BKV is a ubiquitous polyomavirus that is carried by the majority of the human population as a harmless lifelong persistent infection. BKV becomes a dangerous pathogen in immunocompromised individuals, especially those undergoing kidney, bone marrow, or multi- organ transplants. Under these conditions BKV can undergo a rampant infection of the kidney with virus production occurring largely in the tubular epithelial cells. During productive infection BKV undergoes a program of gene expression that results in the synthesis of three early proteins called large T antigen (LT), small T antigen (ST), and truncT. These early proteins act to alter cellular gene expression, capture cellular proteins needed for replication, and to initiate viral DNA replication. Transcription from the viral late promoter is coordinated approximately with the onset of viral DNA synthesis, and results in expression of the capsid genes and the subsequent assembly of new virus particles. Nearly all our knowledge of BKV biology stems from work in cell culture systems in which a homogeneous clonal cell population is infected with a high multiplicity of virions. This allows events such as viral transcription and DNA replication, and changes in cellular transcription to be monitored as infection proceeds. However, each of these measurements is obtained from bulk culture and thus represents an average of the events occurring in individual cells. Furthermore, BKV infections of humans occur in complex tissues, such as the kidney, that consist of multiple cell types in close proximity. There is little knowledge as to how viral infection is influenced by mesenchymal or other cell types that are adjacent to infected cells, or of how infected cells influence the biology of their neighbors. In this application we will develop single-cell transcriptomics coupled with high-throughput droplet microfluidics to address these important questions. The patterns of viral and cellular transcription in individual human kidney proximal tubule epithelial cells infected with BKV will be measured by encapsulating individual cells in microdroplets and extracting RNA, with the droplets serving as reaction vessels. The RNA isolated from each cell will be barcoded such that pooled infected cell populations can be subjected to next generation sequencing (NGS) in bulk. Transcription patterns from individual cells can then be extracted computationally by decoding the barcodes. We will also perform this experiment using infected kidney epithelial cells mixed with uninfected vascular cells. This will allow the coordinate examination of viral and cellular gene expression in mixed cell populations. The successful execution of this research strategy will lead to a novel platform that is broadly applicable to many types of viruses.
Human polyomavirus BKV causes disease as a consequence of productive infection of tissues, such as the kidney, that are composed of a complex mixture of multiple cell types that vary in their susceptibility and response to infection. We propose to study BKV infections of a mixture of primary human kidney cell-types using single cell transcriptomics enabled by high-throughput microfluidics. These studies will lead to a better understanding of the effects of BKV infection of the human kidney, thereby leading to novel prognostic indicators and antiviral therapies.
