Abstract
BackgroundThe 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.ResultsWe 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.ConclusionsGGR 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.
Highlights
The efficiency of CD4/CCR5 mediated Human immunodeficiency virus type 1 (HIV-1) entry has important implications for pathogenesis and transmission
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 fulllength, 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
The Affinofle system and associated VERSA metrics have provided investigators a more quantitative method to characterize viral entry efficiency as a function of CD4 and CCR5 expression. Quantitative comparisons of these three VERSA metrics—Mean infectivity (M), Vector Angle (θ) and Amplitude (Δ)—have facilitated our understanding of how CD4/CCR5 usage efficiencies correspond to distinct envelope glycoproteins (Env) phenotypes associated with resistance to CCR5-inhibitors, and the myriad of in vitro or in vivo selective pressures that result in differential or altered cell tropism [28,34,36,37,39,54,55,56]
Summary
The efficiency of CD4/CCR5 mediated HIV-1 entry has important implications for pathogenesis and transmission. 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. The recent characterization of transmitter/founder (T/F) Envs has indicated that these R5 variants enter and replicate in activated primary T-cells but not macrophages [30], underscoring the increasingly evident notion that CCR5 usage is not equivalent to macrophage-tropism [5,31] Together, these studies show that the efficiency with which a viral Env engages CD4 and/or CCR5 can have an influence on pathogenicity, disease progression and resistance to CCR5 antagonists [5,32,33]. A more refined understanding of how Env-CD4/CCR5 usage develops and differs under alternate evolutionary histories will inform the development and use of HIV-1 vaccines and therapeutics that target HIV-1 entry
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