Bioinformatic analysis of neurotropic HIV envelope sequences identifies polymorphisms in the gp120 bridging sheet that increase macrophage-tropism through enhanced interactions with CCR5.

Bioinformatic analysis of neurotropic HIV envelope sequences identifies polymorphisms in the gp120 bridging sheet that increase macrophage-tropism through enhanced interactions with CCR5.

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Product# 1039 HIV-1 YU2 (M Tropic)Envelope Glycoprotein gp41

Product# 1081 HIV-1 gp120 (ADA)

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Product# 1031 HIV-1 gp120 (YU2)

Abstract

Macrophages express low levels of the CD4 receptor compared to T-cells. Macrophagetropic HIV strains replicating in brain of untreated patients with HIV-associated dementia (HAD) express Envs that adapted to overcome this restriction through mechanisms that are poorly understood. Here, bioinformatic analysis of env sequence datasets together with functional studies identified polymorphisms in the β3 strand of the HIV gp120 bridging sheet that increase M-tropism. D197, which results in loss of an N-glycan located near the HIV Env trimer apex, was detected in brain in some HAD patients, while position 200 was estimated to be under positive selection. D197 and T/V200 increased fusion and infection of cells expressing low CD4 by enhancing gp120 binding to CCR5. These results identify polymorphisms in the HIV gp120 bridging sheet that overcome the restriction to macrophage infection imposed by low CD4 through enhanced gp120-CCR5 interactions, thereby promoting infection of brain and other macrophage-rich tissues.

Keywords: HIV, envelope, macrophage, tropism, CD4, CCR5, bridging sheet, V1/V2 stem

Background

Macrophages are important target cells of Human immunodeficiency virus type I (HIV) infection that contribute to establishment and persistence of viral reservoirs in the brain, spleen, lung, gut, and bone marrow (; ). HIV-infected macrophages in the brain play an important role in the development of HIV-associated dementia (HAD), a complication that can occur in patients with non-suppressed viral replication, and also represent a barrier to virus eradication due to limited penetration by most antiretroviral therapies (; ; ; ; ).

The major determinant of HIV tropism for CD4+ T cells and macrophages is the viral envelope glycoprotein (Env). HIV Env, which consists of a surface-exposed gp120 and a transmembrane gp41 subunit, is organized into trimers on the surface of virions and infected cells (). The gp120 subunit core is composed of an inner domain, a heavily glycosylated outer domain, and four surface-exposed variable loops that extend from the core and partially occlude the coreceptor binding site and neutralization epitopes (; ). HIV entry into cells is initiated by a high affinity interaction between gp120 and CD4, which induces conformational changes in gp120 (). In the CD4-bound state, the inner and outer domains come together via formation of the bridging sheet, which is comprised of two β strands from the inner domain (β2 and β3) and two from the outer domain (β20 and β21), resulting in formation of the coreceptor binding site. In crystal and cryo-EM structures of soluble cleaved HIV Env trimers, the arrangement of the CD4-induced bridging sheet appears to be different compared to how it is shown on monomeric gp120 (; ). In the trimer structures, β2 and β3 are only partially involved in forming the bridging sheet with β20 and β21, and extend toward the trimer apex into the base of the V1/V2 variable loops.

CCR5 is the primary coreceptor for virus entry into macrophages and microglia. However, CCR5 usage per se is not sufficient to predict macrophage tropism (M-tropism) (; ). Furthermore, M-tropism of CCR5-using (R5) HIV isolates is a continuous rather than binary phenotype, with up to 1000-fold variation among different strains in their ability to replicate in primary monocyte-derived macrophages (MDM) (; ; ; ; ). One factor contributing to this wide spectrum of M-tropism is receptor density (; ). Macrophages express lower levels of CD4 than CD4+ T cells in blood () and are preferentially infected by viruses that can utilize low CD4 to mediate fusion and entry (; ; ; ; ; ; ; ). Determinants of reduced CD4-dependence or CD4-independence have been mapped to the HIV and SIV gp120 V1/V2, V3, and V4 variable loops, C2 and C3 regions, and regions of gp41 (; ; ; ; ; ; ; ). These determinants enhance Env interactions with CD4 through different mechanisms that include increased gp120 affinity for CD4 or exposure of the CD4-binding site (; ; ; ; ; ), which in turn can enhance bridging sheet recruitment ().

Another mechanism that allows HIV Envs to overcome the restriction imposed by reduced CD4 on macrophages is enhanced gp120 interactions with CCR5. Although previous studies described M-tropic Envs with reduced CCR5 dependence, genetic determinants that influence this phenotype in M-tropic Envs are poorly understood (; ; ; ). Genetic determinants that enhance gp120-CCR5 interactions in a strain-dependent manner have been mapped to the CCR5-binding site, which includes residues in the V3 loop, inner domain, and bridging sheet formed by the β20/β21 strands and surface-exposed β2/β3 strands, which assemble into a hairpin to form the stem of the V1/V2 loop (; ; ; ). Residues in the bridging sheet can impact gp120 interactions with CCR5 via direct contacts with CCR5 or by repositioning the V1/V2 loops, which occlude the CCR5 binding site in the unliganded conformation (; ; ; ). Mutations in the β20/β21 strands that disrupt the hairpin structure eliminate gp120 binding to CCR5, while genetic determinants in β3 can influence Env-CCR5 binding (). Crystal and cryo-EM structures of a soluble cleaved HIV Env trimer suggest that interactions between V1/V2 and V3 at the top of the trimer stabilizes the trimer apex around the three-fold axis (; ). Amino acids near the trimer apex, including residues in β2 and β3, have the potential to influence Env-CCR5 interactions given proximity to V1/V2 and V3 in the same and adjacent protomers.

