Summary:

We describe an evolutionary pathway taken by HIV-1 to escape from the selective pressure of T20 in a treated patient. Besides the appearance of T20-resistant variants, we report for the first time the emergence of drug-dependent viruses with mutations in both the HR1 and HR2 domains of the envelope glycoprotein 41.

We propose a mechanistic model for the dependence of HIV-1 entry on the T20 peptide. The T20-dependent mutant is more prone to undergo the conformational switch that results in formation of the fusogenic six-helix bundle structure in gp41. A premature switch will generate non-functional envelope glycoproteins ("dead spikes") on the surface of the virion, and T20 prevents this abortive event by acting as a "safety pin" that preserves an earlier pre-fusion conformation.

(Note: If you have problems understanding this you should read "HIV-1 entry and its inhibition by fusion inhibitors" first.

HIV-1 escapes from T20 therapy in a patient

In this study, we have performed a genetic analysis on the entire HIV-1 gp41 ectodomain from a patient that failed on T20 therapy. We analysed the virus population present in this individual at multiple time-points before, during and after T20 therapy.

Figure 1a. Plasma HIV-1 RNA load during T20 therapy. The patient involved in this study received T20 with 90 mg subcutaneous injections twice daily for 50 weeks in combination with RT and protease inhibitors. Start of T20 therapy resulted in a significant decline in HIV-1 viral RNA load from 84377 to 356 cp/ml followed by a rebound to 5352 cp/ml at week 16, suggesting the emergence of a T20-resistant HIV-1 population.

Figure 1b. PCR amplification of the gp41 ectodomain. To determine whether the patient's HIV-1 population had acquired T20-resistance mutations, we sequenced the complete gp41e region. The viral RNA was isolated from plasma and amplified by nested RT-PCR.

Sequencing of the HIV-1 quasispecies revealed the acquisition of mutations in the GIV and SNY sequences of HR1 and HR2 in the course of T20 therapy, but no other changes became fixed in the gp41e sequence (see publication for more details). Changes in the GIV sequence of HR1 have previously been correlated with T20-resistance but the description of the HR2 change is new.

We further performed clonal sequencing of 153 individual clones to obtain a more accurate picture of the relative frequency of the mutational events, which allowed us to build a detailed evolutionary scheme:

Figure 3. Evolution of T20-resistance in the ectodomain of HIV-1 gp41. This schematic of the viral quasispecies in the course of T20 therapy focuses exclusively on the GIV sequence in HR1 and the SNY sequence in HR2. The situation is depicted at week -12 (pre-therapy), weeks 16, 28 and 32 (during T20 therapy) and week 54 (post-therapy). The number of clones that were sequenced per time-point is indicated by n . The initial virus population present at week -12 consists of the fully wild-type sequence (GIV-SNY). The starting virus population is shown as a blue box, all single mutants are shown as hatched-coloured boxes (e.g. GIG -SNY at week 16), and the double mutants in full colour boxes (e.g. GIA-SKY at week 28 and 32). The sizes of the boxes reflect the abundance of that particular variant within the viral population. The actual percentage of that variant in the viral quasispecies and the number of clones with that sequence are indicated within the boxes. The evolution scheme was derived in part from inspection of the actual codon changes that are indicated alongside the arrows. The mutations are ranked according to their likelihood (see publication for more details).

T20-resistant and T20-dependent HIV-1 variants

The major gp41e mutations observed under T20 therapy (GIG , GIW , and GIA in HR1 and SKY in HR2) were introduced in the LAI molecular clone, a CXCR4-using primary HIV-1 isolate from a French AIDS patient, and tested for their impact on in vitro virus replication and resistance to T20. Viral DNA constructs were transfected into the SupT1 T-cell line and cultured in the presence or absence of T20:

Figure 4a. Replication of wild-type and mutant HIV-1 LAI molecular clones. Replication of the wild-type virus was strongly inhibited by T20. In contrast, all HR1 variants (GIG , GIW and GIA ) were highly resistant to T20. Surprisingly, the GIA-SKY double mutant was not only highly resistant to T20, but also critically dependent on T20 for its replication. Figure 4b. We also tested the replication capacity of the GIA and GIA-SKY mutants using concentrations of T20 comparable to levels in patient plasma. The GIA mutant was resistant to relatively high levels of T20 (4 m g/ml). Interestingly, low amounts of T20 seem to have a stimulatory effect on this mutant. However, the GIA-SKY double mutant is critically dependent on T20 for its replication and more resistant to high T20 concentrations (6-8 m g/ml) than the GIA single mutant, which may explain why the GIA-SKY double mutant is selected in vivo.

The T20-dependent Env variant (GIA-SKY) is hyperfusogenic

We next tested the hypothesis that the addition of the K mutation in the GIA-SKY double mutant represented a compensatory mutation to enhance the rate of the structural transition in Env, but in a way that was now partially controlled by the presence of T20.

