Probing the effect of opioids on HIV transcription and latency using a novel dual fluorescent and bioluminescent virus

Abstract Understanding the effect of opioids on Human Immunodeficiency Virus (HIV) infection is increasingly relevant in the current opioid epidemic. One in ten new HIV infections are attributed to injection drug use and people living with HIV also utilize opioids for the management of pain or in harm reduction strategies. Furthermore, HIV is well known to enter the central nervous system where infection of glial cells including microglia and astrocytes establishes a latent viral reservoir. These infected cells contribute to the damage and dysfunctionof surrounding neurons whichmanifest as cognitive, motor, and behavioral abnormalities, collectively termed HIV-associated neurocognitive disorders (HAND). Currently, it is not well understood how opioids influence the progression of this disease within the central nervous system. Previous lines of data suggest that opioids may have stimulatory actions on glial cells, and our preliminary data shows that the opioid methadone can increase HIV proviral transcription in astrocytes. Therefore, the goal of this proposal is to understand the efficacy and mechanism by which opioids may enhance HIV proviral transcription and link this attribute to the reactivation of the latent viral reservoir within the brain. To accomplish this, we will utilize a newly developed virus called EcoHIV NL4-3 Dual Red. This virus contains a dual fluorescent and bioluminescent reporter that is driven by the HIV long terminal repeat that allows for the visualization of active proviral transcription in vivo. We hypothesize that opioids will induce proviral transcription and reactivate latent infection of the viral reservoir in the brain. Aim 1 will focus on the mechanism by which fentanyl, morphine, and methadone influence HIV proviral transcription in EcoHIV NL4-3 Dual Red infected microglia and astrocytes. We will utilize dose response experiments, inhibitors of signal transduction and CRISPR/Cas9 based genetic manipulation of Tat expression to characterize the pathways by which opioids activate HIV proviral transcription. Aim 2 will utilize latent infection of EcoHIV NL4-3 in rats coupled with in vivo imaging systems to longitudinally measure and visualize HIV infection in the brain of animals during opioid administration. Results from this proposal will identify key pathways involved in opioid-mediated transcriptional activation of HIV in glial cells. It will also establish a time-course of viral reactivation in vivo. This information can lead to altered treatment strategies in people living with HIV who use opioids for pain or through harm reduction initiatives.