Here, bioinformatic analysis of HIV env sequence datasets from brain and blood/lymphoid compartments together with functional studies identified rare polymorphisms in the β3 strand of the gp120 bridging sheet that can increase virus entry into macrophages. D197, which eliminates an N-linked glycosylation site, was detected in brain in some HAD patients, while position 200 was estimated to be under positive selection. Mutagenesis studies showed that D197 and T/V200 can enhance macrophage tropism by increasing gp120 interactions with CCR5. These findings identify naturally occurring variants in the β3 strand of the HIV gp120 bridging sheet that can overcome the restriction to macrophage infection imposed by low CD4 by enhancing gp120-CCR5 interactions.

Results

Bioinformatic analysis of HIV env sequence datasets identifies polymorphisms in the gp120 bridging sheet in brain from some patients with HIV-associated dementia

The genetic evolution of HIV variants in brain is distinct from that in lymphoid tissues and other organs (; ; ; ; ; ; ). Diversifying evolution associated with CNS infection can result in nonsynonymous substitutions that affect protein structure and function, and positive selection for polymorphisms that increase viral fitness in brain (; ; ; ). Viruses that can utilize low receptor levels to enter macrophages are expected to have a selective advantage during HIV replication in the brain. We therefore hypothesized that sites under positive selection in the gp120 bridging sheet region of the CCR5-binding site may represent naturally occurring variants that could enhance M-tropism through enhanced interactions with CCR5.

To identify sites under positive selection in the gp120 bridging sheet, we analyzed brain- and blood/lymphoid-derived gp120 sequences using a dataset from 30 patients with or without HAD from 10 published studies (n=336 unique sequences from 19 HAD subjects and 119 from 11 non-HAD subjects) (; ; ; ; ; ; ; ; ; ). Criteria for inclusion in the dataset were availability of gp120 V1-V5 sequences with sequence coverage of all four β-strands of the bridging sheet. Three codon-based maximum likelihood methods (SLAC, FEL, and IFEL, http://datamonkey.org) were used to estimate the dN/dS ratio at each codon position to calculate the probability that codon variation was due to positive selection (; ; ). In HAD patients, a total of 51 codons were estimated to be under positive selection (p<0.05 by at least 2 methods), 29 and 27 codons in brain- and blood/lymphoid-derived sequences, respectively (hereafter referred to as brain and lymphoid sequences) (Figure 1A and Additional File 1). In the bridging sheet, position 200, located in the surface-exposed β3 strand near the trimer apex, was estimated to be under positive selection (p=0.02 and 0.005 in brain and lymphoid sequences from HAD patients, respectively) (Figure 1, Table 1, and Additional File 1). We did not detect positive selection at position 200 in the dataset from non-HAD patients, which contains fewer sequences and subjects (Table 1, and Additional File 1).

Bioinformatic analysis of HIV env sequence datasets identifies position 200 in the β3 strand as a site in the gp120 bridging sheet estimated to be under positive selection in Envs from brain or blood/lymphoid tissues of HAD patients

A) Brain and lymphoid Env sequences from patients with HAD were aligned using ClustalX2 (n=336 brain sequences and 171 blood/lymphoid sequences from 19 HAD patients). dN-dS values were estimated by SLAC, FEL, and IFEL and normalized by total codon tree length (http://datamonkey.org). dN-dS values are shown only for sites under positive selection, with averages calculated for any codon with p<0.05 by at least 2 of the 3 programs. Sites in brain sequences are in red, in lymphoid sequences in blue, and in both brain and lymphoid sequences in black. Locations of β strands in the bridging sheet are indicated by horizontal arrows (β2, 120–125; β3, 196–202; β20, 423–426; β21, 429– 435). Codons were numbered according to codon env position of HIV-1 HXB2 reference strain. Vertical arrow indicates position 200 in the β3 strand. B) Model showing the V1V2 (green), V3 (blue), and β2/β3 regions (red) in a HIV gp120 crystal structure from a soluble cleaved HIV Env trimer () viewed down the trimer axis. Amino acids at positions 197 and 200 in β3 are located near the trimer apex (N and A, respectively, in the published structure; these amino acids and glycans at the N197 N-glycosylation site are colored orange) (; ). C) HIV gp120 bridging sheet region depicted on the CD4-liganded HIV YU2 gp120 core structure (top figure) (). This structure was selected for modeling because it is derived from a macrophage-tropic HIV-1 primary isolate and includes residues at positions 197 and 200. HIV gp120 is shown in blue, CD4 is shown in yellow. Fab 17b was removed for clarity. β3 strand is shown in light blue. A backbone model of the β2/β3 strands of CD4-bound YU2 gp120 is shown in the bottom panel. Clade B consensus amino acids are shown at positions 197 (green, with N-glycan in white) and 200 (orange).

Table 1

Position 200 in the β3 strand in the gp120 bridging sheet is estimated to be under positive selection in HAD patients.

Alignment Codon Region Average dN-dS Average p-valuea
HAD brainb 200 β3 0.8 0.021 (FEL, IFEL)
HAD lymphoidc 200 β3 2.6 0.005 (SLAC, FEL, IFEL)
Non-HAD braind NS NS NS NS
Non-HAD lymphoide NS NS NS NS
aSLAC, FEL, and IFEL analyses (http://datamonkey.org) were used to reconstruct ancestral sequences at each node of a phylogenetic tree. Substitutions are calculated at each branch and terminal node. P-values are averaged for methods in parentheses.
bn=336 unique brain-derived gp120 V1-V5 region sequences from 19 HAD subjects
cn=171 unique blood/lymphoid-derived gp120 V1-V5 region sequences from 15 HAD subjects
dn=119 unique brain-derived gp120 V1-V5 region sequences from 11 non-HAD subjects
en=66 unique blood/lymphoid-derived gp120 V1-V5 region sequences from 7 non-HAD subjects

