The V1-V3 region of a brain-derived HIV-1 envelope glycoprotein determines macrophage tropism, low CD4 dependence, increased fusogenicity and altered sensitivity to entry inhibitors
© Rossi et al; licensee BioMed Central Ltd. 2008
Received: 24 July 2008
Accepted: 06 October 2008
Published: 06 October 2008
HIV-1 infects macrophages and microglia in the brain and can cause neurological disorders in infected patients. We and others have shown that brain-derived envelope glycoproteins (Env) have lower CD4 dependence and higher avidity for CD4 than those from peripheral isolates, and we have also observed increased fusogenicity and reduced sensitivity to the fusion inhibitor T-1249. Due to the genetic differences between brain and spleen env from one individual throughout gp120 and in gp41's heptad repeat 2 (HR2), we investigated the viral determinants for the phenotypic differences by performing functional studies with chimeric and mutant Env.
Chimeric Env showed that the V1/V2-C2-V3 region in brain's gp120 determines the low CD4 dependence and high avidity for CD4, as well as macrophage tropism and reduced sensitivity to the small molecule BMS-378806. Changes in brain gp41's HR2 region did not contribute to the increased fusogenicity or to the reduced sensitivity to T-1249, since a T-1249-based peptide containing residues found in brain's but not in spleen's HR2 had similar potency than T-1249 and interacted similarly with an immobilized heptad repeat 1-derived peptide in surface plasmon resonance analysis. However, the increased fusogenicity and reduced T-1249 sensitivity of brain and certain chimeric Env mostly correlated with the low CD4 dependence and high avidity for CD4 determined by brain's V1-V3 region. Remarkably, most but not all of these low CD4-dependent, macrophage tropic envelopes glycoproteins also had increased sensitivity to the novel allosteric entry inhibitor HNG-105. The gp120's C2 region asparagine 283 (N283) has been previously associated with macrophage tropism, brain infection, lower CD4 dependence and higher CD4 affinity. Therefore, we introduced the N283T mutation into an env clone from a brain-derived isolate and into a brain tissue-derived env clone, and the T283N change into a spleen-derived env from the same individual; however, we found that their phenotypes were not affected.
We have identified that the V1-V3 region of a brain-derived envelope glycoprotein seems to play a crucial role in determining not only the low CD4 dependence and increased macrophage tropism, but also the augmented fusogenicity and reduced sensitivity to T-1249 and BMS-378806. By contrast, increased sensitivity to HNG-105 mostly correlated with low CD4 dependence and macrophage tropism but was not determined by the presence of the brain's V1-V3 region, confirming that viral determinants of phenotypic changes in brain-derived envelope glycoproteins are likely complex and context-dependent.
Human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins (Env), the heavily glycosylated surface gp120 and the non-covalently associated transmembrane subunit gp41, are organized on the virion surface as trimeric spikes and mediate viral entry into susceptible cells. The surface gp120 is composed of a core of conserved regions (C1-C5), shielded by variable loop regions (V1-V5) formed by disulfide bonds (except V5) that retain a large degree of flexibility. The gp41 ectodomain (gp41e) contains the fusion peptide, which is inserted into the membrane of the target cells, as well as two heptad repeat (HR) domains (amino-terminal or HR1 and carboxy-terminal or HR2) that are involved in the formation of a fusion intermediate, the six-helix bundle, through conformational rearrangements following receptor interaction. HIV-1 infection requires two sequential and specific binding steps: first, to the CD4 antigen present in CD4+ T-cells, monocyte/macrophages and other cells; and second, to a member of the chemokine receptor subfamily, within the G protein-coupled, seven-transmembrane domain family of receptors, mainly CCR5 and/or CXCR4.
Structural analysis of unliganded gp120 from the related simian immunodeficiency virus has suggested that the large gp120 region involved in binding to CD4, the CD4-binding site (CD4bs), may only form a stable, binding-competent conformation when gp120 actually engages CD4 . The interaction with CD4 triggers a rather large conformational change in gp120 that results in the formation and/or exposure of highly conserved regions previously folded into the core structure and/or sheltered by the variable loops and the glycans covering the outer domain of gp120 [2–9]. These CD4-induced regions contain discontinuous structures that react with certain human neutralizing monoclonal antibodies (mAbs) (e.g., 17b), which inhibit chemokine receptor binding to gp120 [2, 5, 7–15], and therefore constitute a high-affinity binding site for the co-receptor molecule. Chemokine receptor binding by gp120 has been suggested to occur first through the amino terminus, which then allows interaction with the second extracellular loop, and subsequently triggers further conformational changes on gp120 that are transduced to gp41 and lead to the fusion-active conformation of HIV-1 Env [16–21] and the formation of a fusion pore.
