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Distinct HIV-1 entry phenotypes are associated with transmission, subtype specificity, and resistance to broadly neutralizing antibodies.

Chikere K, Webb NE, Chou T, Borm K, Sterjovski J, Gorry PR, Lee B - Retrovirology (2014)

Bottom Line: First, we profiled a panel of reference subtype B transmitted/founder (T/F) and chronic Envs (n = 12) by analyzing the infectivity of each Env across 25 distinct combinations of CD4/CCR5 expression levels.Lastly, mutations known to confer resistance to VRC01 or PG6/PG19 BNAbs, when engineered into subtypes A-D Envs, resulted in significantly decreased CD4/CCR5 usage efficiency.GGR Affinofile profiling reveals pathophysiological phenotypes associated with varying HIV-1 entry efficiencies, and highlight the fitness costs associated with resistance to some broadly neutralizing antibodies.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Microbiology, Immunology, and Molecular Genetics, Los Angeles, USA. benhur.lee@mssm.edu.

ABSTRACT

Background: The efficiency of CD4/CCR5 mediated HIV-1 entry has important implications for pathogenesis and transmission. The HIV-1 receptor affinity profiling (Affinofile) system analyzes and quantifies the infectivity of HIV-1 envelopes (Envs) across a spectrum of CD4/CCR5 expression levels and distills these data into a set of Affinofile metrics. The Affinofile system has shed light on how differential CD4/CCR5 usage efficiencies contributes to an array of Env phenotypes associated with cellular tropism, viral pathogenesis, and CCR5 inhibitor resistance. To facilitate more rapid, convenient, and robust analysis of HIV-1 entry phenotypes, we engineered a reporter Affinofile system containing a Tat- and Rev-dependent Gaussia luciferase-eGFP-Reporter (GGR) that is compatible with the use of pseudotyped or replication competent viruses with or without a virally encoded reporter gene. This GGR Affinofile system enabled a higher throughput characterization of CD4/CCR5 usage efficiencies associated with differential Env phenotypes.

Results: We first validated our GGR Affinofile system on isogenic JR-CSF Env mutants that differ in their affinity for CD4 and/or CCR5. We established that their GGR Affinofile metrics reflected their differential entry phenotypes on primary PBMCs and CD4+ T-cell subsets. We then applied GGR Affinofile profiling to reveal distinct entry phenotypes associated with transmission, subtype specificity, and resistance to broadly neutralizing antibodies (BNAbs). First, we profiled a panel of reference subtype B transmitted/founder (T/F) and chronic Envs (n = 12) by analyzing the infectivity of each Env across 25 distinct combinations of CD4/CCR5 expression levels. Affinofile metrics revealed that at low CCR5 levels, our panel of subtype B T/F Envs was more dependent on high levels of CD4 for HIV-1 entry compared to chronic Envs. Next, we analyzed a reference panel of 28 acute/early subtype A-D Envs, and noted that subtype C Envs could be distinguished from the other subtypes based on their infectivity profiles and relevant Affinofile metrics. Lastly, mutations known to confer resistance to VRC01 or PG6/PG19 BNAbs, when engineered into subtypes A-D Envs, resulted in significantly decreased CD4/CCR5 usage efficiency.

Conclusions: GGR Affinofile profiling reveals pathophysiological phenotypes associated with varying HIV-1 entry efficiencies, and highlight the fitness costs associated with resistance to some broadly neutralizing antibodies.

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Sensitivity vector metrics further illuminate the phenotype of well-characterized point mutants. (A) Infectivity profile of wt JR-CSF (R5) envelope, and three point mutants: (B) S142N, (C) E153G and (D) K421D, previously shown to enhance or perturb CCR5 or CD4 usage with polar plots (beneath) representing the metrics obtained from mathematical analysis of the infectivity profiles (A-C). The vector angle (θ) is the angle between the x-axis and the dotted line. The vector amplitude (Δ) is represented by the length of the dotted line. The mean infectivity (M) is represented by the size of the circle. Data shown is a representative of two experiments. (E) Table of the average Affinofile metrics obtained from (A-D) and graphically shown in polar plots beneath (A-D). Boxes next to (E) describe the phenotypes indicated by each metric relative to wt JR-CSF. The infectivity profile of each Env was independently repeated twice.
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Figure 3: Sensitivity vector metrics further illuminate the phenotype of well-characterized point mutants. (A) Infectivity profile of wt JR-CSF (R5) envelope, and three point mutants: (B) S142N, (C) E153G and (D) K421D, previously shown to enhance or perturb CCR5 or CD4 usage with polar plots (beneath) representing the metrics obtained from mathematical analysis of the infectivity profiles (A-C). The vector angle (θ) is the angle between the x-axis and the dotted line. The vector amplitude (Δ) is represented by the length of the dotted line. The mean infectivity (M) is represented by the size of the circle. Data shown is a representative of two experiments. (E) Table of the average Affinofile metrics obtained from (A-D) and graphically shown in polar plots beneath (A-D). Boxes next to (E) describe the phenotypes indicated by each metric relative to wt JR-CSF. The infectivity profile of each Env was independently repeated twice.