NS: Not significant (p>0.05)

Next, we performed an exploratory analysis of sequences in the β2/β3 region using the complete dataset of 796 matched brain and lymphoid sequences from 26 patients (n=498 brain sequences, range 3–89 sequences/patient; median=8 sequences/patient; and 298 lymphoid sequences, range 2–57 sequences/patient; median 5 sequences/patient) (; ; ; ; ; ; ; ; ; ; ; ), accounting for the variable number of sequences per patient by weighting amino acid distributions within each patient using stratified 2×2 contingency tables and normalizing for between-patient differences in sequence depth. This exploratory analysis revealed that loss of the N-linked glycosylation site (PNGS) at position 197 near the trimer apex (Fig. 1B) was more frequent in brain than in lymphoid sequences (p=0.03, Fisher’s exact test). Filtering the dataset on one randomly selected sequence per patient followed by analysis of β3 amino acid distributions in the reduced dataset confirmed our initial finding that loss of the PNGS at position 197 was more frequent in brain compared to lymphoid tissues in these patients (Table 2), although this difference was not statistically significant (p>0.05, Fisher’s exact test).

Table 2

Loss of N-linked glycosylation site at position 197 in the HIV gp120 β3 strand is detected in brain in some patients with HAD.

Variant Amino Acid Frequency a
197Ψb A198c T199c T200c V201c A202c
By Tissue
Brain (n=26) 12% (3) 4% (1) 0% (0) 15% (4) 8% (2) 0% (0)
Blood/Lymphoid (n=26) 0% (0) 4% (1) 0% (0) 12% (3) 8% (2) 0% (0)
Brain, by diseased
HAD (n=27) 15% (4) 4% (1) 0% (0) 7% (2) 4% (1) 0% (0)
Non-HAD (n=18) 0% (0) 0% (0) 0% (0) 11% (2) 5% (1) 0% (0)
aVariant amino acid frequency calculated as number of sequences containing most common variant/number of sequences containing any other amino acid (including clade B consensus amino acid) for each position in the reduced sequence dataset of matched brain and lymphoid sequences from 26 subjects after filtering on one randomly selected sequence per patient. Number in parentheses corresponds to the number of sequences containing the variant.
bΨ indicates the presence of any amino acid that results in elimination of the N-linked glycosylation site
cAmino acid designation reflects the most common variant at each position.
dBrain-derived sequences from 27 HAD and 18 non-HAD subjects after filtering on one randomly selected sequence per patient.

To determine if these β2/β3 polymorphisms near the Env trimer apex occur in patients with HAD, we compiled a second dataset containing β2/β3 sequences from brain of patients with or without HAD (n=419 brain sequences from HAD patients, range 1-89 sequences/patient; median=5 sequences/patient; and 163 brain sequences from non-HAD patients, range 1-81 sequences/patient; median 4 sequences/patient) (; ; ; ; ; ; ; ; ). In the complete sequence dataset of 582 sequences from 45 patients, loss of the PNGS at 197 was detected in 10% of brain sequences from 27 HAD patients and 0% of brain sequences from 18 non-HAD patients (p=0.001, Fisher’s exact test). Analysis of β3 amino acid distributions in a reduced dataset of one randomly selected sequence per patient confirmed that loss of the PNGS at position 197 was more frequent in HAD patients (Table 2), although this difference was not statistically significant (p>0.05, Fisher’s exact test)..Thus, loss of the PNGS at position 197 in β3, located near position 200 (Fig. 1C), is a rare variant in the brain in some patients with HAD.

gp120 polymorphisms at 197 and 200 can enhance macrophage entry by reducing dependence on CD4

Many N-linked glycans that comprise the “glycan shield” of the gp120 outer domain are highly conserved and essential for proper protein folding, stability, and interactions between gp120, CD4, and CCR5 (; ). In vitro studies suggest that loss of specific PNGS sites in the V1/V2 loop region, including position 197, can affect viral replication or sensitivity to antibody neutralization (; ; ; ; ). Previous studies investigated the role of the PNGS at position 197 in primary and lab-adapted Envs in determining neutralization sensitivity/resistance (summarized in Table 3); effects of variants at or near this position were strain-dependent, but nonetheless suggested that loss of the PNGS at this position can influence interactions with CD4 and/or CCR5. However, the role of these variants in M-tropism is unknown. To address this question, we introduced N197D (most common variant in brain) and V200T into a brain-derived Env cloned from a weakly M-tropic HIV isolate (MACS2br13, hereafter referred to as M2br) and reciprocal changes to the clade B consensus amino acid (D197N and T200V) into a highly M-tropic Env cloned from autopsy brain tissue (UK1br2-14, hereafter referred to as UK1br) (Figure 2A) (; ; ). Additionally, a double mutant (N197D/V200T) expressing both determinants was introduced into M2br. The parental and mutant Envs were expressed and processed from gp160 to gp120 at similar levels (Figure 2B). Viruses expressing parental and mutant Envs mediated similar levels of virus entry into TZM-bl cells expressing high CD4 and CCR5, and viruses expressing UK1br Envs mediated high levels of entry, comparable to those mediated by the control M-tropic Env, ADA (Figure 2C). M2br N197D and N197D/V200T mediated increased entry into MDM, while M2br V200T mediated similar entry, compared to the parental M2br Env (p<0.01 (Figure 2D). UK1br T200V exhibited increased entry into MDM, while UK1br D197N mediated similar entry, compared to the parental UK1br Env (p=0.05) (Figure 2D). These results suggest that β3 polymorphisms can influence HIV entry into macrophages.