HIV-1 infection of the central nervous system (CNS) seems to occur early after primary infection. Subsequently, HIV-1-infected individuals may develop a neurological syndrome ranging from the mild minor cognitive/motor disorder to HIV-associated dementia, although significant neurological dysfunction and neurodegeneration are typical in advanced stages of disease . Although anti-retroviral therapy has decreased the incidence of HIV-associated dementia, neurological abnormalities continue to be a relevant problem among all HIV-positive individuals [22, 23]. HIV-1 likely enters the CNS as cargo in virus-infected monocytes migrating into the brain to replenish the population of perivascular macrophages. Accordingly, perivascular macrophages and microglia (long-lived, brain resident macrophages) seem to be responsible for most of the viral production within the brain. Multinucleated giant cells, the end product of fusion between infected and uninfected cells, are the principal neuropathological finding of HIV-1 infection in the brain and hallmark of HIV-1 encephalitis [24–27].
Microglia and perivascular macrophages may survive for long time after infection. They express CCR5, CXCR4 and some minor co-receptors , but similarly to macrophages from other tissues, CD4 expression is either undetectable or very low [29–32]. Hence, the nature of the infected cells and the special immunological status of the brain may allow viral persistence and replication in the brain. Several studies have described that viruses isolated from the brain display macrophage tropism and mainly use CCR5 to infect microglia [33–38], suggesting that viral tropism for microglia and macrophages may be determined by similar mechanisms [39–41]. In addition, numerous studies support that HIV-1 evolve independently within the CNS and in particular the brain [42–48] probably leading to compartmentalization and a progressive adaptation to this niche.
We previously described that a primary peripheral isolate adapted in vitro to grow in pure human adult microglial cultures developed increased fusogenicity and an improved ability to use low levels of CD4 on the target cell membrane for fusion and infection (low CD4 dependence) [49–51], which correlated with higher avidity and affinity in the interaction with CD4 . The phenotypic changes mapped to the envelope glycoproteins, which differed only in 8 amino acids, and specifically to four amino acid changes in the V1/V2 region of gp120, which probably result in a more open or partially-triggered gp120 conformation. Subsequently, we hypothesized that HIV-1 could adapt within the brain compartment to specifically improve its ability to use low levels of CD4 for entry/infection on target cells, and therefore viruses with low CD4 dependence and high CD4 affinity might be naturally selected in vivo in the CNS of HIV-1-infected individuals.
We and others have investigated whether viruses or Env with these particular phenotypes are present among primary isolates in the brain/CNS of HIV-1-infected individuals [35, 44, 53–57]. In this sense, Peters et al. have reported that the macrophage tropism of brain-derived Env seems to correlate with reduced sensitivity to inhibition by an anti-CD4 mAb but not by CCR5 antagonists or gp41-targeting antibodies or fusion inhibitor peptides [55, 56]. Thomas et al. have also recently reported that efficient entry into macrophages is more frequent among Env from brain than from lymphoid tissues and seems to correlate with both low CD4 dependence and overall efficiency of fusion . In addition, Dunfee et al. found that asparagine in position 283 (N283) in gp120's C2 region seems to be more common in Env from brain than from lymphoid tissues, associates with the presence of dementia and could contribute to the lower CD4 dependence, slightly increased affinity for CD4 and enhanced macrophage tropism . Likewise, we have previously reported that Env derived from the brain of an individual with HIV-1 encephalitis have lower CD4 dependence and higher avidity for CD4, mediate increased fusion and have significantly lower sensitivity to the fusion inhibitor T-1249, than spleen-derived Env from the same subject . These envelope glycoproteins differed in numerous amino acid residues throughout gp120, with many changes clustering in or around the variable regions V1/V2 and V3, and in the HR2 region of gp41e, but not in HR1.
Several early studies identified the V3 region in gp120 as a primary determinant of macrophage tropism in envelope glycoproteins from CCR5-using peripheral isolates [58–63], although other gp120 regions, and especially the V1/V2 variable loop, have also been shown to influence the ability of viral isolates to infect macrophages [61, 63–67]. Therefore, in this study we investigated the viral determinants for the phenotypic differences observed between the brain- and spleen-derived Env reported above , through the generation of chimeric and mutant Env and their characterization using cell-to-cell fusion and pseudotype infection assays. We found that chimeric Env containing the V1-V3 region of the brain-derived gp120 not only displayed increased macrophage tropism but also had significantly lower CD4 dependence, higher fusogenicity and lower sensitivity to the T-1249 fusion inhibitor and BMS-378806. Interestingly, most low CD4-dependent, macrophage-tropic Env also showed increased sensitivity to a novel allosteric entry inhibitor, the short peptide HNG-105. In addition, we found similar phenotypes with the Env from a brain-derived isolate and obtained evidence that the role that specific amino acid residues in gp120 play on these phenotypes is highly dependent on the Env background.