Mentions: To further define the biological meaning of the three Affinofile metrics, we examined three point mutants in JR-CSF with well-described effects on CD4 and CCR5 binding. S142N[42] and E153G[43] are both V1 loop mutations that increase the ability of JR-CSF to enter cells with low levels of CCR5[20,25] or CD4, respectively, while K421D is a “bridging sheet” mutant that reduces the affinity of gp120 for CCR5[44,45]. Viruses pseudotyped with wild type (wt) JR-CSF, or with S142N or K421D Env mutants were produced and titrated first on Ghost-R5 cells. An equivalent MOI (0.2) of each pseudotype was then used to infect GGR Affinofile cells expressing 25 distinct combinations of CD4 and CCR5 levels. We are cognizant that viral titers are cell-type dependent, but we reasoned that normalizing the infectious inoculum on GGR Affinofile cells using titers obtained from infecting Ghost-R5 cells (where CD4/CCR5 levels are non-limiting) would fairly reveal biologically relevant differences in entry efficiencies when CD4/CCR5 levels do become limiting under certain induction conditions on GGR Affinofile cells.Compared to wt JR-CSF (Figure 3A), the S142N mutant exhibited enhanced entry at every level of CCR5 at or above a specific threshold level of CD4 (0.4 ng/ml Doxy) (Figure 3B, compare the rows of green, yellow, orange and red bars along the CCR5 axis with Figure 3A). A similar increase in infection was observed for E153G, particularly at low CD4 expression (compare blue and green bars in Figure 3C to A), whereas the K421D mutant showed inefficient entry at low CCR5 levels regardless of how much CD4 was present (Figure 3D, note the low infectivity at 0 and 0.25 μM PonA (<20% of maximum) even when CD4 was maximally induced). S142N was more responsive to changes in CCR5 levels than wt JR-CSF, and this phenotype was reflected as an increase in from 30.5° to 38° for wt JR-CSF and S142N, respectively. Recall that a relative increase or decrease in vector angle indicates that an Env’s infectivity is more responsive to changes in levels of CCR5 or CD4, respectively. A summary of the Affinofile metrics is given in Figure 3E, and illustrated in the polar plots below Figures 3A-D.


Distinct HIV-1 entry phenotypes are associated with transmission, subtype specificity, and resistance to broadly neutralizing antibodies.

Chikere K, Webb NE, Chou T, Borm K, Sterjovski J, Gorry PR, Lee B - Retrovirology (2014)

Sensitivity vector metrics further illuminate the phenotype of well-characterized point mutants. (A) Infectivity profile of wt JR-CSF (R5) envelope, and three point mutants: (B) S142N, (C) E153G and (D) K421D, previously shown to enhance or perturb CCR5 or CD4 usage with polar plots (beneath) representing the metrics obtained from mathematical analysis of the infectivity profiles (A-C). The vector angle (θ) is the angle between the x-axis and the dotted line. The vector amplitude (Δ) is represented by the length of the dotted line. The mean infectivity (M) is represented by the size of the circle. Data shown is a representative of two experiments. (E) Table of the average Affinofile metrics obtained from (A-D) and graphically shown in polar plots beneath (A-D). Boxes next to (E) describe the phenotypes indicated by each metric relative to wt JR-CSF. The infectivity profile of each Env was independently repeated twice.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4230403&req=5