Sequence polymorphisms at positions 197 and 200 in the β3 strand of the gp120 bridging sheet can enhance macrophage entry

A) HIV UK1br and M2br gp120 β3 sequences (; ) were aligned against the clade B consensus sequence. Dots represent residues identical to the Clade B consensus. Positions 197 and 200 are shown in red. B) 293T cells transfected with pCR3.1 Env expression plasmids analyzed by Western blotting with goat anti-gp120. ADA and YU2 Envs were included as controls. C) TZM-bl cells expressing high levels of CD4 and CCR5 were infected with HIV luciferase reporter viruses expressing UK1br and M2br parental or mutant Envs, lysed 48 hours after infection, and analyzed for luciferase activity. D) Monocyte-derived macrophages (MDM) were infected with reporter viruses expressing parental and mutant Envs, lysed 6 days after infection, and analyzed for luciferase activity. For C and D, relative luciferase units (RLU) with background subtracted are shown for viruses expressing no Env (mock), ADA, UK1br, and M2br Envs on the left. Data on the right are presented as percentage of wild-type entry and represent duplicate wells of a single experiment. Error bars represent standard deviations. Experiments were repeated a minimum of 3 times for each Env set. *p<0.05, Student’s t-test.

Table 3

Summary of published mutagenesis studies of sequence polymorphisms in the β3 strand of HIV gp120

Env M-tropic Position 197a Position 200a Infectivityb Increased Binding/Neutralizationc Decreased Binding/Neutralizationc Reference
ADA Yes N197S T200 Increased (CD4-independece) sCD4, 17b, 19b, F105 none ()
YU2 Yes N197D V200 ND CD4, CCR5, F105 17b,CG10 ()
89.6 Yes N197Q V200 Increased (CD4-independence) sCD4, 17b, b12, 447-52d none ()
JRCSF No N197A V200 ND 2G12 sCD4, b12, b3 (; )
SF162 Yes N197Q V200 Decreased in macrophaes, JC10 HeLa IgGCD4, b12, patient sera none ()
YU2 Yes N197 V200S ND none sCD4, CCR5, CG10 ()
aMutated residues in each Env are highlighted in blue.
bND, not determined.
cChanges in binding or neutralization sensitivity to soluble CD4, CCR5, or monoclonal antibody 17b are highlighted in red.

Next, we tested the capacity of Envs to mediate fusion with cells expressing low levels of CD4 (Figure 3A) (). The two parental Envs mediated fusion with cells expressing low levels of CD4 at ≤20% of the levels observed for cells expressing high CD4 (Figure 3B). In M2br Env, N197D and N197D/V200T enhanced the overall efficiency of fusion over a range of CD4 levels compared to the parental Env (p=0.14, 0.03, and 0.06 for N197D and 0.03, 0.05, and 0.02 for N197D/V200T, for cells expressing low, intermediate, and high CD4, respectively), whereas the V200T change alone had no significant effect (Figure 3C). Compared to T283N, a C2 variant associated with brain infection and macrophage-tropism in previous studies (), fusion mediated by M2br N197D/V200T at the low CD4 levels was even more efficient (Additional File 2). However, M2br N197D/V200T was not CD4-independent. In UK1br Env, T200V increased fusion with cells expressing intermediate levels of CD4 compared to the parental Env, but no enhancement was observed at low CD4. UK1br D197N exhibited decreased fusion with cells expressing low CD4 compared to the parental Env (Figure 3D). Thus, D197 and T/V200 can increase macrophage tropism by reducing dependence on CD4.

D197 and T/V200 enhance the capacity of HIV Envs to use low CD4 for fusion with CCR5-expressing cells

A) Cell surface expression of CD4 on transfected CF2-Luc cells determined by flow cytometry. (B–D) 293T cells expressing control ADA Env and parental Envs (B), M2br parental and mutant Envs (C), and UK1br parental and mutant Envs (D) were mixed with CF2luc cells expressing low, intermediate, or high levels of CD4 as indicated. Fusion was measured by analyzing luciferase activity at 5 (UK1br) or 8 hours (M2br) depending on the kinetics of fusion. Background luciferase activity, measured as luciferase activity for 293T cells expressing the nonfunctional ΔKS Env, was subtracted from each time point. Results are expressed as mean percentages of fusion relative to the parental Env with cells expressing high CD4 and CCR5 and represent duplicate wells from a single experiment. Error bars represent standard deviations. Experiments were repeated a minimum of 3 times for each Env set. *p<0.05, Student’s t-test.

D197 and T/V200 can enhance fusion and virus entry in cells expressing low CD4

We next used Affinofile cells (), a dually inducible HEK293 cell line with independent regulation of CD4 and CCR5 expression, to investigate whether Envs with enhanced capacity to mediate fusion with cells expressing low CD4 can mediate enhanced entry into cells expressing low CD4 and CCR5. Using 3 serial dilutions each of doxycyline and Ponasterone A to regulate CD4 and CCR5 expression, respectively, created a matrix of 9 conditions to analyze CD4 and CCR5 usage (Figure 4A). Initial experiments were performed using a wide range of CD4 and CCR5 expression levels; on the basis of these experiments, experimental conditions shown in Figure 4 were selected for subsequent experiments because they detected a wide range of infectivity. Furthermore, based on standard curves of Quantibrite beads we estimated antibody binding sites were in the range of 9×104 CD4 molecules on “high” cells, 3×104 CD4 molecules on “intermediate” cells, and 1×104 CD4 molecules on “low” cells, which overlaps the range of values previously detected on human primary activated T lymphocytes, monocytes, and MDM, respectively, and in the range of 5×104 CCR5 molecules for “intermediate” cells, which overlaps the range of values previously detected on MDM. UK1br Env mediated similar entry into cells expressing low CD4 and CCR5 compared to ADA Env, while M2br Env mediated reduced entry into cells expressing low CD4 (Figure 4B and C). The sensitivity of M2br N197D, V200T, and N197D/V200T to changes in cell surface CD4 and CCR5 expression differed from those of the parental M2br Env, with these mutant Envs exhibiting increased entry into cells expressing low CD4 and CCR5 (indicated by decreased blue and/or green areas in the low CD4/low CCR5 quadrant) (Figure 4B). M2br V200T was less sensitive to low CCR5 than N197D, but more sensitive to low CD4 than the double mutant, suggesting that both changes contribute to the ability of M2br N197D/V200T to infect cells expressing low CD4/low CCR5. UK1br T200V mediated increased entry into cells expressing low CD4/low CCR5 compared to the parental UKIbr (indicated by increased green area in the low CD4/low CCR5 quadrant and increased orange area in the low CD4/high CCR5 quadrant). In contrast, UK1br D197N exhibited increased dependence on CD4 and CCR5 (indicated by increased blue area in the low CD4/low CCR5 quadrant and right side of contour plot) (Figure 4B). M2br N197D/V200T and UK1br T200V mediated increased entry into Affinofile cells expressing low CD4/high CCR5 and high CD4/medium CCR5 (p<0.05) (Figure 4C), while M2br N197D and UK1br D197N showed trends toward increased and decreased entry, respectively, with cells expressing low CD4/high CCR5 (p=0.09 and 0.09, respectively), consistent with results from fusion assays (Figure 3).