Materials and methods
Human embryonic kidney 293T cells and quail fibroblasts QT6 cells were cultured in Dulbecco's modified Eagle's medium (DMEM; MediaTech, Hendon, VA) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Clontech, Mountain View, CA) and antibiotics. Human osteosarcoma cells (HOS) stably expressing human CD4 and CCR5 molecules (obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH [ARRRP], from Dr. Nathaniel Landau) [68, 69] were maintained in DMEM supplemented with 10% FBS, puromycin (1 μg/ml), mycophenolic acid (40 μg/ml), xanthine (250 μg/ml) and hypoxanthine (13.5 μg/ml). Human astroglioma U87 cells stably transfected for the expression of human CD4 and CXCR4 (obtained through the ARRRP from Drs. HongKui Deng and Dan Littman)  were cultured in DMEM supplemented with 10% FBS, puromycin (1 μg/ml) and G418 (300 μg/ml) (Invitrogen-Gibco, Carlsbad, CA).
Normal human monocytes were purchased as single cell suspension from the Human Immunology Core of the University of Pennsylvania Cancer Center, counted and plated for differentiation into macrophages by culturing at a density of 2.0 × 104 cells/well in 96-well plates for 7–10 days in DMEM supplemented with 5% FBS and 5% giant cell tumor conditioned media (BioVeris, Gaithersburg, MD), as previously described .
Construction of chimeric and mutant envelope glycoproteins
To evaluate the role of N283 in various Env backgrounds, the Env mutants pSV-BR(N283T) and pSV-SPL(T283N) where the N283 present in the brain Env was mutated to T and the T283 present in the spleen Env was mutated to N, respectively, were created using QuikChange site-directed mutagenesis (Stratagene, La Jolla, CA) following manufacturer's instructions. The presence of the intended mutations was verified by sequence analysis.
Finally, we also characterized several Env from the HIV-1DS-br isolate, recovered from an adult AIDS patients with dementia by Dr. Suzanne Gartner (Johns Hopkins University) through co-cultivation of brain tissue with monocyte-derived macrophages (MDM) [73, 74]. Human adult microglial cells were previously infected with this viral isolate and total DNA was isolated 3 days post-infection and used for env amplification . We have now fully sequenced these env clones (named DS12, DS13 and DS17) (kindly provided by Dr. Francisco González-Scarano, University of Pennsylvania) (GenBank accession numbers, EU850429–EU850431) and used them for phenotypic analyses to determine whether the Env phenotypes are similar to those identified in env amplified directly from brain tissue of HIV-1-infected individuals. In addition, we have used QuikChange site-directed mutagenesis to generate the Env mutant DS17(N283T), in which the N283 residue present in the DS17 clone was mutated to T. The mutation was verified by sequence analysis.
Peptides and other reagents
Fusion inhibitors T-20 (YTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWF) and the more potent T-1249 (WQEWEQKITALLEQAQIQQEKNEYELQKLDKWASLWEWF) are based on the sequence of the HR2 region of gp41e from the HIV-1HxB isolate and inhibit viral entry by targeting the envelope glycoproteins at a fusion intermediate state [75, 76]. T-20 and T-1249, as well as the derived peptides T-20-BR (YTNLIYNLIEKSQNQQEMNEQELLKLDTWASLWNWF) and T-1249-BR (WQEWEQKITALLEQAQIQQEMNEQELQKLDTWASLWEWF) that contain amino acid changes (shown in bold) present in the HR2 region of the previously characterized H0002GH brain-derived env clones , were custom synthesized (BioSynthesis, Inc., Lewisville, TX). In addition, a biotinylated, HR1-derived peptide (N40, biotin-RQLLSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARVL) and a biotinylated scramble peptide (N40scr), to be used as negative control in biosensor experiments, were also custom synthesized. All peptides were dissolved to 1–2 mg/ml final concentration in 10–25% DMSO, aliquoted and stored at -80°C until use. The desired dilutions for inhibition or interaction experiments were freshly made in DMEM or phosphate-buffered saline (PBS), respectively.
The gp120-targeted small molecule BMS-378806 [77, 78], a gift from Dr. Amos Smith (University of Pennsylvania), is an entry inhibitor that has been proposed to function through inhibition of CD4 binding [79, 80]; however, other evidences indicate that it may bind to unliganded gp120 and target the CD4-induced conformational re-arrangement of gp120 and gp41 [81, 82]. The 12p1-derived short peptide HNG-105 (kindly provided by Drs. Hosahudya Gopi and Irwin Chaiken, Drexel University College of Medicine) was generated by click conjugation and functions as an entry inhibitor since it prevents viral infection and has been shown to inhibit interactions of both monomeric and trimeric soluble gp120 with soluble CD4 [83–86]. It has been suggested that HNG-105 works through a novel allosteric mechanism, interacting with a site different from the CD4 or co-receptor binding sites but resulting in a lower affinity of gp120 for either of its receptors. The SK3 anti CD4 mAb was purchased from Becton Dickinson (San Jose, CA).