To test this mechanistic interpretation, we performed additional experiments. First, such a mechanism predicts that fusogenic activity is reduced for the GIA resistant variant, but increased for the GIA-SKY double mutant. This was tested in a cell-cell fusion assay, which measures Env activity before a potential premature switch can occur, because the Env molecules can be engaged in the fusion process as soon as they appear at the cell surface. In this assay, one cell expresses the wild-type or mutant Env protein and the other cell the appropriate receptors (CD4 and CXCR4). Fusion was scored by the formation of syncytia. In addition, we introduced an LTR-luciferase reporter in the acceptor cell that is activated upon cell fusion by Tat protein that is expressed in the donor cell.

We consistently measured reduced fusion activity (syncytia and luciferase counts) for the GIA mutant and increased activity for the GIA-SKY mutant. The representative experiment below shows 126% fusion activity for the GIA-SKY mutant compared to the wild-type control (set at 100%), and similar results were obtained in independent experiments (134%, 136%, 150%, results not shown). This finding confirms the hyper-activity of the GIA-SKY Env mutant, which is an important aspect of our model.

We also tested cell-cell fusion activity in the presence of T20. Not surprisingly, the wild-type Env is inhibited by T20 and GIA is relatively resistant. Interestingly, the GIA-SKY mutant displays a very similar resistance phenotype. These results indicate that the GIA mutation in HR1 is responsible for reduced T20-affinity (resistance) and reduced HR2-affinity (fusion activity), and that the SKY mutation in HR2 creates a hyperfusogenic Env. The latter property may seem to contradict with the observation of impaired virus replication, but a premature switch will result in dead Env spikes on the surface of virus particles. T20 may prevent such premature inactivation, and thus rescue virion infectivity.

Figure 5a. The T20-dependent Env has increased fusogenic activity. SupT1 cells were transfected with the mutants indicated below the x-axis. One day later, transfected cells were mixed with SupT1 cells containing a Tat-responsive LTR-luciferase reporter gene construct. After 24 hours, formation of syncytia was analysed by light microscopy (-, no syncytia; ++++, all cells involved in syncytia) and quantitated by measurement of luciferase activity in cell extracts.

T20 prevents inactivation of the peptide-dependent virus

We produced the GIA-SKY mutant virus particles both in the presence or absence of T20 and scored their infectivity in a single cycle infection assay in which an LTR-luciferase reporter is activated upon successful infection by newly synthesised viral Tat protein. Even though an equivalent amount of HIV-1 virions (based on CA-p24) was used in this assay, we measured a 2- to 3-fold increase in infectivity when virions were produced with T20. When (additional) T20 was added during the infection experiment, we measured a dose-dependent decrease in infectivity of both samples, confirming the resistance phenotype. Thus, T20 needs to be present early during virion production in order to prevent premature switching and the consequent loss of infectivity. When added later during the infection process, T20 does not activate, but rather inhibits the GI -SKY mutant. This temporal separation of positive and negative effects of T20 is consistent with the proposed mechanism.

Figure 5b. T20 prevents the loss of infectivity of GIA-SKY virions. SupT1 cells containing a Tat-responsive LTR-luciferase reporter gene construct were infected with equal amounts of the GIA-SKY double mutant virus, which was produced in the presence (100 ng/ml) or absence of T20 and scored for infectivity in a single cycle infection assay. The virus infectivity is expressed in relative light units (RLU) from a luciferase assay performed on the cell lysate.

Biophysical properties of the T20-resistant and T20-dependent six-helix bundles

The proposed mechanism suggests that the T20-dependent variant undergoes an accelerated transition from the native state to the post-fusion six-helix bundle conformation of gp41 (see publication for details). The pre- and post-transitional slopes and the shape of the main transition are very similar for the wild-type and variant peptides, but there is a profound difference in thermal stability. At a concentration of 10 mM, the midpoint ( Tm ) of thermal denaturation of the wild-type protein is 80°C, as compared with Tm values of 69 and 83°C for the GIA and GIA-SKY mutants, respectively. Taken together, these results indicate that the T20-resistant mutation GIA leads to an appreciable destabilization of the six-helix bundle structure. In contrast, the T20-dependent SKY mutation stabilizes the six-helix bundle conformation. This is striking since the destabilizing GIA mutation is also present in this variant. Thus, the decreased six-helix bundle stability may explain why the T20-resistant virus is less fusogenic while the T20-dependent double mutant, with increased six-helix bundle stability, is hyperfusogenic.

Figure 6. Thermal melts of the wild-type peptide N36(L6)C34 (black fill) and the GIA (white fill) and GIA-SKY (spotted fill) mutants monitored by circular dichroism at 222 nm at 10 m M protein concentration in PBS. We have performed thermal unfolding experiments four times for the wild-type and mutant peptides. The error of Tm determination is ± 0.5°C. It should be emphasized that melting temperature is useful only as a qualitative guide to stability.