D197 and T/V200 enhance the capacity of HIV Envs to use low CD4 and CCR5 for virus entry

A) Cell surface expression of CD4 and CCR5 on induced Affinofile cells determined by flow cytometry. Estimated CD4 and CCR5 antibody binding sites (ABS) on Affinofile cells were calculated using QuantiBrite beads (Invitrogen). (B) Affinofile cells were induced with 2-fold serial dilutions of doxycycline and Ponasterone A for 20 hours, then infected with HIV luciferase reporter viruses pseudotyped with control ADA, M2br and UK1br parental and mutant Envs. Infection was quantified after 48 hours by analyzing luciferase activity. Background luciferase activity, measured as luciferase activity for Affinofile cells infected with env- virus, was subtracted. Results are shown as contour plots depicting percentage of infection relative to cells expressing high CD4/high CCR5 for virus expressing the parental Env. C) Results are presented as percentage of infection relative to cells expressing high CD4 and CCR5 for virus expressing the parental Env. *p<0.05, Student’s t-test.

Polymorphisms at positions 197 and 200 increase gp120 interactions with CCR5

In a previous study, Envs cloned from patient UK1 primary brain isolates were shown to bind with high affinity to CCR5 in vitro (). UK1br T200V mediated increased entry into cells expressing low levels of CCR5 compared to the parental UK1br Env. Therefore, we investigated whether this variant increases binding of UK1br soluble gp120 to cell-surface CCR5. UK1br T200V soluble gp120 bound to CF2th-synCCR5 cells expressing high CCR5 with a 3-fold increase in the presence of sCD4 and 10-fold increase in the absence of sCD4 compared to the parental soluble gp120 (Figure 5). This binding was inhibited by pre-incubation with the CCR5 inhibitor TAK-779 (data not shown). These results suggest that enhanced fusion and infection of cells expressing low CD4 by UK1br T200V is due at least in part to enhanced gp120 binding to CCR5.

V200 enhances macrophage entry mediated by UK1br HIV Env by increasing gp120 binding to CCR5

Soluble gp120 (sgp120) from UK1br wild-type and mutant Envs was produced in 293T cells transfected with Env plasmids containing a stop codon at position 518. Concentrations of sgp120 in the supernatant were determined using ELISA and verified by Western blotting. Concentrated sgp120 was incubated with CF2th-synCCR5 cells with or without preincubation with sCD4. Binding of sgp120 to CCR5 on the cell surface was determined by flow cytometry. Results are representative of 3 independent experiments. *p<0.05, Student’s t-test.

N-linked glycosylation is important for maintaining Env structure and occluding conserved epitopes, including the CCR5-binding site, from neutralizing antibodies (; ; ; ). Loss of the PNGS at position 197 influences HIV sensitivity to neutralizing inhibitors and antibodies, including sCD4, 17b, and b12, in a strain-dependent manner (Table 3) (; ; ; ). Therefore, we measured neutralization sensitivity of the parental and mutant Envs to soluble CD4 (sCD4) and monoclonal antibody (mAb) 17b, which has been used as a surrogate for CCR5-binding, to probe conformational changes in gp120 (). Viruses expressing the M2br parental and mutant Envs were resistant to neutralization by sCD4 at concentrations up to 50 μg/ml (Table 4 and Additional File 3), consistent with published results (; ). UK1br Envs lacking the PNGS at position 197 (UK1br and UK1br T200V) were sensitive to sCD4 neutralization, with IC50 values of 1.3 and 0.4 μg/ml, respectively. As previously reported, the change to Asn at position 197 in the UK1br Env increased resistance to sCD4 neutralization, with a 12-fold increase in the IC50 compared to the parental UK1br (IC50=16.0 μg/ml) (; ; ; ; ). In contrast to previous reports showing that loss of the PNGS at position 197 increased sensitivity of ADA and 89.6 Envs to 17b neutralization (; ; ) (Table 3), only the control virus expressing ADA Env was neutralized by 17b, while those expressing UK1br and M2br parental and mutant Envs were resistant to neutralization by 17b at concentrations up to 40 μg/ml (Table 4 and Additional File 3). Preincubation with a combination of sCD4 and 17b increased neutralization sensitivity of viruses expressing M2br N197D and N197D/V200T, with IC50 values of 26 and 0.22 μg/ml, respectively. M2br Envs containing N197 and all UK1br Envs remained resistant at concentrations of 17b up to 25 μg/ml. These results suggest that changes in β3, including D197 in combination with T/V200, may affect stability and/or exposure of the CCR5-binding site following interactions with CD4.