Pseudotype production and infections
To produce Env-pseudotyped, luciferase-reporter viruses, 293T cells were co-transfected using calcium phosphate precipitation (ProFection Mammalian Transfection System, Promega, Madison, WI) with each Env expression vector and with the Env-deficient, pNL4-3-luc+env- provirus, developed by N. Landau by introducing a frameshift mutation in the env gene of pNL4-3-luc+ . Culture supernatants containing the pseudotyped particles were collected 48–72 hours after transfection, clarified by centrifugation, aliquoted and stored at -80°C until use. Pseudotype particles lacking envelope glycoproteins, which are generated by co-transfecting the luciferase HIV-1 pro-viral backbone with an empty expression vector, are used as negative control for all pseudotype infections. Pseudotype stocks were quantitated using a p24gag antigen capture ELISA (SAIC-National Cancer Institute) and normalized amounts of stocks were used to infect target cells for 5–6 hours at 37°C. At 2–3 days post-infection, the cells were washed with PBS and lysed, and the amount of entry mediated by each Env was measured by detecting luciferase activity (Luciferase Assay System, Promega) in a microplate luminometer (GloMax, Promega), following manufacturer's instructions. Results are obtained as relative light units (RLU) per second. At least three independent experiments were performed for each analysis described below, with 2–8 replicates within each experiment.
To test for CD4 and CCR5 dependencies, QT6 cells were transfected with 5 or 0.5 μg DNA (per 106 cells) of the corresponding expression vectors, since it has been shown that these amounts result in either high or low receptor expression levels, respectively, in the cell surface . Empty pcDNA3.1 vector was used to equal the total amount of DNA used per well. After transfection, target cells were collected with Versene (Invitrogen), re-plated into 96-well plates and incubated overnight before infection.
To investigate the avidity of the interaction between Env trimers in the surface of viral particles and CD4 in the target cell membrane, HOS-CD4-CCR5 cells plated in 96-well plates 24 hours before infection, were incubated for 30–60 minutes at 4°C with 2× the final concentration of SK3 anti-CD4 mAb in a volume of 50 μl; subsequently, 50 μl of viral pseudotype stocks were added and the cells were incubated for 5–6 hours at 37°C before removing the inoculum, washing with PBS and incubating for 2–3 days.
To test for sensitivity to fusion inhibitors, BMS-378806 and HNG-105, the medium from HOS-CD4-CCR5 cells plated in 96-well plates 24 hours before infection, was removed and replaced by fresh medium containing 2× the final concentration of the corresponding inhibitor, and immediately the same volume of viral stocks was added. After 5–6 hours at 37°C, inocula and inhibitors were removed and the cells were incubated for 2–3 days before evaluating the amount of infection.
Finally, macrophage tropism was determined by infecting in parallel MDMs and HOS-CD4-CCR5 cells. After 2–3 days, the extent of infection was measured by testing for luciferase activity in cell lysates (Luciferase Assay System, Promega).
Env-mediated cell-to-cell fusion assays
Cell-to-cell fusion assays were used to test for fusogenicity, receptor utilization and sensitivity to inhibitors. Briefly, 106 QT6 effector cells per well in 6-well plates were infected at a multiplicity of infection of 2 with the recombinant vaccinia virus vTF1.1 that expresses the bacteriophage T7 RNA polymerase  for 1–2 hours at 37°C. After removing the inoculum and washing with PBS, DMEM supplemented with rifampicin was added and the cells were subsequently transfected by calcium phosphate precipitation with an Env expression vector (or empty pcDNA3.1 as a negative control). After 4 hours at 37°C, the cells were washed, DMEM with rifampicin was added, and the cells were incubated overnight at 32°C. Concurrently, 106 QT6 target cells per well in 6-well plates were transiently transfected with 5 μg DNA of each CD4 and CCR5 expression vectors, or CD4 plus empty vector as negative control, and 3 μg of pT7-luc, a luciferase-expression vector where the firefly luciferase gene is under the control of the T7 promoter, for 5 hours at 37°C. DNA was then removed, and cells were collected with Versene and re-plated into 96-well plates and incubated at 37°C overnight. The next day, effector cells were collected, overlaid onto target cells, and incubated at 37°C for 5–6 hours (for receptor utilization and inhibition experiments), or for various time periods between 30 minutes and 7 hours (for fusogenicity experiments). Luciferase activity (indicative of fusion) was measured in cell lysates as described above. At least two independent experiments were performed for testing the sensitivity to the SK3 anti-CD4 mAb, the T-1249 and T-1249-BR fusion inhibitors and the small molecule BMS-378806, with samples at least in triplicate within each experiment. Results were obtained as RLU per second.
