Although it is widely accepted that resting, latently-infected CD4+ T-cells are a cART-resistant reservoir for HIV, little attention has been devoted to alternative reservoir sites. Using an ultrasensitive Fluorescent in situ Hybridization (FISH)-based system for detecting HIV mRNA by flow cytometry we mapped the distribution of HIV in the lung of chronically infected, asymptomatic HIV-seropositive individuals and found the virus to be present at greater abundance in alveolar macrophages (AMs) than in lymphocytes. AMs are unusual in that they are long-lived cells derived from a self-sustaining cell lineage retained within the lung. Recently, we extended our study to a cohort of patients on ART and found that those AMs positive for HIV mRNA persisted at sustained levels despite some individuals having been on ART for up to 9 years. The goal of this proposal is to assess the significance of these HIV-positive AMs as a potential reservoir with the capacity to reconstitute peripheral viremia upon cessation or failure of ART, and to identify those HIV ORFS critical to the maintenance of this cell population.

1. Are HIV mRNA-positive AMs productively infected? We will determine whether HIV mRNA-positive AMs can generate infectious virions with the potential to seed CD4+ T-cells and reconstitute viremia. We have engineered a viral reporter cell line that detects production of infectious virions from infected AMs in Malawi.

2. Are HIV-infected AMs capable of contributing to peripheral viremia upon ART failure? We are using our FISH platform to sort cells positive for HIV mRNA, enabling RNASeq analysis of viral transcriptomes, in parallel with PCR-based sequencing of viral env gene diversity. We propose generating a molecular signature of the HIV population in the AMs of individuals on suppressive cART to compare with peripheral virus isolated from those individuals who subsequently go on to fail therapy. Can we demonstrate a founder contribution?

3. Utilization of CRISPR/Cas9 for functional analysis of HIV ORFs required to sustain AM infection. Recent advances in the lab with expression of foreign genes in primary human macrophages through the use of synthetic mRNA have opened the way to functional analysis of HIV ORFs in HMDMs and AMs infected with HIV. We propose systematic analysis of HIV non-structural ORFs to define those viral proteins required to maintain HIV infection, promote cellular viability and minimize immune recognition.

4. Can we model the HIV/AM interactions with NHP/SIV infections? Our collaborators, Dr. Charles Scanga, University of Pittsburgh and Dr. Shelby O’Connor, University of Wisconsin, are conducting a NHP study of SIV/M.tb co-infection. We will analyze these macaques during early SIV infection to determine whether NHP infected with SIV exhibit efficient seeding of the AM population. Further analysis of these infected cells will be guided by the RNASeq data gained in Aim 2 and the CRISPR/Cas9 editing data from Aim 3.