Table 4

Neutralization of parental and mutant HIV Envs by sCD4 and 17b.a

Virus sCD4 17b sCD4 + 17b
ADA 1.7 4.4 ND
M2br >50 >40 >25
N197D >50 >40 26.0
V200T >50 >40 >25
N197D/V200T >50 >40 0.22
UK1br 1.3 >40 >25
D197N 16.0 >40 >25
T200V 0.4 >40 >25
aValues (μg/ml) represent IC50 values calculated from neutralization curves.

Discussion

In this study, integration of bioinformatic analysis of env sequence datasets with functional studies identified naturally occurring rare variants in the β3 strand of the HIV gp120 bridging sheet that can increase macrophage tropism by enhancing gp120-CCR5 interactions. D197, which results in loss of a PNGS located at the HIV Env trimer apex that determines neutralization sensitivity/resistance in several primary isolates (; ; ; ; ; ), is a rare polymorphism that was detected in brain in some patients with HAD. Codon 200 was estimated to be under positive selection in both brain and lymphoid sequences from HAD patients. D197 and T/V200 enhanced fusion and infection of cells expressing low CD4, including primary macrophages. In UK1br, T200V exhibited increased sgp120 binding to CCR5, reaching levels 10-fold higher than those of the parental Env. Requirements for CD4 and CCR5 are interdependent, with requirements for each receptor being increased when the other component is present at limiting levels (). Together, these findings suggest that β3 polymorphisms can enhance macrophage-tropism by increasing gp120 interactions with CCR5 to overcome the restriction imposed by low CD4 on macrophages.

The location of D197 and T/V200 in the gp120 bridging sheet, their proximity to V3 of adjacent protomers at the trimer apex, and our finding that these polymorphisms can increase HIV infection of cells expressing low levels of CD4 and CCR5, suggests that enhancement of infection in cells expressing low CD4 is most likely due to increased affinity of gp120 for CCR5. This model is also supported by increased binding of monomeric UK1br T200V sgp120 to cell-associated CCR5, and 10-fold increase in binding to CCR5 in the absence of CD4 compared to the parental Env (Figure 5). However, UK1br viruses with T200V did not mediate CD4-independent fusion or infection, a finding that may reflect structural and functional differences between sgp120 monomers and native Env trimers. In crystal and cryo-EM structures of the HIV Env trimer (; ), the V3 crown is buried under the N197 glycan from an adjacent protomer. Data from these structures suggest that the N197 glycan helps to stabilize the Env by occluding V3 and inhibiting its premature release prior to CD4 binding. Thus, loss of the glycan in D197 may affect V3 exposure upon CD4 binding, thereby influencing gp120-CCR5 interactions.

The PNGS at 197 is highly conserved among clade A, B, C, and D Envs (>88%) (). In the present study, sequence polymorphisms that eliminate this PNGS were found in only 8% of brain and 1% of blood/lymphoid Envs in the complete dataset of 796 sequences, indicating this variant is uncommon. Based on previous studies (; ; ; ), we expected to find that loss of the PNGS at 197 would increase exposure of the CCR5-binding site and corresponding neutralization epitopes (Table 3). Unexpectedly, viruses expressing UK1br and M2br Envs containing D197 were not neutralization sensitive to mAb 17b even at high concentrations. In contrast, incubation with both sCD4 and 17b increased neutralization sensitivity of viruses expressing M2br N197D and N197D/V200T (IC50=26.0 and 0.2 μg/ml, respectively), but not the UK1br Envs. In M2br, D197 and T200 may enhance gp120 interactions with CCR5 by stabilizing the 17b epitope following binding to CD4, consistent with a model proposed by O’Connell et al (). UK1br Envs were sensitive to neutralization by sCD4, with increased neutralization resistance associated with addition of the PNGS at position 197 (IC50=16.0 and 1.3 μg/ml for UK1br D197N and UK1br, respectively). These strain-dependent differences in sCD4 neutralization sensitivity may relate to differences in intrinsic reactivity and propensity to sample different conformations, which in turn may influence sCD4-induced sgp120 shedding and virus inactivation (; ).

The strain-dependent effects of the PNGS at position 197 found in the present study are consistent with previous studies reporting strain-dependent effects of this PNGS on sensitivity to the entry inhibitor BMS-806, several monoclonal antibodies (e.g., 17b, b3, b12, F105, 2G12), and patient sera, as well as binding to sCD4 and CCR5 (; ; ; ; ; ; ; ; ). Furthermore, our results suggest that effects of D197 on fusion and entry into cells expressing low CD4 can be additive with those of T/V200. Structural models suggests that specific residues in β3 (D197, T198, and V200) may affect interactions with the variable V3 loop in a cooperative manner (; ; ). Topological relationships of these amino acids to each other, and to V1/V2 and V3 elements near the trimer apex and inter-protomer interface, may help to explain some of the unexpected findings for V200T and T200V; these structural relationships are complex, so amino acid variation at these positions could exert different phenotypes depending on sequence variation in nearby residues. Together, these observations suggest that strain-dependent effects of β3 residues on neutralization sensitivity and macrophage-tropism may reflect amino acid covariation and/or context dependence in these regions.