Figure 3: Sensitivity vector metrics further illuminate the phenotype of well-characterized point mutants. (A) Infectivity profile of wt JR-CSF (R5) envelope, and three point mutants: (B) S142N, (C) E153G and (D) K421D, previously shown to enhance or perturb CCR5 or CD4 usage with polar plots (beneath) representing the metrics obtained from mathematical analysis of the infectivity profiles (A-C). The vector angle (θ) is the angle between the x-axis and the dotted line. The vector amplitude (Δ) is represented by the length of the dotted line. The mean infectivity (M) is represented by the size of the circle. Data shown is a representative of two experiments. (E) Table of the average Affinofile metrics obtained from (A-D) and graphically shown in polar plots beneath (A-D). Boxes next to (E) describe the phenotypes indicated by each metric relative to wt JR-CSF. The infectivity profile of each Env was independently repeated twice.
Mentions: To further define the biological meaning of the three Affinofile metrics, we examined three point mutants in JR-CSF with well-described effects on CD4 and CCR5 binding. S142N[42] and E153G[43] are both V1 loop mutations that increase the ability of JR-CSF to enter cells with low levels of CCR5[20,25] or CD4, respectively, while K421D is a “bridging sheet” mutant that reduces the affinity of gp120 for CCR5[44,45]. Viruses pseudotyped with wild type (wt) JR-CSF, or with S142N or K421D Env mutants were produced and titrated first on Ghost-R5 cells. An equivalent MOI (0.2) of each pseudotype was then used to infect GGR Affinofile cells expressing 25 distinct combinations of CD4 and CCR5 levels. We are cognizant that viral titers are cell-type dependent, but we reasoned that normalizing the infectious inoculum on GGR Affinofile cells using titers obtained from infecting Ghost-R5 cells (where CD4/CCR5 levels are non-limiting) would fairly reveal biologically relevant differences in entry efficiencies when CD4/CCR5 levels do become limiting under certain induction conditions on GGR Affinofile cells.Compared to wt JR-CSF (Figure 3A), the S142N mutant exhibited enhanced entry at every level of CCR5 at or above a specific threshold level of CD4 (0.4 ng/ml Doxy) (Figure 3B, compare the rows of green, yellow, orange and red bars along the CCR5 axis with Figure 3A). A similar increase in infection was observed for E153G, particularly at low CD4 expression (compare blue and green bars in Figure 3C to A), whereas the K421D mutant showed inefficient entry at low CCR5 levels regardless of how much CD4 was present (Figure 3D, note the low infectivity at 0 and 0.25 μM PonA (<20% of maximum) even when CD4 was maximally induced). S142N was more responsive to changes in CCR5 levels than wt JR-CSF, and this phenotype was reflected as an increase in from 30.5° to 38° for wt JR-CSF and S142N, respectively. Recall that a relative increase or decrease in vector angle indicates that an Env’s infectivity is more responsive to changes in levels of CCR5 or CD4, respectively. A summary of the Affinofile metrics is given in Figure 3E, and illustrated in the polar plots below Figures 3A-D.

Bottom Line: First, we profiled a panel of reference subtype B transmitted/founder (T/F) and chronic Envs (n = 12) by analyzing the infectivity of each Env across 25 distinct combinations of CD4/CCR5 expression levels.Lastly, mutations known to confer resistance to VRC01 or PG6/PG19 BNAbs, when engineered into subtypes A-D Envs, resulted in significantly decreased CD4/CCR5 usage efficiency.GGR Affinofile profiling reveals pathophysiological phenotypes associated with varying HIV-1 entry efficiencies, and highlight the fitness costs associated with resistance to some broadly neutralizing antibodies.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Microbiology, Immunology, and Molecular Genetics, Los Angeles, USA. benhur.lee@mssm.edu.

ABSTRACT

Background: The efficiency of CD4/CCR5 mediated HIV-1 entry has important implications for pathogenesis and transmission. The HIV-1 receptor affinity profiling (Affinofile) system analyzes and quantifies the infectivity of HIV-1 envelopes (Envs) across a spectrum of CD4/CCR5 expression levels and distills these data into a set of Affinofile metrics. The Affinofile system has shed light on how differential CD4/CCR5 usage efficiencies contributes to an array of Env phenotypes associated with cellular tropism, viral pathogenesis, and CCR5 inhibitor resistance. To facilitate more rapid, convenient, and robust analysis of HIV-1 entry phenotypes, we engineered a reporter Affinofile system containing a Tat- and Rev-dependent Gaussia luciferase-eGFP-Reporter (GGR) that is compatible with the use of pseudotyped or replication competent viruses with or without a virally encoded reporter gene. This GGR Affinofile system enabled a higher throughput characterization of CD4/CCR5 usage efficiencies associated with differential Env phenotypes.

Results: We first validated our GGR Affinofile system on isogenic JR-CSF Env mutants that differ in their affinity for CD4 and/or CCR5. We established that their GGR Affinofile metrics reflected their differential entry phenotypes on primary PBMCs and CD4+ T-cell subsets. We then applied GGR Affinofile profiling to reveal distinct entry phenotypes associated with transmission, subtype specificity, and resistance to broadly neutralizing antibodies (BNAbs). First, we profiled a panel of reference subtype B transmitted/founder (T/F) and chronic Envs (n = 12) by analyzing the infectivity of each Env across 25 distinct combinations of CD4/CCR5 expression levels. Affinofile metrics revealed that at low CCR5 levels, our panel of subtype B T/F Envs was more dependent on high levels of CD4 for HIV-1 entry compared to chronic Envs. Next, we analyzed a reference panel of 28 acute/early subtype A-D Envs, and noted that subtype C Envs could be distinguished from the other subtypes based on their infectivity profiles and relevant Affinofile metrics. Lastly, mutations known to confer resistance to VRC01 or PG6/PG19 BNAbs, when engineered into subtypes A-D Envs, resulted in significantly decreased CD4/CCR5 usage efficiency.

Conclusions: GGR Affinofile profiling reveals pathophysiological phenotypes associated with varying HIV-1 entry efficiencies, and highlight the fitness costs associated with resistance to some broadly neutralizing antibodies.

Show MeSH
Related in: MedlinePlus