Limitations of dual‐fluorescent HIV reporter viruses in a model of pre‐activation latency

INTRODUCTION: HIV latency can be established in vitro following direct infection of a resting CD4+ T cell (pre‐activation latency) or infection of an activated CD4+ T cell which then returns to a resting state (post‐activation latency). We modified a previously published dual‐fluorescent reporter virus seeking to track the establishment and reactivation of pre‐activation latency in primary CD4+ T cells. METHODS: A previously published dual‐fluorescent reporter virus was modified so that expression of enhanced green fluorescent protein (GFP) was under control of the elongation factor 1 alpha (EF1α) promoter to detect latent infection, and E2 crimson (E2CRM) was under control of the nef promoter to detect productive infection. NL4.3 that expressed GFP in place of nef was used as a positive control. We infected the Jurkat T‐cell line and primary CD4+ T cells that were either unstimulated or stimulated with either the chemokine CCL19 or phytohaemagglutinin (PHA)/IL‐2 and quantified the expression of both fluorescent proteins by flow cytometry. The study was carried out over a period of two years from September 2016 to October 2018. RESULTS AND DISCUSSION: Expression of both fluorophores was detected following infection of the Jurkat T‐cell line while only low levels of the latent reporter were observed following infection of primary CD4+ T cells. In unstimulated and CCL19‐treated CD4+ T cells, expression of the GFP latent reporter, increased after further activation of the cells with PHA/phorbol 12‐myristate 13‐acetate (PMA). CONCLUSIONS: Our findings demonstrate that the EF1α promoter has poor constitutive expression in resting CD4+ T cells. Therefore, dual‐fluorescent reporter viruses with the EF1α promoter may underestimate the frequency of latent infection in resting CD4+ T cells.