Genetic evolution of HIV Env occurs in response to in vivo selection pressures that include adaptation to target cell populations and immune evasion. The specific selection pressures that drive changes in M-tropism of R5 Envs in vivo are poorly understood (). Analysis of diversifying selection in the bridging sheet region of the CCR5-binding site in gp120 identified position 200 as a codon in the bridging sheet under positive selection in both brain and blood/lymphoid sequences in sequences from HAD patients. We did not detect positive selection at this position in non-HAD patients; however, this difference could be a result of the different numbers of sequences between HAD (336 sequences from 19 subjects) and non-HAD groups (119 sequences from 11 subjects), which is a limitation of the study. Position 200 is located in several overlapping immunodominant cytotoxic T lymphocyte (CTL) epitopes (http://www.hiv.lanl.gov/content/immunology/index.html); diversifying selection at this position may therefore be a consequence of immune evasion. A better understanding of host and immune selection pressures that shape the genetic evolution of HIV in brain and other tissues is important not only for understanding mechanisms of viral persistence in the CNS reservoir, but also for developing new strategies to prevent HIV-related neurocognitive dysfunction.

Conclusions

In summary, polymorphisms in the β3 strand of the HIV gp120 bridging sheet can increase macrophage tropism by enhancing gp120 interactions with CCR5. T/V200 can enhance fusion and virus entry when combined with D197, suggesting covariation and sequence context influence this phenotype. Requirements for CD4 and CCR5 are interdependent, with the requirement for each receptor being increased when the other is present at limiting levels (). By enhancing Env interactions with CCR5, these polymorphisms may enable viruses to overcome the restriction to macrophage infection imposed by low CD4, thereby promoting infection of the CNS and other macrophage-rich tissues.

Methods

Sequence Analysis

Nucleotide sequences from published studies and Genbank were aligned using ClustalX2. HIV gp120 sequences spanning the V1-V5 region were examined using three different methods to identify codons under positive selection: single likelihood ancestor counting (SLAC), fixed-effects likelihood (FEL), and internal fixed effects likelihood (IFEL) (http://datamonkey.org). SLAC and FEL detect sites under selection at external branches of the phylogenetic tree, while IFEL identifies sites along internal branches (; ; ). Sites were classified as positively selected when a significant p-value (p<0.05) was estimated by at least 2 of these methods. To identify viral determinants associated with brain compartmentalization or dementia, gp120 β2 and β3 amino acid sequences were aligned using ClustalX2. Exploratory analyses of amino acid distributions using large sequence datasets accounted for the variable number of sequences per patient using stratified 2×2 contingency tables to weight sequence variants within each patient, and normalizing for differences in sequencing depth between patients. Statistical analysis of β3 amino acid distributions was then performed using a filtered dataset consisting of one randomly selected sequence per patient. P values were assigned using Fisher’s exact test, with p<0.05 considered significant. Previously unpublished sequences were deposited in Genbank [Genbank:HQ122390-HQ122408; HM355533-HM355546].

Cells

293T and TZM-bl cells, a HeLa cell clone engineered to express CD4 and CCR5 that contains integrated reporter genes for firefly luciferase and E. coli β-galactosidase under control of HIV-1 LTR, were cultured in DMEM media supplemented with 10% (vol/vol) fetal bovine serum (FBS) and 100 μg/ml penicillin and streptomycin. CF2-Luc cells, which are derived from canine thymocyte cell line CF2th and stably express firefly luciferase under control of HIV-1 LTR (), and CF2th-synCCR5 cells, which express a codon-optimized version of human CCR5 containing a C-terminal nonapeptide (TETSQVAPA) tag derived from bovine rhodopsin (), were cultured in medium supplemented with 0.7 mg/ml G418 (Mediatech, Herndon, VA). Affinofile cells were cultured in DMEM supplemented with 10% dialyzed FBS (Gibco, Grand Island, NY), 100 μg/ml penicillin and streptomycin, and 50 μg/ml blasticidin S HCl (Invitrogen, Grand Island, NY) (). Monocyte-derived macrophages (MDM) were derived from peripheral blood mononuclear cells (PBMC) isolated from healthy HIV-negative donors. As previously described, MDM were isolated from PBMC by plastic adherence and cultured in RPMI 1640 medium supplemented with 10% FBS, 100 μg/ml penicillin and streptomycin, and 10 ng/ml macrophage colony stimulating factor (M-CSF, R&D Systems, Minneapolis, MN) (). TZM-bl cells were provided by Norman Letvin. Affinofile cells were provided by Benhur Lee.

Viruses and Mutagenesis

Primary clade B env genes from AIDS patients with HAD were cloned into pCR3.1 as previously described (; ). Env expression and processing was verified by Western blotting with goat anti-gp120 (AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH, Bethesda, MD) (; ). Mutant Env plasmids with changes at positions 197 and 200 (HXB2 numbering) were created by PCR-based mutagenesis and verified by DNA sequencing.

Entry Assays

HIV luciferase reporter viruses were generated by cotransfection of 293T cells with pNL4-3env-luc, an HIV provirus with env deleted and nef replaced with luciferase, and an Env-expressing plasmid as described (). TZM-bl cells were seeded into 96-well plates in media supplemented with 15 μg/ml DEAE-dextran and infected with 104 3H cpm reverse transcriptase (RT) units of virus stock. Cells were lysed 48 hours post-infection and assayed for luciferase activity. MDM were prepared in 48-well plates and infected overnight with 2 × 104 RT units of virus stock in media supplemented with 2 μg/ml polybrene. Cells were lysed 6 days post-infection and assayed for luciferase activity. Affinofile cells were seeded into 96-well plates and treated for 20 hours with serial dilutions of 3 to 0.09 ng/ml doxycycline (Clontech, Mountain View, CA) to induce CD4 expression and 2 to 0.06 μM Ponasterone A (Invitrogen) to induce CCR5 expression (). Cells were then infected with 104 RT units of virus stock, lysed 2 days post-infection, and assayed for luciferase activity. Mock infected cells were used as negative controls. Background luciferase levels were subtracted from all results.

