Limits...
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.

Show MeSH

Related in: MedlinePlus

Generation and characterization of the GGR Affinofile Cell Line. (A) Schema of the tat-rev dependent Gaussia luciferase (gLuc)-IRES-GFP reporter vector as described in the text. (B) and (C) GGR cells were maximally induced with doxycyline (Doxy, 4ng/ml) and ponasterone A (PonA, 4 μM) at the time of their seeding in 96-well plates. 16–21 hours post-seeding/induction, cells were infected with wt JR-CSF virus at varying multiplicities of infection (MOI). The titer of the virus was previously determined on stable CD4/CCR5-expressing GHOST cells where CD4/CCR5 levels are non-limiting. At 17, 24, 48, and 72 hpi, 10 μL (out of 150) of the infected cell supernatant was removed and analyzed for gLuc activity as per manufacturer’s instructions. Luciferase activity (measured as relative light units, RLU), and the corresponding signal:noise ratios at each data point are shown in (B) and (C), respectively. Mock-infected cell supernatant served as the background signal. (D) and (E) GGR cells were induced at high (3.2ng/mL Doxy, 2 μM PonA), medium (1.6ng/mL Doxy, 1μm PonA), and low (0.4ng/mL Doxy, 0.25μM PonA) levels, and infected as above with pseudotyped virus at an MOI of 0.25. Three days post-infection, supernatant was collected and analyzed for gluc expression (E), while cells from each well were individually processed for intracellular p24 staining (D) as described in methods. Data shown is representative of two independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Generation and characterization of the GGR Affinofile Cell Line. (A) Schema of the tat-rev dependent Gaussia luciferase (gLuc)-IRES-GFP reporter vector as described in the text. (B) and (C) GGR cells were maximally induced with doxycyline (Doxy, 4ng/ml) and ponasterone A (PonA, 4 μM) at the time of their seeding in 96-well plates. 16–21 hours post-seeding/induction, cells were infected with wt JR-CSF virus at varying multiplicities of infection (MOI). The titer of the virus was previously determined on stable CD4/CCR5-expressing GHOST cells where CD4/CCR5 levels are non-limiting. At 17, 24, 48, and 72 hpi, 10 μL (out of 150) of the infected cell supernatant was removed and analyzed for gLuc activity as per manufacturer’s instructions. Luciferase activity (measured as relative light units, RLU), and the corresponding signal:noise ratios at each data point are shown in (B) and (C), respectively. Mock-infected cell supernatant served as the background signal. (D) and (E) GGR cells were induced at high (3.2ng/mL Doxy, 2 μM PonA), medium (1.6ng/mL Doxy, 1μm PonA), and low (0.4ng/mL Doxy, 0.25μM PonA) levels, and infected as above with pseudotyped virus at an MOI of 0.25. Three days post-infection, supernatant was collected and analyzed for gluc expression (E), while cells from each well were individually processed for intracellular p24 staining (D) as described in methods. Data shown is representative of two independent experiments.

Mentions: We modified a previously published Tat/Rev-dependent vector[40,41] by cloning the Gaussia luciferase (GLuc) gene upstream of an eGFP reporter gene, linked via an internal ribosomal entry site (IRES) (Figure 1A). Judiciously placed splice donor and acceptor sites, in addition to the Rev-responsive element (RRE) placed downstream of the eGFP reporter gene, ensures that only the full-length, unspliced reporter mRNA will be translated in the presence of Tat and Rev, which is provided by commonly used HIV-1 reporter vectors and replication-competent HIV-1. Lentiviral VSV-G pseudotypes containing this GLuc-eGFP Reporter (GGR) vector were used to transduce early passage Affinofile cells. Stable GGR Affinofile cell lines with optimal properties were single cell cloned as described in methods.To determine the ability of GGR Affinofile cells to detect HIV-1 infection, we infected a stable clone of GGR Affinofile cells (at maximum CD4/CCR5 induction) using a range of viral inoculums (JR-CSF, MOI = 0.5 – 0.0625) and serially sampled the infected cell culture supernatant for GLuc activity. GLuc activity could be detected at 20-fold above background as early as 17 hpi depending on the amount of viral inoculum used (Figure 1B-C). Furthermore, we observed that GLuc activity in the infected culture supernatant mirrored the level of infection as reported by intracellular p24 staining (Figure 1D-E), especially at low MOIs (e.g. 0.2) that ensure a single infectious event per cell.


