High-throughput High Content Quantification of HIV-1 Viral Infectious Output

Infection with human immunodeficiency virus (HIV-1) remains a global health issue and still drives the development of significant pathology and various comorbidities. Antiretroviral therapy (ART) can effectively suppress viral replication but is often initiated months or years after initial infection, leaving a substantial period in which viral replication progresses unchecked. While ART suppresses HIV-1 replication, it does not prohibit the development of HIV-1-associated comorbidities, highlighting a lack of understanding in the connection between replication and HIV-1-associated pathogeneses. Thus, it is critical to better define HIV-1 replication dynamics to more effectively target different stages of the viral replication cycle in distinct cell populations. Here, we show a high-content imaging reporter assay that uses modified human osteosarcoma cells expressing HIV-1 receptors (GHOST cells) which fluoresce in response to HIV-1 infection. These cells have been previously used to assess HIV-1 infectivity and tropism, but this modified assay enables rapid evaluation of large numbers of samples with consistency and replicability, while also easily integrating into existing experimental pipelines that analyze p24 secretion in collected supernatants. This also allows for direct correlation between infectivity and p24 secretion, resulting in a deeper interrogation and more robust understanding of HIV-1 infection kinetics. Institutional PermissionsAll of the primary cells used in this study were obtained from donors in accordance with Institutional Review Board protocols at the New York Blood Center and Drexel University. All studies using primary cells were performed with cells from deidentified donors and were found by the Institutional Review Board at Drexel University to be exempt from human subjects protocols (protocol number 2208009386). All experiments using in vitro cell lines or primary human cells were approved by the Institutional Biosafety Committee and at Drexel University. HighlightsO_LIThe current toolkit for evaluating in vitro HIV-1 infection dynamics does not currently include an assay providing direct, efficient assessments of infectivity in large numbers of samples. C_LIO_LIDirect assessment of viral infectivity longitudinally alongside other measurements of viral infection such as p24 secretion or amount of proviral DNA enables correlation of different stages of viral replication efficiency over time. C_LIO_LIThe high-content viral titer assay described here correlates with widely used surrogate measures of viral infectivity, such as p24 secretion, and integrates easily into existing experimental pipelines that collect supernatant from infected cultures. C_LIO_LIAdapting fluorescent reporter assays to a high-content imaging and analysis pipeline creates a high-throughput assay of direct infectivity that enables evaluation of changes in infectivity across multiple treatments or timepoints and integrates easily into existing experimental pipelines that collect supernatant from infected cultures. C_LI