Quantifying CD4/CCR5 Usage Efficiency of HIV-1 Env Using the Affinofile System

Abstract

Entry of HIV-1 into target cells involves the interaction of the HIV envelope (Env) with both a primary receptor (CD4) and a coreceptor (CXCR4 or CCR5). The relative efficiency with which a particular Env uses these receptors is a major component of cellular tropism in the context of entry and is related to a variety of pathological Env phenotypes (Chikere et al. Virology 435:81-91, 2013). The protocols outlined in this chapter describe the use of the Affinofile system, a 293-based dual-inducible cell line that expresses up to 25 distinct combinations of CD4 and CCR5, as well as the associated Viral Entry Receptor Sensitivity Assay (VERSA) metrics used to summarize the CD4/CCR5-dependent infectivity results. This system allows for high-resolution profiling of CD4 and CCR5 usage efficiency in the context of unique viral phenotypes.

The Effects of Statistical Multiplicity of Infection on Virus Quantification and Infectivity Assays

“…The luciferase reporter assay is commonly used to measure the infectivity of a viral strain. Here, the ratio m ¼ N/M of total infections over the number of plated cells is estimated by measuring the transcription activity of viral proteins (14)(15)(16). The reporter employs an oxidative enzyme luciferase that facilitates a reaction when introduced to the substrate luciferin, resulting in bioluminescence.…”

“…Once these parameters are assigned, viral and cell population dynamics and their statistical properties can be predicted. Among the different experimental assays, one often seeks to evaluate the number of virus particles in a stock solution or the number of viruses that have successfully infected host cells (6,(14)(15)(16)(17)(18)(19).…”

“…Infectivity assays (IAs), on the other hand, aim to quantify the number of viruses that have successfully infected host cells under varying antiviral drug environments (14)(15)(16). IAs may measure the total transcription activity across all cells, such as the luciferase reporter assay (15,26), or may count the number of host cells that were successfully infected, such as the enzyme-linked immunosorbent assay and the immunofluorescence assay with fluorescence-activated cell sorting (4,14,15,18).…”

The Effects of Statistical Multiplicity of Infection on Virus Quantification and Infectivity Assays

“…The luciferase reporter assay is commonly used to measure the infectivity of a viral strain. Here the ratio µ = N/M of total infections over the number of plated cells is estimated by measuring the transcription activity of viral proteins [14][15][16]. The reporter employs an oxidative enzyme luciferase that facilitates a reaction when introduced to the substrate luciferin, resulting in bioluminescence.…”

“…Infectivity assays (IA), on the other hand, aim to quantify the number of viruses that have successfully infected host cells under varying antiviral drug environments [14][15][16]. IAs may measure the total transcription activity across all cells, such as the luciferase reporter assay [15,25], or may count the number of host cells that were successfully infected, such as the enzyme-linked immunosorbent assay (ELISA) and the immunofluorence assay with fluorescence activated cell sorting (FACS) [4,14,15,26].…”