of sequence variation in the nearby V1/V2 variable regions indirectly influences the susceptibility to these drugs . Therefore, it can be expected that the natural variation in these regions that exist among different subtypes can significantly influence baseline susceptibility to certain of these compounds for some subtypes/CRFs. Moreover, a high intra-patient diversity is also seen in env sequences over time.
After binding of the HIV-1 gp120 envelope glycoprotein to CD4, conformational changes occur in the gp120 that translocate the variable regions V1/V2 and V3 of gp120 to create or expose a binding site for co-receptor. An interesting target in HIV entry is the co-receptor binding phase, and current research is focused on designing compounds that interact with the CCR5 and CXCR4 receptors. Resistance to co-receptor antagonists can be the result of either a shift in co-receptor usage or from other changes in the envelope gly-coproteins that alter the interaction with co-receptor. Multiple mutations in V3, but also in V2, C2 and V4 seem to account for drug resistance. These regions are known to be highly variable among different subtypes, and, moreover, different co-receptor usage has also been reported for certain subtypes, e.g., CXCR4 variants are rarely observed among subtype C , whereas these variants seem to occur at higher frequencies in subtype D . Although preliminary results have not yet identified co-receptor switch as a main resistance pathway, this has to be monitored closely since such shifts could have serious consequences in disease progression. Independent of subtype-related differences, the presence of X4 viruses as minor quasi-species in an individual could allow selection of this variant.
The final step in the viral entry pathway is fusion of the viral envelope with the target surface membrane, and the gp41 ectodomain is the key structure responsible for membrane fusion. Enfuvirtide, previously known as T-20, is the first fusion inhibitor to be approved for the treatment of AIDS. Enfuvirtide is a 36-amino acid peptide derived from a continuous sequence within HR2 from the gp41 from the HXB2 HIV-1 subtype B prototype strain. The peptide binds to the HR1 region and prevents gp41-mediated fusion with the host cell membrane (see Chapter 6 for details). Overall, enfuvirtide should be considered as a drug with a low genetic barrier to resistance . Drug resistance to this new drug seems to occur in the HR1 region and, more precisely, genetic changes within the 36-45 amino acid region of HR1 have been shown to confer resistance to enfuvirtide, especially mutations in the highly conserved 3-amino acid motif at codons 36-38 (GIV) . Other common substitutions observed in phase II and III studies, including Q40H and N42T, and in vitro studies showed that site-directed HR1 mutants G36D, V38A, Q40H, N42T, N43D, N43S and N43K are significantly resistant to enfuvirtide. Due to its cutaneous route of administration and its high cost, the use of this drug remains limited to the US and Europe, where subtype B HIV-1 strains predominate. Data on sensitivity and resistance are thus derived mostly from subtype B-infected patients. However, the majority of HIV-1 infections worldwide are with other HIV variants and the proportion of non-B strains is increasing in the western hemisphere. Several studies have examined baseline susceptibility to enfuvirtide using genotypic and/or phenotypic methods. Natural enfuvirtide resistance is rare in B and non-B HIV-1 group M strains . No resist ance-associated mutations were seen as natural variants on non-B group M HIV-1 strains. However, other polymorphisms were seen, e.g., Q39L and Q40K, but these mutations were not documented to be associated with resistance, although it has to be further examined to what extent they might affect the accessibility of the drug to its target sequence. On the other hand, the N42S polymorphism has been previously observed in 15% of baseline isolates and is associated with mild hypersusceptibility . This latter mutation is present at the baseline in a large majority of non-B strains, e.g., in 80% of 185 strains studied from Cameroon, and could be detected in almost all the subtypes/CRFs . Analysis of the HR2 domain, from which the peptide is derived, indicated a much greater genetic variability as compared to HR1. Only certain amino acid positions are highly conserved between the different HIV-1 variants and correspond mainly to the amino acids involved in the 5-helix interaction and binding. Despite this high genetic diversity in the HR2 region, the efficacy of enfuvirtide to inhibit replication of such polymorphic strains seems not to be influenced. This was shown by a few studies analyzing the in vitro efficacy of T-20 on non-B samples (C, CRF01, CRF02) from Africa and India with more than half of the loci harboring amino acids that are different from the enfuvir-tide peptide, suggesting that the HR2-HR1 interaction can tolerate significant genetic changes . Although, the in vitro observations together with the highly conserved HR1 regions suggest a broad applicability of T-20 against very diverse HIV-1 group M strains, T-20 is not effective against HIV-2 . Unexpectedly, despite the high genetic diversity in HR1 and HR2, HIV-1 O isolates were as sensitive as group M viruses to inhibition by T-20 in vitro and in vivo [28, 33]. These findings suggest that T-20 has a broad antiretroviral activity against a large diversity of HIV-1 strains. Other fusion inhibitors targeting HR1 are in clinical development, for example T-1249. Although, group O viruses are susceptible to T-20, polymorphisms in gp41 seem to affect the sensitivity of HIV-1 O to second-generation fusion inhibitors like T-1249. On the other hand, mutations in HR1, known to be associated with resistance to T-20, are not resistant to T-1249. More in vitro and in vivo studies will be necessary using a larger panel of non-B subtypes to determine the impact of subtypes on enfuvirtide efficacy.
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