Establishment and characterization of a cell model for HIV-1 latency with a Tat inducible expression system

Abstract

Highly Active Anti-Retroviral Therapy (HAART) reduces the viral load in the patients to undetectable titres but it is incapable of eradicating the virus from the patient's system. HIV is notorious for having the propensity to hide inside a patient's memory T cells and silencing its own transcription thus going into a state called latency. Several theories have been proposed and tested towards how this latency is established and maintained, one of which states that the viral protein Tat has a very important role to play. Studies have shown that Tat positive feedback loop supersedes the cell-driven silencing of the viral LTR transcription thus preventing the virus from staying in its latent phase. HIV-1C is known to have three functional NFκB TFBS but a recent study has shown that a strain of subtype C virus having an additional functional NFκB site is fast evolving. In this study, we separated Tat from the HIV-1C viral genome so as to have an extrinsic control over its expression which is dependent on the addition of Doxycycline. We generated isogenic variants of the virus which differ in the number of NFκB sites (0-5 sites). Using a dose response study, we found out the optimum concentration of doxycycline needed to drive Tat expression. Thus we developed a Tat inducible latency model which on addition of the viral LTR can be used to study the establishment and maintenance of latency in HIV-1C. Since we have 5 constructs varying in the number of NFκB sites, this model can further be used study how their reactivation kinetics vary.