Optical biosensor binding experiments
Interaction analyses were performed on a Biacore® 3000 optical biosensor (GEHC, Biacore, Piscataway, NJ) with simultaneous monitoring of four flow cells. Immobilization of the HR1-based peptides N40 and N40scr (a scramble peptide to be used as negative control that was generated at http://us.expasy.org/tools/randseq.html) was achieved through interaction of an amino-terminus biotin molecule on the peptides with the streptavidin-coated SA sensor chips (GEHC, Biacore) following standard protocols. The N40scr surface served as a reference.
After immobilization, increasing concentrations of T-1249 and T-1249-BR were passed over the N40 and N40scr surfaces at 25°C using a flow rate of 25 μl min-1. Due to the hydrophobic nature of the peptides, 8% DMSO was included in the running buffer (25 mM Tris-HCl, 150 mM NaCl, pH 7.4) to keep them in solution. Regeneration of the peptide surfaces during runs was achieved by pulses of 0.05% SDS, followed by extensive washes of the integrated microfluidics cartridge.
Inhibition curves obtained by plotting the relative infection (expressed as a percent of untreated) versus the concentration of the inhibitor were analyzed by non-linear regression with a sigmoidal dose-response model (variable slopes) using GraphPad Prism software, version 4.02 (GraphPad Software, San Diego, CA); this analysis uses a four parameter logistic equation, Y = Bottom + (Top-Bottom)/() where Y is the response, Bottom is 0 and Top is 100, X is the logarithm of the concentration, and Slope is the slope of the curve. 50% inhibitory concentrations (IC50) and 95% confidence intervals were estimated from the non-linear regression analysis. Fusogenicity data were analyzed by non-linear regression with a one-phase exponential model (using GraphPad Prism) that uses the equation Y=Ymax(1-e(-kX)), where Ymax is the maximum response and k is the observed rate. CD4 dependence data were analyzed with SPSS, version 16, using the non-parametric Mann-Whitney test.
Results and discussion
Construction of chimeric and mutant Env
To investigate the viral determinants for the phenotypic differences reported between the brain- and spleen-derived Env of a patient with HIV-1 associated dementia and encephalitis , we constructed chimeric env genes as described in Materials and Methods. There were 13 amino acid differences between the wild-type BR and SPL Env clones in V1/V2, 8 in V3 and 9 in V4, in addition to less numerous changes in more conserved Env regions. Six amino acid differences were also found absolutely conserved between all brain and spleen clones from this individual in the HR2 region of gp41e. First, we generated the BS and SB chimeras (brain gp120-spleen gp41 and spleen gp120-brain gp41, respectively) in pcDNA3.1 (Figure 1). These chimeric Env were functional in cell-to-cell fusion assays but, as with the parental wild-type Env, they did not result in a highly efficient production of infectious pseudotypes. To test whether we would be able to produce pseudotype viruses and perform infection experiments utilizing a different expression vector, we sub-cloned most of the gp160 coding regions, except for the first 11 amino acids after the signal peptide in gp120 and the last 132 amino acids in gp41 (which were identical in the brain and spleen derived parental Env), from the BR, SPL, BS and SB env clones in pcDNA3.1 into the pHXB2-env vector  in which efficient expression is obtained from an SV40 promoter. Therefore, we generated HxB-BR, -SPL, -BS and -SB chimeras, which will be referred to as BR, SPL, BS and SB, respectively. Additional chimeric Env containing the brain V1/V2-C2-V3 fragment in the context of spleen Env and vice versa (Bv1v3 and Sv1v3, respectively) were generated, since numerous amino acid differences between the brain and spleen Env cluster in this gp120 region, which has previously been shown to contain the determinants for increased fusogenicity and low CD4 dependence in the Env of the in vitro microglia-adapted isolate HIV-1Bori-15 [49, 50]. Furthermore, we have also constructed chimeric Env containing only the brain-derived V1/V2 region in the context of the spleen Env and vice versa (Bv1v2 and Sv1v2, respectively), and we have also generated the mutant Env SPL(T283N) and BR(N283T) to evaluate the potential role of N283 vs. T283 in the phenotype of these Env, as described recently by Dunfee et al. .