Fusion Assays

293T cells were cotransfected with 2 μg pCR3.1 Env-expressing plasmid and 0.2 μg pLTR-Tat using calcium phosphate. Cf2-Luc cells were cotransfected with 0.1 (low), 1 (intermediate), or 10 μg (high) amounts of pcDNA3-CD4 and 10 μg (high) pcDNA3- CCR5 using Lipofectamine 2000 (Invitrogen). 2.5 × 104 293T cells and 2.5 × 105 CF2-Luc cells were mixed in 48-well plates and incubated for 5–8 hours. Cells were then lysed and analyzed for luciferase activity. 293T cells cotransfected with a nonfunctional Env (pSVIII-ΔKSenv) and pLTR-Tat were used to determine background levels of luciferase.

Flow Cytometry

Cell surface expression of CD4 and CCR5 on induced Affinofile and transfected CF2-Luc cells was analyzed by collecting cells with 5 mM EDTA in PBS and staining with anti-CD4-PE (BD Biosciences, San Jose, CA) and anti-CCR5-PE (BD Pharmingen, San Diego, CA). Cell surface staining was analyzed using a FACSCantoII flow cytometer (BD Biosciences). The approximate number of CD4 and CCR5 molecules per cell was calculated using FACS analysis of Quantibrite beads according to manufacturer’s instructions (BD Biosciences).

ELISA

ELISAs were based on previously described methods (; ; ) with several modifications. Briefly, 96-well microplates were coated overnight with sheep antibody D7324 at 5 μg/ml in PBS (AALTO Bioproducts, Ltd., Ireland). Wells were washed 3 times with PBS supplemented with 2.5% Tween-20 (PBS-T) and blocked for 1 hour at room temperature with PBS-T supplemented with 3% BSA. Supernatants were incubated in the wells for 3 hours, followed by 10 washes with PBS-T and 1 hour incubation with polyclonal rabbit anti-gp120 (American Biotechnologies Inc.). After 10 additional washes with PBS-T, wells were incubated with anti-rabbit-HRP (GE Healthcare). The reaction was developed using BM chemiluminescence ELISA substrate (Roche) according to the manufacturer’s instructions. The reaction was read on a luminometer (Viktor2, Perkin Elmer, Waltham, MA). Calculations of sgp120 concentrations were based on a standard curve of purified gp120 (). Supernatant from cells transfected with empty pcDNA3 vector was used to determine background.

CCR5-Binding Assays

Plasmids expressing soluble gp120 (sgp120) glycoproteins were constructed by introducing a frameshift in gp120 that resulted in a truncation at position 518 (HXB2 numbering). Supernatants from 293T cells transfected with the Env-expressing plasmids were collected 48 hours post-transfection and cleared by centrifugation at 2000 rpm for 5 minutes. Supernatants were stored at −80ºC. Supernatants were concentrated by centrifugation using Amicon Ultra columns with a 30-kD cutoff (Millipore, Billerica, MA). Sgp120 concentrations in concentrated supernatants were determined by ELISA. Equal concentrations of sgp120 were incubated with or without 1 μg/ml soluble CD4 (sCD4, Immunodiagnostics, Woburn, MA) for 30 minutes at 37ºC prior to incubation with CF2-synCCR5 cells. Binding of sgp120 to cells was detected by staining with mAb C11, followed by anti-human-PE (Bio-Rad, Hercules, CA), and was analyzed by flow cytometry. Specificity of gp120 interaction with CCR5 was verified by pre-incubating CF2-synCCR5 cells with 100 nM TAK-779 (obtained through the AIDS Research and Reference Reagents Program, Bethesda, MD) () for 1 hour at 37ºC prior to incubation with sgp120.

Neutralization Assays

HIV luciferase reporter viruses were incubated with a range of concentrations of sCD4, monoclonal antibody (mAb) 17b (NIH AIDS Research and Reference Reagent Program, donated by Dr. James E. Robinson), or 0.1 μg/ml sCD4 and a range of concentrations of 17b in combination for 1 hour at 37ºC prior to infection of TZM-BL cells. Cells were harvested 48 hours post-infection and assayed for luciferase activity ().

Statistical Analysis

Statistical significance in experiments comparing parental and mutant Envs was determined using Student’s unpaired 2-tailed t-test, with results presented as means of duplicate wells from a single experiment. Bars represent standard deviations. P-values < 0.05 were considered significant.

  • We analyze HIV Env sequences and identify amino acids in beta 3 of the gp120 bridging sheet that enhance macrophage tropism.

  • These amino acids at positions 197 and 200 are present in brain of some patients with HIV-associated dementia.

  • D197 results in loss of a glycan near the HIV Env trimer apex, which may increase exposure of V3.

  • These variants may promote infection of macrophages in the brain by enhancing gp120-CCR5 interactions

Supplementary Material

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Acknowledgments

We thank J. Cunningham, J. Sodroski, and B. Chen for helpful discussions, R. Bosch for helpful discussions regarding statistical analysis of HIV sequences, N. Letvin for TZM-bl cells, B. Lee for Affinofile cells, and Will (Po-Jen) Yen for help with structural modeling. The following reagents were obtained through the NIH AIDS Research and Reference Reagent Program, Division of AIDS, NIAID: monoclonal antibody 17b from Dr. James E. Robinson, goat anti-gp120, and TAK-779. This work was supported by NIH Grant MH83588 and MH 97659 to D.G. M.E.M. was supported in part by NIH fellowship F31NS060611. Core facilities were supported by Harvard Medical School Center for AIDS Research (CFAR) and DFCI/Harvard Cancer Center grants P30 AI060354 and P30 CA06516.

Footnotes

 

Competing Interests

The authors declare that they have no competing interests.

 

Authors’ Contributions

M.E.M. and D.G. designed research; M.E.M. performed research; M.E.M. and D.G. analyzed the data; K.K. and S.M.W. provided sequence data; M.E.M. and D.G. wrote the paper. All authors reviewed the manuscript and approved the final version.

 

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Jawahar Raina

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