The current ART can reduce plasma HIV-1 viremia to undetectable levels, but do not eliminate persistent latent HIV-1 reservoirs. A major form of HIV-1 latency is due to transcriptional silencing of the integrated provirus. Current efforts to eliminating these latent reservoirs include a ?wake and kill? strategy, where a Latency Reversing Agents (LRA) is used to transcriptionally activate the virus so that the reactivated cell can be recognized either by host defense system or drugs to kill them. However, the current LRA are ineffective in reversing latency. The mechanistic basis for this inefficient reversal of latency is unknown. It is hypothesized that there could be transcriptional as well as post-transcriptional blocks in these latent cells and that the LRAs may vary in their potential to overcome the post-transcriptional blocks. To facilitate ?wake and kill? approach, we will develop strategies to investigate the nature of blocks in latent cells in patient samples by applying a multiplex, single cell RNA-FISH+IF methodologies. This will be done in collaboration with world's expert in RNA-FISH technology, Dr. Robert Singer at Albert Einstein College. We will develop a temporal and subcellular spatial map of reactivation of latent HIV-1 from transcription initiation to virus particle production by investigating expression of three major specifies of mRNA and Gag protein using a combination of RNA-FISH and IF. Using this approach we will be able to determine (i) transcription and expression of three primary transcripts (un-spliced, singly and multiply spliced RNAs); (iii) nuclear export of RNA; (iv) accumulation of viral RNAs in the cytoplasm; (v) p24 protein expression; and (vii) trafficking of viral proteins to plasma membrane. We will apply these techniques to primary T-cell model of latency (Siliciano model) and resting CD4+ memory T-cells from a set of aviremic HIV-infected individuals on suppressive therapy to get a glimpse of different stages of blocks to reactivation in latent cells. These will be done in collaboration with Dr. Siliciano and Dr. Anastos. We will use PerkinElmer Pannoramic 250 Flash II Scanning microscope to detect rare reactivated cells that are positive for RNA-FISH+IF. One of the application for the single cell method is the ability to use this method to simultaneously analyze transcriptional and post-transcriptional events in small amount of sample at single cell level. We will apply this method to test the effect of LRAs not only on their intended use (transcriptional activation) but also on post-transcriptional events in primary latent cell models and in patient derived exVivo latent cells, upon reactivation. It has not been possible to investigate early stages of transcriptional and post-transcriptional mechanisms in reactivated latent cells due to their small numbers and because the current approaches use pooled samples and late time points. We believe that using single cell approach and early time points, a comprehensive account of various blocks in latent cells can be analyzed. These studies are likely to provide novel insight into the nature of blocks to reactivation in latent cells to facilitate future development of effective agents to reverse latency as part of ?wake and kill? approach.

Public Health Relevance

HIV-1 remains hidden in a latent state in immune cells of infected individuals. In order to cure infected individuals, such latently infected cells need to be eliminated by a ?Wake and Kill? approach. Understanding of various forms of latency that may exist in primary T-cell models and patient samples, is likely to provide insight into the mechanisms. This application employs sophisticated single cell procedure to study the molecular roadblocks to ?waking? latent HIV.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Exploratory/Developmental Grants (R21)
Project #
5R21AI127326-02
Application #
9298589
Study Section
Special Emphasis Panel (ZRG1-AARR-E (56)R)
Program Officer
Kuo, Lillian S
Project Start
2016-06-16
Project End
2018-05-31
Budget Start
2017-06-01
Budget End
2018-05-31
Support Year
2
Fiscal Year
2017
Total Cost
$250,500
Indirect Cost
$100,500
Name
Albert Einstein College of Medicine, Inc
Department
Type
Domestic Higher Education
DUNS #
079783367
City
Bronx
State
NY
Country
United States
Zip Code
10461