Finally, since there is the potential that the env genes amplified by PCR from total DNA isolated from brain tissue might not fully represent a truly replicating virus present in that tissue, we decided to similarly characterize several env genes obtained by PCR from microglial cells that had been infected with HIV-1DS-br , a viral isolate recovered through co-cultivation of brain tissue derived from an adult, demented AIDS patient with normal MDMs [73, 74]; in addition, one of these clones (DS17) was mutated to generate DS17(N283T) to test as well the potential role of N283 in an additional Env background.
Analysis of CD4 and CCR5 dependence
Analysis of the relative pseudotype infection in cells expressing low levels of CD4.
Analysis of inhibition of pseudotype infection in the presence of anti-CD4 antibody.
IC50 values obtained in inhibition experimentsa.
SK3 anti-CD4 (ng/ml)
Surprisingly, the env clones obtained from genomic DNA extracted from microglial cells infected with an isolate recovered from brain tissue co-cultured with MDMs (DS12, DS13 and DS17) [38, 73, 74] displayed a mixed phenotype, since they all showed a similar ability than BR to infect cells expressing low levels of CD4 but had a much greater sensitivity, similar to that observed with SPL, to inhibition of infection by the SK3 anti-CD4 mAb (Figure 2 and Tables 2 and 3; only DS17 is shown but the other two clones had the same phenotype). In addition, the mutant Env generated in the background of the DS17 env clone, DS17(N283T), had an identical phenotype to the parental DS17 Env in both CD4 dependence and sensitivity to anti-CD4 mAb. Thus, while some Env with a greater capacity to utilize low levels of CD4 for infection may also feature a high avidity for CD4 (as shown by the ability to outcompete the presence of an anti-CD4 mAb), some other Env may be able to acquire low CD4 dependence by a different mechanism not involving an increased avidity for CD4 in the context of the trimeric Env:CD4 interaction.
Although it has been reported that brain-derived Env may also have reduced CCR5 dependence and/or increased affinity for CCR5, albeit in the absence of differences in sensitivity to the CCR5 inhibitor TAK-779 , we did not find such phenotypic differences in our initial characterization of these brain- and spleen-derived Env . Concurrently, we did not observe any difference in CCR5 dependence among the wild-type, chimeric and mutant Env evaluated in this study. In addition, contrary to CD4, CCR5 levels in microglia and macrophages seem to be comparable to those in other target cells for HIV-1 such as CD4+ T-cells [30, 33, 89, 90], and therefore it may not be surprising that acquisition of reduced CCR5 dependence by neurotropic Env may occur less frequently than that of low CD4 dependence.
In addition to using single-round, Env-pseudotyped viruses to test for the ability of various Env to mediate infection, Env-mediated cell-to-cell fusion assays are also commonly used to evaluate their ability to interact with receptors and mediate fusion in the context of cell-cell interaction, rather than virus-cell interaction. We evaluated CD4 dependence and sensitivity to the SK3 anti-CD4 mAb with wild-type, chimeric and mutant Env using cell-to-cell fusion and obtained similar results to those described above with pseudotype infection (data not shown), confirming similar functionality in both types of assays. The presence of multinucleated giant cells is the principal neuropathological finding of HIV-1 infection in the brain and the hallmark of HIV-1 encephalitis [24–27], and high fusion was found to be characteristic of the Env of an in vitro microglia-adapted isolate [50, 51] and of env clones derived from brain tissues [54, 55, 57]. In addition, asparagine in position 362 in gp120's C3 region, which has been found in a higher proportion of CCR5-using Env derived from AIDS patients than from individuals with asymptomatic infection, has been shown to contribute, albeit in a strain-dependent manner, to increased fusogenicity and higher sensitivity to an anti-CD4bs mAb, perhaps by promoting greater exposure of the CD4bs and/or stabilizing the CD4-bound Env conformation . However, wild-type BR and SPL Env, as well as DS12, DS13 and DS17, all have N362 and thus any difference in fusogenicity will be due to other determinants.
Analysis of Env-mediated fusogenicitya.
Log10 [Ymax] (95% confidence limit)
Time to reach 1.0E+4 RLU/secb
Time to reach 1.0E+5 RLU/secb
Sensitivity to fusion inhibitors
In addition, most pseudotypes showed similar sensitivity to both T-1249 and T-1249-BR, suggesting that the fusion inhibitor modified with amino acids from brain Env's HR2 region does not usually have higher efficiency than parental T-1249. However, as shown in Table 3, the SB and DS17 pseudotypes had statistically significant greater sensitivity to T-1249-BR than to T-1249, since an F test performed as part of non-linear regression analysis rejected the null hypothesis of a single best fit curve for both inhibitors with each pseudotype (p < 0.05 and p < 0.001, for SB and DS17, respectively). Thus, it is interesting that only in the context of the SB chimera (but not with the wild-type brain-derived Env) and DS17, T-1249-BR seemed to show a slightly, but significant, higher potency than parental T-1249. Since the SB chimera does not have low CD4 dependence, high avidity for CD4 or increased fusogenicity, but does contain the brain gp41 subunit in which the T-1249-BR sequence is based upon, it is possible that only in this context of limited efficiency in the interaction with CD4 and limited fusogenicity, T-1249-BR will be able to demonstrate a greater efficiency than T-1249 at blocking fusion mediated by the brain-derived gp41.
