Quantitative and high-throughput receptor affinity profiling system reveals distinct pathophysiological HIV-1 phenotypes
HIV-1 affinity for CD4 and CCR5 is associated with differential pathogenicity. Therefore, we pioneered a dually inducible cell line based system that could quantitatively and comprehensively characterize viral entry efficiency as a co-dependent function of CD4 and CCR5 expression levels. This receptor affinity profiling system (Affinofile) has revealed biologically relevant phenotypes in Envelopes (Envs) with differential CD4/CCR5 usage efficiencies, providing a more refined understanding of receptor and coreceptor affinities, which can have an impact on the development and use of HIV-1 therapeutics.
To facilitate a more rapid and refined analysis of CD4 and CCR5 usage efficiencies with even greater sensitivity, we engineered a reporter Affinofile system containing a tat-rev dependent eGFP-Gaussia luciferase Reporter (GGR). Using this GGR Affinofile system, we (1) characterized the phenotypic and biological consequences for Envs with defined mutations that modulate CD4 or CCR5 binding, (2) determined Transmitter/Founder (T/F) Envs have differential CD4/CCR5 usage compared to Chronic Env, (3) revealed phenotypically distinct CD4/CCR5 usage patterns among the prevalent HIV-1 subtypes (A,B, C and D), and (4) uncovered that mutations that confer resistance to Broadly Neutralizing Antibodies (BNAbs) often compromised the efficiency of CD4/CCR5 usage and entry.
Analysis of mutations known to only modulate CCR5 (K421D, S142N) binding demonstrated that CD4 and CCR5 usage is an inter-related process as mutations that affect CD4 binding influenced the efficiency of CCR5 usage and vice versa. The relative entry efficiencies defined in GGR Affinofile system were also reflected in their entry efficiencies into primary CD4+ T cell subsets. In addition, we show that transmitter/founder and chronic envelopes have distinct entry efficiencies that yield characteristic vector metrics. Next, we analyzed over 28 pseudotyped HIV-1 viruses from four different subtypes, and noted that subtype C envelopes could be distinguished from the other subtypes based on their greater efficiency of CD4/CCR5 usage which was reflected in their vector metrics (increased vector angle and mean infectivity). Lastly, envelopes with engineered mutations known to confer resistance to BNAbs, VRC01 and PG6/PG19, invariably resulted in a decreased CD4/CCR5 usage efficiency.
Our results suggest that our GGR Affinofile system can quantify and reveal biologically relevant differences in CD4/CCR5 usage patterns in Envs that reflect their genetic-epidemiological differences, pathogenicity, cell tropism, and even fitness cost as a result of resistant-mutations to BNAbs.