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)

Generation and characterization of the GGR Affinofile Cell Line. (A) Schema of the tat-rev dependent Gaussia luciferase (gLuc)-IRES-GFP reporter vector as described in the text. (B) and (C) GGR cells were maximally induced with doxycyline (Doxy, 4ng/ml) and ponasterone A (PonA, 4 μM) at the time of their seeding in 96-well plates. 16–21 hours post-seeding/induction, cells were infected with wt JR-CSF virus at varying multiplicities of infection (MOI). The titer of the virus was previously determined on stable CD4/CCR5-expressing GHOST cells where CD4/CCR5 levels are non-limiting. At 17, 24, 48, and 72 hpi, 10 μL (out of 150) of the infected cell supernatant was removed and analyzed for gLuc activity as per manufacturer’s instructions. Luciferase activity (measured as relative light units, RLU), and the corresponding signal:noise ratios at each data point are shown in (B) and (C), respectively. Mock-infected cell supernatant served as the background signal. (D) and (E) GGR cells were induced at high (3.2ng/mL Doxy, 2 μM PonA), medium (1.6ng/mL Doxy, 1μm PonA), and low (0.4ng/mL Doxy, 0.25μM PonA) levels, and infected as above with pseudotyped virus at an MOI of 0.25. Three days post-infection, supernatant was collected and analyzed for gluc expression (E), while cells from each well were individually processed for intracellular p24 staining (D) as described in methods. Data shown is representative of two independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Generation and characterization of the GGR Affinofile Cell Line. (A) Schema of the tat-rev dependent Gaussia luciferase (gLuc)-IRES-GFP reporter vector as described in the text. (B) and (C) GGR cells were maximally induced with doxycyline (Doxy, 4ng/ml) and ponasterone A (PonA, 4 μM) at the time of their seeding in 96-well plates. 16–21 hours post-seeding/induction, cells were infected with wt JR-CSF virus at varying multiplicities of infection (MOI). The titer of the virus was previously determined on stable CD4/CCR5-expressing GHOST cells where CD4/CCR5 levels are non-limiting. At 17, 24, 48, and 72 hpi, 10 μL (out of 150) of the infected cell supernatant was removed and analyzed for gLuc activity as per manufacturer’s instructions. Luciferase activity (measured as relative light units, RLU), and the corresponding signal:noise ratios at each data point are shown in (B) and (C), respectively. Mock-infected cell supernatant served as the background signal. (D) and (E) GGR cells were induced at high (3.2ng/mL Doxy, 2 μM PonA), medium (1.6ng/mL Doxy, 1μm PonA), and low (0.4ng/mL Doxy, 0.25μM PonA) levels, and infected as above with pseudotyped virus at an MOI of 0.25. Three days post-infection, supernatant was collected and analyzed for gluc expression (E), while cells from each well were individually processed for intracellular p24 staining (D) as described in methods. Data shown is representative of two independent experiments.
Mentions: We modified a previously published Tat/Rev-dependent vector[40,41] by cloning the Gaussia luciferase (GLuc) gene upstream of an eGFP reporter gene, linked via an internal ribosomal entry site (IRES) (Figure 1A). Judiciously placed splice donor and acceptor sites, in addition to the Rev-responsive element (RRE) placed downstream of the eGFP reporter gene, ensures that only the full-length, unspliced reporter mRNA will be translated in the presence of Tat and Rev, which is provided by commonly used HIV-1 reporter vectors and replication-competent HIV-1. Lentiviral VSV-G pseudotypes containing this GLuc-eGFP Reporter (GGR) vector were used to transduce early passage Affinofile cells. Stable GGR Affinofile cell lines with optimal properties were single cell cloned as described in methods.To determine the ability of GGR Affinofile cells to detect HIV-1 infection, we infected a stable clone of GGR Affinofile cells (at maximum CD4/CCR5 induction) using a range of viral inoculums (JR-CSF, MOI = 0.5 – 0.0625) and serially sampled the infected cell culture supernatant for GLuc activity. GLuc activity could be detected at 20-fold above background as early as 17 hpi depending on the amount of viral inoculum used (Figure 1B-C). Furthermore, we observed that GLuc activity in the infected culture supernatant mirrored the level of infection as reported by intracellular p24 staining (Figure 1D-E), especially at low MOIs (e.g. 0.2) that ensure a single infectious event per cell.

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