Interaction between HR1- and HR2-derived peptides
In addition, we developed a surface plasmon resonance assay to evaluate whether the modified T-1249-BR might differ from parental T-1249 in the interaction with the HR1 region in gp41. In this assay, a biotinylated, HR1-derived peptide (N40) was synthesized and immobilized onto the surface of a streptavidin-coated chip to evaluate binding of the HR2-derived T-1249 and T-1249-BR using a Biacore® 3000 optical biosensor. Under the experimental conditions outlined in Materials and Methods, and over a concentration range of 81–650 nM, both HR2-derived peptides interacted in a similar manner with the immobilized HR1-derived peptide resulting in an almost identical amount of mass bound at the sensor surface (1 response unit = 1 pg/mm2). Moreover, the interaction was specific since no binding was observed to the immobilized, biotinylated N40scr, scramble peptide (data not shown). Thus, we did not find a qualitatively appreciable difference between the two peptides.
In summary, the amino acid changes found in the HR2 region of gp41 in brain-derived Env from H0002GH did not seem to affect the interaction between HR2- and HR1-derived peptides in biosensor experiments, and did not seem to contribute to the increased fusogenicity or to the reduced sensitivity to fusion inhibitors, as compared to the spleen-derived Env. Furthermore, low CD4 dependence and reduced sensitivity to the anti-CD4 mAb, and increased fusogenicity and reduced sensitivity to peptide fusion inhibitors seem to be determined by the same viral determinants in gp120.
Sensitivity to the entry inhibitors BMS-378806 and HNG-105
Analysis of inhibition of pseudotype infection by BMS-378806
In this regard, it has been shown in a very recent paper by Liu et al  that changes associated with CD4-binding seem to open up the trimeric Env structure significantly, and this occurs primarily by conformational changes affecting the variable domains. In the context of the sensitivity to HNG-105, a slightly more "triggered" conformation in the BR Env might allow access of the peptide to the inner domain, whereas with the SPL Env, the more "closed" and compact structure would not allow it, perhaps resulting in the differences in sensitivity that we have observed. Considering the dynamism and flexibility of gp120, this could be explained by the possibility that a gp120 that is further away from the CD4-bound conformation would sample CD4-bound conformations less frequently than another gp120 that is closer or partially triggered.
Therefore, low CD4 dependence seems to correlate not only with fusogenicity and with the sensitivity to fusion and other entry inhibitors, but also with the macrophage tropism of Env. This is in agreement with recent work by Thomas et al.  reporting that Env conferring macrophage tropism display low CD4 dependence and higher efficiency of fusion. Our results are also comparable to those recently published by Peters et al.  in that there is a relationship between macrophage tropism and sensitivity to certain entry inhibitors. Similar to us, they found that decreasing sensitivity to an anti-CD4 mAb and to BMS-378806 correlated with increasing macrophage tropism. However, they did not find a reduced sensitivity of macrophage-tropic Env to the fusion inhibitor T-20 (in fact, a trend towards increased sensitivity to T-20 with increasing macrophage tropism was observed), while in our study, we did find that macrophage-tropic Env had significantly lower sensitivity to the more potent, second-generation fusion inhibitor T-1249 (but not to T-20), than non-macrophage-tropic Env. It is possible that this discrepancy could be due to the difference in potency between the two fusion inhibitors.
Modeling studies are always to some extent speculative since they are based on previous crystal structure data and sequence information. However, we evaluated the reliability of these molecular models using Verify3D http://nihserver.mbi.ucla.edu/Verify_3D/, which determines the goodness of a model based on an available structure [96–99]. This analysis has confirmed the goodness of our models for DS17 and SPL Env, since for both we obtained scores greater than zero throughout gp120 on the Verify3D plot. Regions of bad fitting and loop regions tend to score zero or close to zero, and in fact a large proportion of BR gp120 (corresponding to the structured inner domain), which we could not model, had values close to zero with the published gp120 crystal structures. Therefore, we are confident with the models presented above.
