Abstract
Understanding the mechanisms involved in HIV infection and latency, and development of a cure, rely on the availability of sensitive research tools such as indicator cells, which allow rigorous quantification of viral activity. Here we describe the construction and validation of a novel dual-indicator cell line, Sup-GGR, which offers two different readouts to quantify viral replication. A construct expressing both Gaussia luciferase and hrGFP in a Tat- and Rev-dependent manner was engineered into SupT1-CCR5 to create Sup-GGR cells. This cell line supports the replication of both X4 and R5-tropic HIV as efficiently as its parental cell line, SupT1-CCR5, and allows repeated sampling without the need to terminate the culture. Sup-GGR demonstrates comparable sensitivity and similar kinetics in virus outgrowth assays (VOA) to SupT1-CCR5 using clinical samples. However the Gaussia luciferase reporter is significantly less labor-intensive and allows earlier detection of reactivated latent viruses compared to the conventional HIV p24 ELISA assay. The Sup-GGR cell line constitutes a versatile new tool for HIV research and clinical trials.
Highlights
Understanding the mechanisms involved in HIV infection and latency, and development of a cure, rely on the availability of sensitive research tools such as indicator cells, which allow rigorous quantification of viral activity
Gaussia luciferase is secreted into the growth media; as such these supernatants can be harvested for reporter readout and replaced with fresh media so that the same culture can be maintained for subsequent harvests at different time points
The bicistronic reporter cassette contains Gaussia luciferase (GLuc) and humanized Renilla GFP (hrGFP) coding sequences separated by an internal ribosome entry site (IRES), and is flanked by HIV major splice donor and acceptor sequences
Summary
Understanding the mechanisms involved in HIV infection and latency, and development of a cure, rely on the availability of sensitive research tools such as indicator cells, which allow rigorous quantification of viral activity. A construct expressing both Gaussia luciferase and hrGFP in a Tat- and Rev-dependent manner was engineered into SupT1-CCR5 to create SupGGR cells This cell line supports the replication of both X4 and R5-tropic HIV as efficiently as its parental cell line, SupT1-CCR5, and allows repeated sampling without the need to terminate the culture. We report construction and characterization of a novel dual-indicator cell line, Sup-GGR (Gaussia GFP Reporter) that overcomes this limitation These cells are derived from SupT1-CCR5, a T-lymphoblastic lymphoma cell line that stably expresses the HIV-1 receptor CD4, and the co-receptors CCR5 and CXCR4, allowing entry of both X4 and R5 tropic viruses[3]. We made a head to head comparison, and found that the novel Sup-GGR cell line is comparably efficient to SupT1-CCR5 in supporting the replication of a range of laboratory and clinical strains of HIV, while maintaining equivalence in virus outgrowth kinetics.
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