Recent advances in single-cell technologies uncovered a significant degree of functional and phenotypic heterogeneity between individual cells. How such cell-to-cell variation affects HIV-1 transmission by virus- producing cells is unclear. We have developed single-cell assays to quantify virus production and transmission by individual virus-infected cells in vitro. Our preliminary studies using both quantitative RT-PCR and single-cell viral transmission assays revealed substantial single-cell variations in both virus yield and transmissibility. Namely, a fraction of individual cells produced a large number of virions whereas there was a significant portion of single cells that barely release virions. Such immense cellular heterogeneity should have broad implications for HIV-1 biology, from viral transmission, dynamics to latency. The major goal of this proposal is to gain quantitative, mechanistic and biological insights into cell-to-cell variation in HIV-1 transmissibility at the single-cell level.
In specific aim 1, we will determine cell fate upon HIV-1 infection with single-cell resolution by using reporter virus and the single-cell virion production assay. Longitudinal monitoring of chronically infected cells at the single cell level will allow us to characterize temporal changes of virus production, providing important information about how individual virus-infected cells shut off virion production before transitioning to a transcriptionally latent state.
In specific aim 2, we will track individual cells to analyze their ability to produce and transmit HIV-1. Rates of virion production by single cell can be studied by time-course analysis. We will also estimate the extent of cell-to-cell variation in viral transmission by utilizing a barcoded viral library for high- throughput tracking of single cell infection events in vitro and also in vivo by using a humanized mice model.
In specific aim 3, we will use single-cell assays to address how virally induced diversity and cell-intrinsic heterogeneity contribute to cellular differences in infectiousness.
Individual virus-infected cells may differ from each other in their ability to produce virions and transmit the virus to nave target cells. The goal of this project is to provide a quantitative and mechanistic understanding of cell- to-cell heterogeneity in HIV-1 replication at the single-cell level. New findings will provide novel insights into initial events during viral transmission and the establishment of viral latency.