Model for T20-dependence

We propose that part of the GIA-phenotype (weakened HR1-HR2 interaction and reduced fusion) is reversed by the SKY mutation in HR2. This mutation creates an Env variant that is more prone to undergo the conformational switch leading to six-helix bundle formation. Premature switching of GIA-SKY will make a dead Env spike and thus effectively kill virus infectivity, which is what we measured. More importantly, this scenario easily explains the observed T20-dependency, as HR1-T20 binding will block the premature switch and Env-inactivation. This T20-blockade should obviously be transient, and release of the T20 peptide may be facilitated by the reduced HR1-T20 affinity due to the GIA resistance mutation. This mechanism also explains why the GIA-SKY double mutant is inhibited at high T20 levels, which freezes Env in a pre-fusion conformation.

The key mutation for the T20-dependent phenotype is the SNY to SKY change within HR2. This change creates a hyperfusogenic Env; approximately 1.5-fold more active than the wild-type control, but around 2-fold more active than the GIA precursor form. We propose that the GIA-SKY double mutant does hardly replicate because this Env variant is too aggressive, leading to a premature switch of the spring-loaded Env, which results in non-functional six-helix bundles ("dead spikes") on the virus particle. The SKY mutation is solely responsible for this enhanced fusogenicity and lack of replication capacity because these properties are not observed for the GIA single mutant (in fact, reduced fusogenicity is measured). Furthermore, the SKY single mutant is replication-impaired (results not shown). This also means that evolution of the GIA-SKY mutant must follow the precise two-step scenario; first GIA-mediated resistance and then SKY-mediated dependence. The GIA mutation is also important for the ability of the double mutant to replicate in the presence of T20. The GIA-SKY double mutant exhibits a T20-resistance phenotype that is very similar to that of the GIA single mutant, the difference being that T20 is required for GIA-SKY to maintain a pre-fusion Env conformation, but the presence of T20 during the infection process partially inhibits virus entry. In other words, the T20 peptide acts as a "safety pin" that locks or stabilises a pre-fusion conformation of the gp41 protein, but it presumably has to be removed at the right time to ensure correct Env function.

Figure 7. Proposed model for T20-dependent viral entry. Each box depicts one of three scenarios: T20-sensitive (eg. GIV-SNY), T20-resistant (eg. GIA -SNY) and T20-dependent (eg. GIA -SKY). A simplified gp41 ectodomain comprised of only one subunit of HR1 (shaded cylinder) and HR2 (white cylinder) joined by a loop region (black line) is used to depict a pre-fusion and post-fusion state of the peptide. The thickness of the arrows represents the speed of the conformational switch between pre- and post-fusion conformations. A white star represents the GIA mutation in HR1 and a black star represents the S K Y mutation in HR2. Explanations for each reaction are provided on the right hand side.

We anticipate that our T20-dependent virus will provide a useful tool to dissect molecular details of the Env-mediated entry process and such studies are ongoing. Further research could yield novel information on structural intermediates that may facilitate Env-based vaccine research and the construction of improved immunogens. The T20-dependent phenotype may be a useful tool to control or to synchronize the entry of viral vectors. Conditional viral entry may also add to the safety of a doxycycline-dependent HIV-1 variant that was constructed as a live-attenuated virus vaccine. In fact, we have been successfull in making a virus that can be controlled from the outside both at the level of cell entry (T20) and viral gene expression (doxycycline). (See publication list)

Finally, the evolution of drug-dependent HIV-1 variants has an obvious clinical relevance. The appearance of such variants during antiviral therapy may be an indication to modify the drug regime. It is therefore important to test if the next generation of T20-like compounds are able to trigger the replication of the GIA-SKY mutant. It is already known that patients harbouring T20-resistant viruses do not show cross-resistance to the related T1249 compound, and we have evidence that cross-dependence of the GIA-SKY double mutant does not occur either (results not shown).

An interesting difference between drug-resistant and drug-dependent viruses is at the level of the human population and virus transmission. Drug-resistant viruses are known to spread within the current epidemic, but this would seem impossible for T20-dependent viruses because the antiviral inducer T20 is not available in the newly infected individual.

Find a lot of HIV research literater at Chris Baldwin's personal blog.

Relevant publications

Baldwin CE, Sanders RW, Deng Y, Jurriaans S, Lange JM, Lu M, Berkhout B. Emergence of a Drug-Dependent Human Immunodeficiency Virus Type 1 Variant during Therapy with the T20 Fusion Inhibitor. J Virol. 2004 Nov;78(22):12428-37.

Das AT, Baldwin CE, Vink M, Berkhout B. Improving the Safety of a Conditional-Live Human Immunodeficiency Virus Type 1 Vaccine by Controlling both Gene Expression and Cell Entry. J Virol. 2005 Mar;79(6):3855-8.

Baldwin CE, Sanders RW and Berkhout B. Inhibiting HIV-1 entry with fusion inhibitors. Current Medical Cemistry, 2003, Sep;10(17):1633-42.

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