We had previously found that brain-derived Env from an individual with HIV-1 encephalitis have lower CD4 but not CCR5 dependence, increased fusogenicity and reduced sensitivity to inhibition by anti-CD4 mAbs and the fusion inhibitor T-1249, but not by the CCR5 antagonist TAK-779, than their splenic counterparts . We hypothesized that the brain gp120 would contain the determinants for low CD4 dependence and higher avidity for CD4, while gp41 could likely play a role in the increased fusogenicity and reduced sensitivity to fusion inhibitors. In the present study, we have used chimeric and mutant Env and have identified that the V1-V3 region of the brain gp120 contains the determinants not only for low CD4 dependence, but also for the increased fusogenicity, enhanced macrophage tropism and reduced sensitivity to fusion inhibitors and the small molecule BMS-378806. Macrophage tropism had already been linked to low CD4 dependence and increased fusogenicity [31, 39, 41, 53, 55, 57, 67] and our study identified the specific gp120 region from a brain-derived, CCR5-using, macrophage-tropic Env conferring these phenotypes. The chimera containing the V1/V2 region of the brain-derived Env in the context of the spleen Env displayed an intermediate phenotype with reduced low CD4 dependence, macrophage tropism and fusogenicity, as compared to BR, but still different from SPL. This confirms that brain gp120's V3 region plays a role in the macrophage tropism of this Env, but also suggests that it is the V1/V2 region the main determinant for these phenotypes. However, regarding the sensitivity to entry inhibitors, although there is a clear relationship between macrophage tropism and diminished sensitivity to BMS-378806 (similarly to Peters et al. ), this is not the case with HNG-105, since some macrophage-tropic Env (Bv1v3, Bv1v2, BaL) show reduced sensitivity similar to the non-macrophage tropic Env SPL and SB, and quite different from the other macrophage-tropic Env, BR, BR(N283T), BS, DS17 and DS17(N283T). This could relate to the proposed allosteric mechanism of action of HNG-105.
Potentially, differences in the level of expression between wild-type, chimeric and mutant Env could have an impact on the phenotypes observed; however, it is not likely that this is the case since luciferase activity in cell-to-cell fusion assays was always between 100 and 1000-fold higher than the background levels, and pseudotype infection of HOS-CD4-CCR5 cells showed limited variability between pseudotype stocks but always remained at least 1000-fold higher than background levels. In addition, the fact that many pseudotypes display an opposite sensitivity to HNG-105 than to anti-CD4 mAb, BMS-378806 and fusion inhibitors supports the specificity of the results.
Diminished sensitivity to co-receptor and fusion inhibitors has been shown to correlate with higher affinity of gp120 for the co-receptor molecule CCR5 and with faster fusion kinetics [100, 101]. In this set of Env, reduced sensitivity to T-1249 appears to correlate with lower CD4 dependence, increased fusogenicity and enhanced macrophage tropism, but not with a difference in the interaction with CCR5. Resistance to fusion inhibitors is primarily acquired both in vitro and in vivo through mutations in HR1 and the association between reduced sensitivity and mutations in HR2 has rarely been reported, and always in the presence of additional mutations within HR1 [102, 103]. While there were no amino acid differences in the HR1 region of gp41e between brain and spleen clones, HR2 contained several amino acid changes which involved residues that can be considered polymorphic (moderately to highly variable). Thus, it was possible that the conserved amino acid sequence in the HR2 region of brain Env could result in an alteration/improvement of the interaction between HR2 and HR1, potentially increasing the fusogenicity of brain-derived Env and making the fusion process more resistant to the action of fusion inhibitors. This could be of great relevance since it might relate to the frequent finding of multinucleated giant cells in the CNS (while they are absent in most other tissues in vivo) and to the potential role of Env-mediated cell-to-cell fusion of infected and uninfected cells in the development of HIV encephalitis and the onset of neurodegeneration. Our results however demonstrate that, at least in these Env backgrounds, the determinants for increased fusogenicity and reduced sensitivity to fusion inhibitors lie in gp120 and overlap with those conferring low CD4 dependence, increased avidity for CD4 and enhanced macrophage tropism. It is possible that the genetic changes resulting in an altered Env conformation that confers lower CD4 dependence, higher avidity for CD4 and increased fusogenicity, may also affect the interaction with certain, but not all, entry inhibitors, with the difference potentially being on changes on the accessibility of the key residues that interact with those inhibitors due to the conformational alterations.
This work was partially supported by National Institutes of Health/National Institute of Neurological Disorders and Stroke research grant NS047970 (to JMG) and by Intramural Research Funds of Drexel University College of Medicine. We would like to thank Drs. Hosahudya Gopi and Irwin Chaiken (Drexel University College of Medicine) for providing us with HNG-105, and Drs. Francisco González-Scarano and Amos Smith (both at the University of Pennsylvania) for sharing env clones from the HIV-1DS-br brain-derived isolate, and for providing BMS-378806, respectively.
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